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UNDERSTANDING THE MOLECULAR MECHANISM OF TRP CHANNEL

ACTIVATION/INHIBITION BY STRUCTURAL ANALYSIS

by

AMRITA SAMANTA

Submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Advisor: Vera Moiseenkova-Bell

Department of Physiology and Biophysics

CASE WESTERN RESERVE UNIVERSITY

August 2018

CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

AMRITA SAMANTA

Candidate for the degree of Physiology and Biophysics*

Witold Surewicz (Committee Chair)

Sudha Chakrapani

Vera Moiseenkova-Bell

Phoebe Stewart

Derek Taylor

Isabelle Deschenes

May 29, 2018

*We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents

List of Tables vii

List of Figures viii

Acknowledgments xi

List of Abbreviations xiii

Abstract xv

Chapter 1: Introduction

1.1 The Transient Potential (TRP) family of channels 2

1.2 TRPC subfamily 2

1.2.1 TRPC1 3

1.2.2 TRPC4 & TRPC5 5

1.2.3 TRPC2, TRPC3, TRPC6 & TRPC7 6

1.3 TRPV Subfamily 7

1.3.1 TRPV1 7

1.3.2 TRPV2 10

1.3.3 TRPV3 & TRPV4 11

i

1.3.4 TRPV5 & TRPV6 12

1.4 TRPM Subfamily 13

1.4.1 TRPM1 & TRPM3 14

1.4.2 TRPM4 & TRPM5 15

1.4.3 TRPM2, TRPM6 & TRPM7 16

1.4.4 TRPM8 17

1.5 TRPA Subfamily 18

1.6 TRPML Subfamily 20

1.6.1 TRPML1 21

1.6.2 TRPML2 & TRPML3 22

1.7 TRPP Subfamily 23

1.7.1 Polycystin family 25

1.7.2 TRPP2, TRPP3 & TRPP5 27

1.8 Purpose of this study 29

1.9 Cryo Electron Microscopy 30

Figures 32

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Chapter 2: Understanding the molecular mechanism of

the TRPV2 channel gating during activation by cryo-EM

2.1 Introduction 37

2.2 Materials and Methods

2.2.1 Stable cell line generation and flux assay 38

2.2.2 expression and purification 40

2.2.3 Cryo-EM data collection 40

2.2.4 Image processing 41

2.2.5 Model building 42

2.3 Results

2.3.1 Role of the pore turret domain in TRPV2 activation and large 43 organic uptake

2.3.2 Architecture of the TRPV2 in apo- and CBD-activated states 44

2.3.3 Conformational differences between apo- and CBD-activated 47 TRPV2

2.4 Discussion 49

Figures 52

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Chapter 3: Understanding the ligand induced conformational changes of mouse TRPA1 channel during activation and inhibition

3.1 Introduction 71

3.2 Materials and Methods

3.2.1 Expression and purification of TRPA1 74

3.2.2 Limited proteolysis of TRPA1 75

3.2.3 LC-MS analysis 75

3.3 Results

3.3.1 Limited proteolysis and in- mass spectrometry to 77 determine ligand induced conformational changes in TRPA1

3.3.2 Analysis of NMM (electrophilic ) induced 79 conformational change in TRPA1

3.3.3 Analysis of PF-4840154 (non-electrophilic agonist) induced 80 conformational change in TRPA1

3.3.4 Analysis of (non-electrophilic modulator) induced 82 conformational change in TRPA1

3.3.5 Analysis of A-967079 (non-electrophilic antagonist) induced 83 conformational change in TRPA1

3.4 Discussion 84

iv

Table 89

Figures 90

Chapter 4: Discussion and future directions

4.1 Summary 103

4.2 Impact of this study

4.2.1 Application for structural studies of other memorable 105

4.2.2 Application on health and disease 106

4.3 Future directions

4.3.1 Permeation of large organic cations through TRP channels 107

4.3.2 Charecterization of TRPV2 specific blocker and 108 understanding the mechanism of TRPV2 inhibition

4.3.3 Resolving structure of full length TRPA1 reconstituted into 109 nanodisc by using cryo EM

4.3.4 Regions of TRPA1 affected by other electrophilic 110 and non-electrophilic ligands

4.4 Concluding remarks 110

Figures 112

Appendix 117

v

Reference 119

vi

List of Tables

Table 3.1. Modulators of TRPA1 used in this study 89

vii

List of Figures

A schematic representation of the TRP superfamily of ion Figure 1.1 32 channels

High resolution structures for TRP channels initially Figure 1.2 33 resolved

A schematic representation of the TRP channel pore Figure 1.3 34 showing the two regions of constriction (gates)

A schematic representation of the TRPP1 and TRPP2

Figure 1.4 interacting with each other through their C-terminal region and 35

forming the receptor-channel complex

Functional characterization of wild type and truncated

Figure 2.1 TRPV2 indicates the pore turret region is essential for 52

passage of both Ca2+ and large cations

Figure 2.2 Summary flowchart of TRPV2 data processing 53

Figure 2.3 Resolution data for TRPV2 refinement 54

Cryo-EM analysis of apo and CBD-activated TRPV2 Figure 2.4 55 channel

Figure 2.5 Structural details of apo and CBD-activated TRPV2 56

Structural comparison of 4.6Å apo TRPV2 and previously Figure 2.6 58 published ~5Å apo TRPV2 channel

Figure 2.7 Putative CBD binding site 59

viii

Cryo-EM density map comparison of apo and CBD-

Figure 2.8 activated TRPV2 channel indicates large conformational 60

change in the TMD but not in the ARD

Comparison of apo and CBD-activated TRPV2 models and Figure 2.9 62 TMD

Figure 2.10 Architecture of the apo and CBD-activated TRPV2 pores 64

Structural comparison of full length apo TRPV2, truncated Figure 2.11 65 apo TRPV2 and apo TRPV1 pores

Figure 2.12 Comparison of TRPV pore architectures 67

Figure 2.13 Comparison of the pore region of the TRPV channels 68

Electron density corresponding to regions of interest in the Figure 2.14 69 TRPV2 structures validates model assignment

Figure 3.1 TRPA1 purification and limited proteolysis of pure TRPA1 90

Construction of a representation of the full-length TRPA1 Figure 3.2 91 channel

Figure 3.3 Effect of NMM on TRPA1 conformation 92

Nano- LC-MS/MS spectrum of the modified after Figure 3.4 94 Asp-N digestion of NMM-activated TRPA1

Effect of non-electrophilic modulators on TRPA1 Figure 3.5 96 conformation

Figure 3.6 Effect of A-967079 on TRPA1 conformation 97

Dimer representations of TRPA1 showing additional Figure 3.7 99 regions of miscleavage upon ligand interaction

ix

Cartoon representation of TRPA1 showing the regions Figure 3.8 101 involved in channel gating

Cartoon representation of gating mechanism of TRPV2 in Figure 4.1 112 presence of CBD

A tree representing the mammalian TRP superfamily of ion Figure 4.2 113 channels with most of the high resolution structures

Schematic representation of the principle of limited Figure 4.3 114 proteolysis followed by mass spectrometry

Inhibition of TRPV2 activity by PL and preliminary cryo-EM Figure 4.4 115 data of PL-inhibited TRPV2

Cryo-EM analysis of CBD-activated and PL-inhibited Figure 4.5 116 TRPV2 channel

x

ACKNOWLEDGEMENTS

I am especially thankful to my PhD advisor, Dr. Vera Moiseenkova-Bell, for her professional and personal support and guidance during my training as a graduate student in her lab. I would like to thank the previous and present lab members of the Moiseenkova-Bell lab: Dr. Matthew Cohen, who taught me some of the molecular biology techniques and helped me to think critically about projects during the initial few years of my journey; Dr. Kevin Huynh, who taught me a lot of the molecular biology techniques that I used to complete this dissertation; Monica

Kane, who was an undergraduate in the lab and brought a lot of fresh energy to the lab; Taylor Hughes, who is a great lab mate and colleague I enjoyed working and learning new techniques together with in the lab; Dr. Ievgen Ignatenko, a great lab mate who is always ready to help. I was also fortunate to have the opportunity to train an undergraduate student, Connor Dawedeit and a high school student,

Fatema Uddin, both of whom brought a lot of young energy to the lab. I would like to thank my dissertation committee members (Dr. Witold Surewicz, Dr. Sudha

Chakrapani, Dr. Phoebe Stewart, Dr. Derek Taylor and Dr. Isabelle Deschenes) for guiding me to think critically, forcing me to stay focused and supporting me in completion of my dissertation. Additionally, I would like to thank Heather Holdaway and Dr. Sudheer Molugu, who trained me in cryo-EM microscopy; Dr. Janna

Kiselar who helped me with mass spectrometry; Dr. George Dubyak who trained me in cell culture and Ca2+ flux assay measurements; Dr. David Lodowski for his collaboration and support, Dr. Yuhang Liu and Dr. Seungil Han for their collaboration and allowing to use their electron microscope. Moreover, I would like

xi to acknowledge Dr. Sudha Chakrapani who taught me the technique of and gave me space in her lab during the last few months of my graduation when my lab relocated to UPenn. In addition, I would also like to thank

Chakrapani lab members for being great colleagues to work with and good friends.

Thank you to my family for believing in me; Diptendu konar – I could not have completed this journey without you and your constant support and Adhrit for being such a patient and understanding child. Finally, I would like to thank all the professors, friends and colleagues whom I did not mention here but who were essential for my success in graduate school.

xii

List of Abbreviations

ADPKD Autosomal Dominant Polycystic Disease

AITC Allyl

ARD Repeat Domain

CBD

Cryo-EM Cryo Electron Microscopy

DADS Diallyl Disulphide

GBM Glioblastoma Multiforme

ICRAC Release-Activated Calcium Current

IP3 Inositol Triphosphate

ISOC Store Operated Calcium Current

LEL Late Endosome and

LC-MS Liquid Chromatography Mass Spectrometry

NaV channels Voltage gated channels

NHERF Na+/H+ exchanger regulatory factor

NOMPC No Mechanoreceptor Potential C

PKD Polycystic Kidney Disease

PL Piperlongumine

xiii

REJ Receptor for Egg Jelly

RTX/DkTx /Double-knot Toxin

S1-S6 Transmembrane helices 1 through 6

SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel

Electrophoresis

SOCE Store-Operated Calcium Entry

TMD Transmembrane Domain

TMZ Temozolomide

TRP Transient Receptor Potential

TRPA Transient Receptor Potential Ankyrin

TRPC Transient Receptor Potential Cannonical

TRPM Transient Receptor Potential Melastatin

TRPML Transient Receptor Potential Mucolipin

TRPP Transient Receptor Potential Polycystin

TRPV Transient Receptor Potential Vanilloid

VSD Voltage Sensing Domain

xiv

Understanding the Molecular Mechanism of TRP Channel

Activation/Inhibition by Structural Analysis

Abstract

By

AMRITA SAMANTA

Transient Receptor Potential (TRP) superfamily of ion channels are evolutionarily conserved integral membrane proteins. The 28 members of the mammalian TRP superfamily are cation permeable channels which are grouped into six subfamilies based on their . This superfamily of ion channels is involved in a vast array of physiological and pathophysiological processes including , neuropathic , , , diabetes and polycystic kidney disease. Therefore, the study of these channels is imperative to our better understanding of subcellular biochemistry. The latter half of the 20th century marked the discovery of TRP channels and a lot is known about their physiological characteristics and functions. But until the last couple of years, not much was known about their structure and molecular mechanisms underlying their biophysical and physiological properties. Here I utilized our ability to heterologously express and purify full length, stable and functional mammalian

xv

TRP channels to gain insight into the molecular mechanism of gating of two of the

TRP channels: TRPV2 and TRPA1. Using cannabidiol (CBD), an activator of

TRPV2 channel, we tested the hypothesis that the pore turret region of the channel is important for opening of its ion conduction pore and could also capture the open state structure of TRPV2 using cryo electron microscopy (cryo-EM) revealing the gating mechanism of this channel during activation. In case of TRPA1, it was known that certain residues are critical for channel activation with electrophilic ligands, however the mechanism of channel gating was unknown.

Moreover, not much was known about effect of non-electrophilic modulators on this channel. In second part of this study I illustrated the regions of TRPA1 that undergoes ligand induced conformational changes using limited proteolysis coupled with mass spectrometry. The results showed that in case of

TRPA1, irrespective of the ligand, the linker region and the N-terminal ankyrin repeats experience conformational changes leading to activation or inhibition of the channel.

xvi

Chapter 1

Introduction

Portions of this chapter were published in:

Samanta A, Hughes TET, Moiseenkova-Bell VY. Transient Receptor Potential

(TRP) Channels. Subcell Biochem. 2018;87:141-165. 1.1 The Transient Receptor Potential (TRP) family of ion channels

In order to survive, organisms have adapted to rapidly and accurately sense the environment around them. One group of biomolecules that play a key role in interpreting these environmental stimuli are a class of integral called Transient Receptor Potential (TRP) channels. TRP channels are a class of cationic channels that act as signal transducer by altering membrane potential or intracellular calcium (Ca2+) concentration. The TRP channel era began in 1969 when Cosens and Manning discovered a phenotype in that exhibited as blindness in the presence of constant bright light [1]. This mutant strain was named trp, transient receptor potential, and cloning of the mutated trp identified the first member of the TRP superfamily [1]. This superfamily constitutes a diverse group of polymodal ion channels that are mostly conserved from nematodes to . Based on sequence homology the mammalian TRP channel superfamily is classified into 6 subfamilies (Figure 1.1): TRPC (Canonical),

TRPV (Vanilloid), TRPM (Melastatin), TRPA (Ankyrin), TRPML (Mucolipin), and

TRPP (Polycystic). The first four subfamilies are categorized as group 1 and the last two constitute group 2.

1.2 TRPC subfamily

The first subfamily of mammalian TRP channels studied is known as the

TRPC subfamily or the canonical TRP channels. The founding member TRPC1 was cloned in 1995 and was discovered based on its sequence homology to the

Drosophila trp gene [2]. The seven members of this subfamily are commonly

2 divided into four groups based on sequence homology (I) TRPC1; (II) TRPC2; (III)

TRPC3, TRPC6, TRPC7; and (IV) TRPC4 and TRPC5 (Figure 1.1).

All seven members of this subfamily are structurally similar having 6 transmembrane helices, a putative hydrophobic pore forming loop, three to four ankyrin repeats, coiled-coil domains in the N- and C-terminus, a C-terminal proline rich region, a /IP3 binding region and what is known as the TRP motif

[3, 4]. TRPC channels have been shown to form both heterotetramers and homotetramers within the TRP channel superfamily, with different members having certain preferences, for example, TRPC1 forms physiologically relevant functional channels with several TRPC channels including TRPC4, as well as, TRPV1, and

TRPP2 [5, 6].

The TRPC subfamily channels, like most other TRP channels, function as

Ca2+ permeable plasma membrane channels with varying Ca2+ selectivity [4]. The mechanism of regulation of these channels is contentious, with the two main hypotheses being store-operated channel activation and receptor-activated channel regulation [4]. It appears, based on the current body of scientific research, that different members of this subfamily are activated based on one or both of these two methods depending on the expression system, expression level and the used. Other research has also suggested mechanosensitive gating mechanisms [6]. In spite of these experimental limitations, much is known about this subfamily of TRP channels.

1.2.1 TRPC1

3

The founding member of this subfamily can also be considered the founding member of the mammalian TRP channel superfamily. TRPC1 was first cloned by the Montell group in 1995 because of its high sequence homology (~40%) to the

Drosophila trp gene [2]. This invertebrate trp gene was proposed to be a store- operated Ca2+ channel and therefore mammalian homologues had potentially the same function. TRPC1 was originally found in fetal , liver and kidney tissues as well as adult , testes, and brain [2]. Since then, it has been found to be broadly expressed in mammalian tissues [6]. TRPC1 knockout mice have emerged as a highly successful tool for research of this channel in vivo [6]. These models have implicated TRPC1 in differentiation, organism growth and development, immune regulation, tumor and Parkinson disease

[6]. In spite of being involved in these critical biological functions, TRPC1-/- mice have been reported to live a healthy and normal life [6].

TRPC1 monomers have been shown to create functional channels with not only other TRPC members, but also with TRP members outside its subfamily. For example, Tsiokas et al. showed that TRPC1/TRPP2 heterotetrameric channels form and operate in membrane bilayers [6, 7]. TRPC1 has also been shown to interact with members of the TRPV subfamily [6]. TRPC1 undergoes alternative splicing and there are currently five known splice variants with only three that have been shown to be translated to functional proteins [6]. Different splice variants have been reported to affect channel functionality especially in heteromeric channels [6]. Recently, there has also been a start codon found upstream of the

4 currently used start codon for TRPC1. This has produced an extended form of

TRPC1, which could be of interest for future studies of the protein [6].

As mentioned previously, TRPC1 was originally discovered as a potential store-operated Ca2+ channel, but characterization of this channel did not show it to

2+ have the predicted traits of a store operated Ca channel or ICRAC activity [5]. Still,

TRPC1 has been shown to be involved in store-operated Ca2+ entry (SOCE) through its interactions with Orai1 and STIM1 thus designating it as having ISOC activity [5]. SOCE is a signaling cascade that is critical for an abundance of biological processes. When Ca2+ is released from the the cytosolic Ca2+ concentration increases which triggers activation of plasma

2+ membrane Ca channels (ICRAC or ISOC). Evidence suggests that TRPC1 colocalizes with STIM1 and Orai1 in endoplasmic reticulum-plasma membrane

(ER-PM) junctions wherein these two membranes exist in close proximity to one another [5]. Orai1 has been shown to be necessary for TRPC1 activation, though different splice variants of TRPC1 seem to be modulated by Orai1 differently [5].

Mechanosensitivity has also been discussed as a potential regulation method for this channel, but various expression systems have provided conflicting results to date [6].

1.2.2 TRPC4 & TRPC5

TRPC4 and TRPC5 are often grouped with TRPC1 because they, like

TRPC1, are inwardly rectifying and were originally hypothesized to be involved in

SOCE [4]. What distinguishes these two channels is their extended C-terminus that contains a PDZ binding domain. This domain has been shown to interact with

5

NHERF, a scaffold protein that links plasma membrane proteins to the actin [4, 8]. Through this PDZ domain, TRPC4 has been shown to be activated downstream of Gq-coupled receptors and receptor tyrosine [9].

The function of the PDZ domain in TRPC5 has not been as thoroughly explored.

TRPC5 has generally been found to behave as a receptor-operated [4]. Both TRPC4 and TRPC5 channel currents are potentiated by lanthanides in contrast to the inhibitory effects seen in store-operated channels and other TRPC channels [4].

1.2.3 TRPC2, TRPC3, TRPC6 & TRPC7

TRPC2 is a and therefore is not translated in humans, but it is expressed as a functional protein in other . Here, it is grouped with

TRPC3, TRPC6 and TRPC7 because these four members can all be directly activated by lipids, specifically diacylglycerol (DAG), a product of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) degradation [10]. Though the other TRPC family members have been shown to be regulated by lipids, seen through their localization to lipid rafts (LRDs) and other lipid targeted cellular locations, for these four members lipid binding is necessary and sufficient for channel activation [5, 10]. A range of affinities for lipids has been reported for all

TRPC subfamily channels, as well as a multitude of interaction mechanisms.

Currently, both direct binding of lipids to cause activation, and indirect binding of lipids using adaptor proteins are possible for activation and/or localization for

TRPC2, TRPC3, TRPC6 and TRPC7, since an exact lipid binding pocket has yet to be elucidated [10].

6

1.3 TRPV Subfamily

Transient receptor potential vanilloid (TRPV) channels were named based on the activation of the founding member of this group by , a vanilloid- like molecule. This original TRPV channel was initially known as VR1 [11]. In the

1997 study, it was shown that VR1, expressed in sensory , is activated by capsaicin, the active ingredient in chilies, by higher than 42˚C and by . Due to the structural similarity of VR1 with other known TRP channels it was later renamed TRPV1. Subsequently five additional members of this subfamily were cloned and named TRPV2, TRPV3, TRPV4, TRPV5 and TRPV6 (Figure 1.1)

[12-17]. Initially all the members of TRPV subfamily were thought to be heat like TRPV1, but extensive physiological studies and knockout mice later revealed that although TRPV2-6 have more than 50% sequence homology with

TRPV1, they do not all respond to temperature stimuli. Moreover, TRPV1-4 channels have nonselective cation conducting pores while the pores of TRPV5 and

TRPV6 are highly calcium selective. Recently, high resolution structures of TRPV1

[18-20] (Figure 1.2A and 1.2B), TRPV2 [21-23] (Figure 1.2C and 1.2D) and TRPV6

[24] (Figure 1.2E) have been resolved using cryo-electron microscopy (cryo-EM) and X-ray crystallography which has helped to elucidate the physiological characteristics of this subfamily as a whole as well as the finer details and differences of the individual members. These structures also provide the first glimpse into the architectural details of the TRP channel superfamily.

1.3.1 TRPV1

7

TRPV1 is expressed in sensory neurons and is activated by capsaicin, protons, toxins and temperature in the noxious range (>42˚C), making it physiologically important for thermal and chemical . To date, TRPV1 is the most well characterized and extensively studied mammalian TRP channel.

These studies have provided a wealth of information about its physiological and biophysical properties as well as its role in disease and its potential as a therapeutic target.

Functional TRPV1 is a homotetramer, with each monomer consisting of six ankyrin repeats in the cytosolic N-terminal domain, a transmembrane domain containing 6 transmembrane helices (S1-S6) with a pore forming P-loop between

S5 and S6 and a TRP domain in the cytosolic C-terminal domain. The crystal structure of the domain was solved in 2007 by Rachelle Gaudet’s group [25] and the first full length single particle cryo-EM structure of TRPV1 was resolved to 19Å resolution by Moiseenkova-Bell in 2008 [26].

The detailed architecture of a TRP channel was first elucidated in 2013 when the structure of a truncated, functional TRPV1 channel, called “minimal TRPV1”, was resolved to 3.4Å resolution [20] (Figure 1.2A). This was also the first time that cryo-EM was used to reconstruct the 3D structure of a small membrane protein to near atomic resolution. Additionally, the structure of TRPV1 in the presence of the vanilloid agonist capsaicin and resiniferatoxin/Double-knot toxin (RTX/DkTx) were also resolved by the same group at that time to resolutions 4.2Å and 3.8Å respectively [19]. This advancement in structural analysis of small proteins was possible due to the development of direct electron detectors, improved image

8 processing algorithms that allowed correction of motion induced blurring, and the improvement of signal to noise ratio in cryo-EM data.

The near atomic resolution structure of minimal TRPV1 in the apo state displayed a four-fold symmetry along a central ion permeating pathway. The ion conducting pore is formed by transmembrane segments 5 and 6 (S5 and S6) and the pore forming P loop, which has an overall similarity to that of voltage gated Na+ and K+ channels. However, unlike the voltage gated ion channels where S1 – S6 constitutes the voltage sensing domain and undergoes significant conformational rearrangement during channel gating, the S1-S4 of TRPV1 remains fairly static between closed, partially open and fully open states. Although the overall topology of the pore region is analogous to NaV channels, TRPV1 has a flexible selectivity filter due to the absence of hydrogen bonding within and between adjacent pore helices [20].

The flow of through the pore is controlled by a dual gating mechanism consisting of an upper gate located in the selectivity filter region and a lower gate formed by residue Ile679 (Figure 1.3). Comparison of the apo state structure with the capsaicin bound and RTX/DkTx bound structures further illustrate the dual gate architecture. In the apo state, the pore is constricted both at the selectivity filter region and at the lower gate (I679). In the capsaicin bound state, capsaicin does not affect the selectivity filter region, but the lower gate expands significantly. In the RTX/DkTx bound structure there is no apparent constriction in the ion- conducting path [19]. Recently, the structure of TRPV1 reconstituted in nanodiscs was resolved at a higher resolution by the same group [18]. The nanodisc-

9 stabilized structures elucidated some of the important putative regions of lipid interaction with TRPV1 in a membrane bilayer system. The fully open RTX/DkTx bound state showed formation of a lipid, channel and toxin tripartite complex, suggesting how toxin binding stabilizes the open state. Aromatic side chain residues, tryptophan and phenylalnine, from finger 1 and finger 2 of the doubleknot toxin forms hydrophobic interaction with the aliphatic tail of a whose polar head group interacts with channel residues Arg534, present in the extracellular loop connecting S3 and S4, and Ser629, present in the pore loop domain, respectively. Additionally, the study also suggests the vanilloid-binding pocket, between S3, S4, S4-S5 linker and the TRP domain, to be a plausible phosphatidylinositide-binding region in the apo channel [18].

1.3.2 TRPV2

The second member of the TRPV subfamily, TRPV2, was cloned because of its homology to TRPV1 [12, 17]. Like TRPV1, TRPV2 was described as a heat , specifically as a noxious heat sensor (>52˚C) in heterologous expression systems. However, later studies and knockout mice models suggested otherwise since TRPV2 knockout mice displayed normal thermo sensation and had prenatal lethality [27].

TRPV2 is ubiquitously expressed in various tissues types including both neuronal and non-neuronal tissues. This channel has been implicated in various physiological processes, such as nerve growth, and a variety of disease states, including cancer. The Moiseenkova-Bell lab has recently shown that TRPV2 has a punctate distribution in DRG neurons and other neuronal cell lines and it co-

10 localizes with Rab7, a late endosomal marker in these cell lines [28]. Although

TRPV1 and TRPV2 have 50% sequence homology and the overall structures at low resolution looks very similar, the near atomic resolution structures show some differences.

The structure of full length TRPV2 was first resolved at ~13Å resolution in

2014 and was later resolved to ~5Å in 2016 (Figure 1.2C) [21, 22]. This was the first time a full length TRP channel was resolved to near atomic resolution. Another group published ~4Å resolution structure of a truncated TRPV2 construct (Figure

1.2D) recently [23]. The pore region of TRPV2, like that of TRPV1, demonstrated two regions of constriction (Figure 1.3), one in the selectivity filter region and another at the distal part S6 helix. However, the pore of full length apo TRPV2 was shown to be much wider than the pore of apo TRPV1, suggesting apo TRPV2 may be able to accommodate partially hydrated cations and other large organic ions.

Interestingly, the lower gate of apo TRPV2 is wider than that of RTX/DkTx bound

TRPV1. The truncated structure of TRPV2 in the apo state, however, had a pore similar to that of apo TRPV1, with both the gates closed. This difference in the structures of TRPV2 can be attributed to either the truncation of the pore turret region or to the differences in channel properties between different mammalian orthologous (mouse vs rabbit).

1.3.3 TRPV3 & TRPV4

TRPV3 and TRPV4 were also cloned and hypothesized to be heat sensors due to their homology to TRPV1. TRPV3 was found to be responsible for detecting innocuous warm temperature ranging from 31˚C to 39˚C. It is expressed in various

11 tissues and organs, but the most pronounced expression of TRPV3 is in the epithelial cells of , the oral cavity and the . TRPV3 knockout mice had strong deficit in response to innocuous heat sensitivity [29].

However, a later study demonstrated that this deficit in thermo sensation was dependent on the background strain used for developing the knockout mice [30].

Recently, TRPV3 has gained importance as a channel essential in maintaining skin health.

The role of TRPV4 in thermo sensation is also controversial with studies done in heterologous expression systems, native tissues and knockout mice giving conflicting result [30-32]. TRPV4 has a wide and varied expression pattern in the body [33]. It has been shown to respond to osmotic changes in cellular environment and mechanical stress [15, 34-37]. Therefore, it has an established role in osmoregulation and in the body. A few months back the structure of truncated and mutated TRPV4 named “TRPV4cryst” was resolved by employing both cryo-EM and x-ray methodologies to a resolution of 3.8Å resolution [38].

1.3.4 TRPV5 & TRPV6

TRPV5 and TRPV6 are unique from other TRP channels in that they are highly calcium selective. Therefore, they have a significant contribution in calcium in the body. TRPV5 and TRPV6 have high sequence homology

(~75%) at the amino level and can form both homotetrameric and heterotetrameric functional units [39]. TRPV5 is exclusively expressed in the kidney, while TRPV6 has a wide tissue distribution, including in intestinal,

12 pancreatic and placental tissues. Both TRPV5 and TRPV6 are present in the apical membrane of the epithelial cells and act as the entryway for Ca2+ during absorption and reabsorption. They are constitutively active channels when present in the plasma membrane, but are inactivated in the presence of high Ca2+, preventing

Ca2+ poisoning of the cells.

Recently the crystal structure of TRPV6 was resolved with some modifications (TRPV6cryst) at 3.2Å [24] (Fig. 1.2E). Overall the structure is very similar to that of TRPV1 and TRPV2 [19, 21]. The ion conducting pathway has two gates, one in the selectivity filter region formed by Asp541, which is critical for calcium selectivity, and the other at the lower end of the pore formed by Met577, similar to that of full length TRPV2 structure [21]. It was proposed that the selectivity filter of TRPV6 is more static in contrast to other TRPV channels. The flexibility of the selectivity filter was thought to be required for pore dilation as seen in ligand bound TRPV1 channel structures[19]. A highly electronegative outer region of the pore likely assists in the recruitment of Ca2+ ions to the pore. It was further proposed that calcium permeation of TRPV6 occurs via a “knock-off” mechanism similar to that of Cav channels and the rate-limiting step in this process is knocking off the Ca2+ ion from D541 site [24].

1.4 TRPM Subfamily

The melastatin-related TRP (TRPM) subfamily contains similar structural regions compared to the other TRP subfamilies, including the presence of six membrane spanning regions, cytoplasmic C- and N-terminal domains and a C- terminal TRP motif [40]. TRPM channels also contain a C-terminal tetrameric

13 coiled-coil domain, the structure of which was solved by the Minor Jr. group in 2008 using X-ray crystallography [41]. Interestingly, each member of this subfamily contains an N-terminal ‘TRPM homology region’ that is involved in channel assembly and trafficking that is not seen in other TRP channels [40]. Another defining characteristic of this subfamily is the lack of N-terminal ankyrin repeats that is commonly seen in other subfamilies [40]. The large C-terminal sections of

TRPM channels are highly varied between subfamily members with TRPM2,

TRPM6 and TRPM7 containing active enzymatic domains in this region.

Originally, most members of this subfamily were cloned from cancerous tissues and were therefore implicated in tumorigenesis, proliferation and differentiation [40]. After nearly two decades of research, members of this subfamily have also been found to be involved in temperature sensation, magnesium (Mg2+) homeostasis and [41, 42]. The TRPM subfamily has been shown to have vastly differing modes of activation, cation selectivities and tissue distributions [43]. Here, the TRPM subfamily is presented in four groups based on structural similarity (I) TRPM1 & TRPM3 (II) TRPM4 & TRPM5 (III) TRPM2,

TRPM6 & TRPM7 and (IV) TRPM8 (Figure 1.1).

1.4.1 TRPM1 & TRPM3

The first member of the TRPM subfamily, TRPM1, originally named melastatin, is a Ca2+ permeable ion channel that was first cloned from benign [44]. This protein was found to be an indicator of aggressiveness since it was found to be expressed at higher levels in non- metastatic melanomas [44]. Due to this correlation, TRPM1 was found to be a

14 tumor suppressor and a potential prognostic marker for metastatic melanomas

[45]. In noncancerous tissues, TRPM1 is found only in the brain, melanocytes, and heart, all at very low levels [46].

TRPM3 is a Ca2+ permeable, non-selective cation channel that can function as a homotetramer or in complex with TRPM1 [47]. Molecular activators of TRPM3 include sphingolipids, sulfate (PS), and [47]. Other stimuli of TRPM3 include cell swelling and heat (40°C) [47]. Although TRPM3 was originally thought to be involved in induced release in the β-cells of the , TRPM3 knockout mice showed normal glucose metabolism [47].

Other studies have shown that physiologically TRPM3 plays an important role in heat sensation in the somatosensory system [47]. in TRPM3 have been found in hereditary diseases such as cataracts and some high-tension glaucomas [47].

1.4.2 TRPM4 & TRPM5

Both TRPM4 and TRPM5 are distinct from the other members of the TRPM subfamily, and the TRP family of channels in general, because they are impermeable to Ca2+ [48]. Rather, they are only permeable to monovalent cations including sodium (Na+) and potassium (K+) [48]. Because of this, TRPM4 and

TRPM5 are considered to be Ca2+ activated, non-specific cation (CAN) channels

[40]. Voltage modulation of both of these channels has been reported and the sensitivity to Ca2+ gating of these channels can be altered by changes in temperature, calmodulin binding, PIP2 binding and channel phosphorylation [48].

15

TRPM4 is highly expressed throughout the body with high levels present in the intestine and [46]. TRPM5, on the other hand, is expressed in discrete tissues with high expression in the intestines and taste buds [46, 48]. In the taste buds, TRPM5 has been shown to be a transducer of bitter, sweet and taste sensation [49]. This was confirmed using mice models lacking functional TRPM5.

These mice exhibited a drastic reduction in response to those , while sour and salty taste sensations were unaffected [49]. Bitter, sweet and umami taste sensations are regulated by G-protein coupled receptor signal cascades and

TRPM5 is thought to be a downstream target of these pathways [49]. The role of

TRPM5 in the intestines is thought to be related to postingestion chemosensation

[49].

1.4.3 TRPM2, TRPM6 & TRPM7

TRPM2, TRPM6 and TRPM7 are unique because they are bifunctional proteins. In addition to being cation permeable channels, these TRPM members also contain functional enzymatic domains in their C-terminal segments [40]. The catalytic domain of TRPM2 has sequence homology to Nudix hydrolases. TRPM2 has been shown to be activated by ADP-ribose and

(ROS), which implicates this channel in cellular redox sensation [40]. TRPM2 is permeable to Ca2+, Mg2+, and monovalent cations [40]. In human tissues, TRPM2 was found to be widely expressed with the highest levels in the brain, macrophages and bone marrow [46].

TRPM6 and TRPM7 are highly sequence homologous and hence both have the ability to permeate Mg2+, (Zn2+) and Ca2+, though they are most often

16 studied for their role in whole organism and cellular Mg2+ homeostasis [50]. The enzymatic domains of these two channels are classified as atypical alpha protein kinases [40]. These two TRPM members have been shown to form functional heterotetrameric channels [51]. A lack of either TRPM6 or TRPM7 was found to be embryonically lethal and several magnesium-related diseases have been linked to mutations in these channels. For example, autosomal-recessive hypomagnesemia with secondary hypocalcemia (HSH) is correlated to mutations in the TRPM6 gene [52].

In spite of these similarities, the tissue distributions of TRPM6 and TRPM7 are widely divergent. TRPM6 is found at highest levels in the intestines where it has been shown to be involved in dietary Mg2+ uptake [42, 46]. It is also found at relatively high levels in the brain, pituitary, and the (DCT) of the kidney [46]. TRPM7, on the other hand, is ubiquitously expressed in the human body [46].

1.4.4 TRPM8

One of the most thoroughly explored members of this subfamily is TRPM8, which is best known for its cold sensing ability. In addition to thermosensation,

TRPM8 has also been shown to be involved in pain sensation, , bladder function and cancer [53]. TRPM8 is a nonselective, Ca2+ permeable, outwardly rectifying cation channel [53, 54]. Of the TRPM subfamily, TRPM8 is the

2+ most selective for Ca with a selectivity ratio (PCa/PNa) of 3.3 [48]. This channel was originally cloned from a prostate cDNA library screen [55]. It was found to be involved in , specifically there was found to be an

17 dependent overexpression of TRPM8 in androgen receptor positive prostate [53]. TRPM8 is involved in a variety of other cancers including , melanomas, as well as and breast cancers [53]. In 2002, two independent research groups identified TRPM8 as a cold (<28°C) and menthol activated ion channel [56, 57]. Other chemical for TRPM8 include cooling agents such as and [53]. The temperature threshold of TRPM8 can be modulated changes in membrane potential, , lysophospholipids and PIP2 [53]. There is also evidence that G-proteins are involved in regulation of

TRPM8, specifically, Gαq is involved in the inflammation mediated inhibition of

TRPM8 [53].

1.5 TRPA Subfamily

Transient receptor potential ankyrin 1 (TRPA1) is the solitary member of the mammalian TRPA subfamily (Figure 1.1). It was discovered as an ankyrin-like , with similarities to existing TRP channels [58]. Like other

TRP channels, TRPA1 also has a cytosolic N-terminal domain, a transmembrane domain containing six transmembrane helices with a transmembrane loop between S5 and S6 and a cytosolic C-terminal domain. It is a unique channel within the superfamily in that it lacks the TRP motif and it contains 14 to 17 ankyrin repeats in the N-terminal domain. This abundance of ankyrin repeats is why it is known as the ankyrin subfamily. TRPA1 was proposed to be a putative noxious cold sensor, but it is now best described as chemo-, making it an ideal target for [59]. The TRPA1 channel is expressed in both peptidergic and non-peptidergic neurons like Aδ and C-fiber and in some myelinated Aβ-fibers

18

[60]. It is also expressed in TRPV1 expressing neurons and in non-neuronal cells such as epithelial cells, melanocytes, mast cells, fibroblasts, and enterochromaffin cells [60].

TRPA1 is a polymodal channel in a true sense. It is a homotetrameric, non- selective cation channel, activated by a long list of exogenous and endogenous compounds. The most well-known activators of TRPA1 are various cysteine and reactive electrophilic molecules like (AITC), the active ingredient in oil and , which is an extract from , from extract and from fume exhaust [61-63].

Additionally, TRPA1 is also activated by non-electrophilic molecules like menthol and [64, 65]. It is also modulated by various endogenous ligands like , H2O2, and nitrated lipids like nitro oleic acid [62, 66, 67]. Mutational studies along with other biochemical, electrophysiological and biophysical methods have established that the reactive ligands interact with TRPA1 via a cluster of cysteine and lysine residues present in the N-terminus of the channel,

[68, 69] but the molecular mechanism of TRPA1 channel modulation by non- reactive ligands is still largely unknown.

The first glimpse into the structure of TRPA1 was provided by a ~16Å resolution EM structure resolved by the Moiseenkova-Bell lab in 2011 [70].

Although this structure provided insight regarding the boundaries of the transmembrane domain and cytoplasmic domain, it gave no information about the molecular architecture of TRPA1. In 2015, a near atomic resolution structure of

TRPA1 at ~4Å was resolved using cryo-EM (Figure 1.2F) [71]. Although full length

19 protein was used for cryo-EM, 3D reconstruction was only successful for ~ 50% of the channel. Overall, the transmembrane domain structure resembles those of

TRPV1 and V2 structures, containing six transmembrane helices and a reentrant pore loop, with some differences in the pore region. The ion path is gated by two restriction points, the upper gate is formed by Asp915 (Gly643 and Met644 in

TRPV1) and the lower gate is made up of two hydrophobic residues Ile957 and

Val961 (Ile679 in TRPV1) (Figure 1.3). The outer region of the pore has two short pore helices similar to those seen in Nav channels. In the cytoplasmic region, the

C-terminus forms a central stalk-like domain, which is flanked by the ankyrin repeats of the N-terminus. Human TRPA1 is thought to have at least 16 ankyrin repeats, out of which only 5 were resolved in this ~4Å structure, with the other 11 ankyrin repeats remaining unresolved (Figure 1.2F) [71]. 3D reconstructions of TRPA1 both in the presence of agonist and antagonists were performed, but no differences between the open and closed structures were identified. Therefore, these structures have served to elucidate the general molecular structure of TRPA1, but a full length TRPA1 structure and the molecular mechanisms of channel activation have remained elusive.

1.6 TRPML Subfamily

The mucolipin transient receptor potential (TRPML) subfamily consists of

2+ three Ca permeable cation channels known as TRPML1 (MCOLN1), TRPML2

(MCOLN2) and TRPML3 (MCOLN3) (Figure 1.1). Physically, these channels are the smallest in the TRP superfamily with each channel measuring <600 amino acids in length [72-74]. Though they have the typical TRP six transmembrane

20 segments with cytosolic N- and C-termini, this subfamily has not been reported to contain any ankyrin repeats or a complete TRP motif [72-74]. Additionally, these channels unlike others in the family are mostly localized to intracellular compartments instead of the plasma membrane [75]. The localization of these channels is controlled by two di-leucine motifs, one in the N-terminus and the other in the C-terminus [76]. Their function in intracellular compartments is not completely understood, but they are implicated in a variety of vesicular trafficking events [75]. Another distinguishing feature of this subfamily is the presence of a large highly N-glycosylated luminal loop between the first and second transmembrane segments [76]. Homomeric and heteromeric complexes for all three channels have been reported [77].

1.6.1 TRPML1

The first member of the TRPML subfamily was discovered because of its role in the autosomal-recessive lysosomal storage disease (LSD) known as type IV mucolipidosis (ML-IV) [78]. Patients with ML-IV have a in the TRPML1 gene which manifests on a cellular level as an accumulation of late endosomes and (LELs) and a build up of lysosomal storage materials [76]. These patients show cognitive impairment and compromised motor skills beginning in years 1-2 of life and this disorder has been shown to have a degenerative component [75]. Research regarding TRPML1 tends to focus on understanding this disease in order to produce treatments for these patients.

TRPML1 is an inwardly rectifying Ca2+ permeable cation channel that is localized to LELs [76]. This channel is ubiquitously expressed and has been shown

21 to be involved in membrane trafficking, , and LEL ion homeostasis [76]. Currently there are no known splice variants of human TRPML1

[76]. There is a predicted phosphatidylinositol 3,4-bisphosphate (PI(3,5)P2) binding site in the N-terminus of this protein and, like TRPML2 and TRPML3, the presence of PI(3,5)P2 activates the channel [76]. For TRPML1, it has also been reported that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) can inhibit activation [75]. This is interesting because PI(3,5)P2 is found in higher abundance in intracellular vesicles and PI(4,5)P2 is present mostly on the plasma membrane [75, 76]. This could potentially mean that even though TRPML1 can be found on the plasma membrane, it may not be active due to the presence of PI(4,5)P2 [75, 76]. TRPML1 is permeable to a wide range of divalent and monovalent cations including Ca2+,

Fe2+, Na+ and K+. While TRPML1 is not permeable to protons, it is pH sensitive

[76].

1.6.2 TRPML2 & TRPML3

TRPML2 is the least studied of this subfamily because it has not been implicated in any disease or phenotype [77]. Trafficking of TRPML2 is mediated via the ADP-ribosylation factor (ARF)-6 associated pathway [75]. This channel is found primarily in lymphoid and myeloid tissues and due to this distribution, recent studies have focused on the role of TRPML2 in the immune system [77]. TRPML2 co-localizes with several immune-associated proteins such as MHC-I, TLRs and

Fc-εRI and is thought to be involved in trafficking these proteins [77]. Unlike many other TRP channels TRPML2 is not regulated by changes in Ca2+ levels or linoleic

22 acid, but it is as mentioned previously activated by the presence of PI(3,5)P2 and is pH sensitive [76, 77].

While TRPML3, like TRPML2, does not have any known role in human diseases, this channel is of interest because of its involvement in varitint-waddler

(Va) mouse phenotypes [77]. In short, the gain of function mutation A419P in

TRPML3 causes a Ca2+ overload in melanocytes which results in a loss, vestibular disfunction and abnormal pigmentation in mice [77]. Additionally, a correlation has been shown between downregulated TRPML3 and viral and bacterial infections such as cytomegalovirus infection, hemophilus infection and chlamydial infection [79].

TRPML3 is localized to early endosomes, recycling endosomes, and LELs

[79]. The tissue distribution of TRPML3 is broad with higher expression levels in the organs of the endocrine system as well as the kidney, intestines and [79].

High sodium levels and low pH have been shown to inactivate this channel [79].

The pH sensitivity of this channel is controlled by regions in the luminal loop between first and second transmembrane region [79]. Several splice variants of human TRPML3 have been reported [79]. TRPML3 knockout mice have been shown to live healthy normal lives, though the model is limited due to the differences in tissue expression of TRPML3 in mice and humans [79].

1.7 TRPP Subfamily

The transient receptor potential polycystin subfamily of TRP channels constitutes of integral membrane proteins, mutations in which attribute to the pathological condition called autosomal dominant polycystic kidney disease

23

(ADPKD). The founding members of this group, TRPP2 (PKD2) and TRPP1

(PKD1) were identified by positional cloning of disease causing of ADPKD

[80-82].

ADPKD is one of the most common, monogenic, progressive disorders affecting 1 in 400 – 1000 humans. It is characterized by formation of fluid filled cysts and enlargement of the kidneys gradually leading to renal failure. ADPKD is also associated with formation of cysts in extra-renal tissues like the liver, kidney stone formation, as well as and cardiovascular abnormalities.

Mutation of the PKD1 gene accounts for 85% of the disease and the PKD2 gene mutation account for all other cases. These two mutations have indistinguishable pathologies.

The TRPP sub-family of TRP channel is present throughout the animal kingdom and , making it likely the most ancient member of the TRP family

[83]. TRPP2 is structurally homologous to other TRP channels in having 6 transmembrane helices and intracellular amino and carboxyl termini. However,

TRPP1 has a large extracellular N-terminus, 11 transmembrane helices and a relatively small cytoplasmic C-terminus. It does not have much semblance to the prototypical TRP channel structure and is also not a functional ion channel.

Therefore, it was renamed as Polycystin1 (PC1), an integral membrane glycoprotein. Polycystin1 together with TRPP2 form a receptor-channel signaling complex, which plays important physiological roles from maintaining left-right symmetry to tubular . After the discovery of the two founding members, two other TRPP proteins, TRPP3 and TRPP5 (Figure 1.1) and four other

24

Polycystin1-like proteins, PKD1L1, PKD1L2, PKD1L3 and PKDREJ (TRPP4) were identified.

1.7.1 Polycystin family

Polycystin1 (PC1), one of the founding members of the TRPP subfamily, is a 462kDa integral membrane protein whose domain architecture suggests that it is involved in receptor signaling and cell adhesion. Polycystin1 has a wide tissue distribution and is highly expressed in the tubular epithelial cells of the kidney, pancreas, liver and brain. Perinatal lethality of knockout mice due to large cystic kidneys suggests that Polycystin1 plays an important developmental role in the kidney [84]. In the fetal kidney, Polycistin1 is present in both the apical and basolateral side of the tubular epithelial cells. However, in the adult kidney it is localized to the basolateral side at the interface between cell contact and [85]. Polycystin1 forms multiprotein complexes with various cell adhesion proteins thereby participating in cell adhesion. It also forms a signaling complex with TRPP2 where Polcystin1 may act as a receptor for the TRPP2 ion channel. Although, Polycystin1 was initially named as TRPP1, due to its lack of significant structural and functional characteristics of a TRP channel, its nomenclature has been changed to Polycystin1.

To date, no structure of Polycystin1 is available, but from sequence analysis it is predicted to have a large extracellular N-terminus with various motifs for protein-protein and protein-ligand interaction, a transmembrane domain containing

11 transmembrane helices and a relatively small C-terminus. The extracellular domain contains two cysteine flanked, leucine rich domains that have been shown

25 to interact with various extracellular matrix proteins like collagen type 1, laminin and fibronectin [86]. It also has a putative C-type lectin domain similar to those found in selectin and might play role in protein carbohydrate interaction and Ca2+ sensitivity. The most interesting feature of the large extracellular domain of

Polycystin1 is the presence of 16 immunoglobulin (IG) like PKD domains (Figure

4). The 10th PKD domain is highly conserved between humans and Fugu fish suggesting a role in ligand binding. The most distal part of the extracellular domain contains a stretch of 1000 residues that is homologous to the sea urchin receptor for egg jelly (REJ). The REJ domain is required for proteolytic cleavage of the extracellular domain of polycistin1 in a G-protein coupled receptor proteolytic site [87]. The transmembrane domain of Polycystin1 has 11 transmembrane helices (S1-S11) out of which S6-S11 share high sequence homology to the transmembrane domain of TRPP2 protein and contains an equivalent loop domain between S6 and S7. The short C-terminus of Polycystin1 has multiple potential phosphorylation sites and is involved in protein-protein interaction. It has a putative coiled coil domain that interacts with TRPP2, forming the receptor-channel complex (Figure 1.4). The C-terminus can be cleaved and translocated to the nucleus in a process resembling Notch mediated signaling via regulated intramembrane proteolysis [88].

Four other proteins that are related to Polycystin1 were identified by homology cloning and are now part of the Polycystin family. The members of polycystin family have high sequence homology in their transmembrane segment but lower sequence similarity in the C-terminus and N-terminus. However, the

26 family can be further categorized into two subsets based on their structural and physiological characteristics. PC1 and PKD1L1, both have a coiled coil domain in their C-terminus, but lack a functional ion channel-forming motif. On the contrary,

PKD1L2, PKD1L3 and PKDREJ contain an ion transport motif but lack the coiled coil domain in the C-terminus.

1.7.2 TRPP2, TRPP3 & TRPP5

TRPP2, a founding member of the TRPP subfamily, is the quintessential

TRPP protein with an overall structural homology to other TRP ion channels. It has a ubiquitous expression pattern in the body although its precise sub-cellular localization and intracellular trafficking is debated, but there is general agreement about its presence in the primary cilia. TRPP2 is a tetrameric, nonselective monovalent and divalent cation permeable channel implicated in a wide array of cellular functions including fertilization, proliferation, and polarity [89]. Each monomer of TRPP2 is a ~110kDa protein containing a cytosolic

N-terminal domain, a transmembrane domain made of 6 transmembrane helices

(S1-S6) with a pore forming loop between S5 and S6 and a cytosolic C-terminus.

The N-termini of TRPP channels are devoid of ankyrin repeats, but the C-terminal region contains a coiled coil domain, which is likely involved in protein-protein interaction, and an EF-hand motif. A unique feature of TRPP channels is the presence of a large extracellular loop between S1 and S2 consisting of 245 amino acid residues in TRPP2, 224 residues in TRPP3 and 225 amino acid residues in

TRPP5 proteins.

27

Recently the structure of a “minimal” TRPP2 (hPKD2:198-703) construct reconstituted in nanodiscs and “truncated PC2 construct” in detergent were resolved to 3Å and 4.2Å resolution respectively, using cryo-EM (Figure 1.2G and

1.2H) [90, 91]. This structure gives a detailed understanding of the architecture of

TRPP transmembrane domain. Overall the topology of this region is similar to other known Group1 TRP protein structures (TRPV1, TRPV2 and TRPA1) with S1 – S4 forming the voltage sensing domain (VSD) and S5 and S6 along with the reentrant pore loop forming the ion-conducting pathway. The pore of the TRPP2 channel also has two points of restriction, one residing in the selectivity filter region (Ile641,

Gly642 and Asp643) and the second one located at the distal end of the pore

(Leu677 and Asn681) near the inner leaflet of the bilayer (Figure 1.3). The archetypical extracellular loop between S1 and S2 of TRPP proteins is named as the “polycystin” domain by the authors and acquires a novel three-layered fold; with the top layer comprising three α helices, the second layer made up of a five- stranded antiparallel β-sheet and the third layer consisting of two fingers (finger 1 and finger 2). This polcystin domain contributes to channel assembly via interaction with adjacent polycystin domain of other subunits and might also be instrumental in allosterically modulating the channel due its strategic location above the VSD.

TRPP3 was the first homologue of TRPP2 to be identified [92] followed by

TRPP5. TRPP3 and TRPP5 have high levels of sequence homology to the transmembrane segment of TRPP2, 78% and 71% respectively. However, the amino and the carboxyl termini have a very low degree of sequence homology.

28

The major difference between TRPP2 and TRPP3 is in their amino termini where

TRPP3 lacks a 100 amino acid segment. TRPP3 is expressed in a wide variety of tissues including neurons, testis, kidney and non-myocyte cardiac tissue. The precise physiological function of TRPP3 and TRPP5 is still elusive.

1.8 Purpose of this study

TRP channels are polymodal in true sense in that most of the superfamily members can be activated by a multitude of stimuli. They were known to be vital for sensory physiology, but recent studies have established that TRP channels are crucial role players in various other physiological and pathological conditions like cancer, renal physiology, cardiac health and neuronal development. Therefore, most TRP channels are important therapeutic targets. Though there is much known about these channels, much controversy exists in this field regarding their physiological functions. This may be a result of majority of the TRP channels originally being identified through sequence homology rather than function derived identification. Recent advancement in the field of cryo-EM has enabled in resolving near atomic resolution structures of a lot of the TRP channels giving a glimpse into their molecular architecture. The structural information will help in establishing some of the functional characteristics of these channels and will also be beneficial for drug development. Resolving more structures and answering more structure related queries will be important in better understanding this family of ion channels.

In this dissertation, in the first part we have resolved the structure of full length apo TRPV2 at a better resolution than previously reported and the structure of CBD-activated full length TRPV2. Comparisons between these two structures

29 revealed a potential gating mechanism of channel activation at molecular level.

Additionally, in the second part of the dissertation, we investigated the effect of electrophilic and non-electrophilic compounds on TRPA1 architecture. Our study revealed that irrespective of their chemical structure or nature, these modulators cause conformational changes in the ankyrinn repeats and linker region comprised of the pre-S1 helix, the TRP-like domain, the linker between TMD and N- and C- terminus of the TRPA1 ion channel.

1.9 Cryo Electron microscopy

One of the major technique used in this study was cryo-EM. This is a powerful technique that is routinely used to elucidate the structural architecture of biological macromolecules in a physiologically relevant environment. The fundamental theme of structural biology is to understand and provide the mechanistic details of how these macromolecules perform their intricate task in a living cell. Cryo-EM allows us to study proteins and their complexes in a dynamic state, giving insight into how these molecules behave in their various functional states. The methodology of cryo-EM involves freezing purified protein in its aqueous buffer into a thin layer of vitreous ice. The protein molecules are embedded in various orientation in this thin layer of ice which are then imaged under an electron microscope. The images or raw data are then processed using various software algorithms available resulting in high resolution structures of the protein molecule or complex.

Electron microscopy has been used since a long time but in the mid- seventies the era of cryo electron microscopy successfully started [93]. The first

30 atomic resolution structure of a protein was resolved in 1990 [94, 95]. However, the high resolution structures were limited for macromolecules and soluble proteins. But in the year 2013, the field of cryo EM witnessed a resolution revolution. This was the first time that a membrane protein was resolved to near atomic resolution by cryo EM [19, 20]. The two major contributing factors for this revolution were: 1) The development of Direct electron Detector Devices (DDD).

The direct electron detection method allows to compensate for radiation damage to the sample and also increases the signal-to-noise ratio allowing for better resolution; 2) Application of Maximum-likelihood approach for data processing algorithms. With these developments in the field high resolution structures of many proteins that were previously unresolved, has been resolved in the past couple of years.

31

Figure 1.1. A schematic representation of the TRP superfamily of ion channels. The 28 members of the mammalian TRP channels are divided into 6 subfamilies. Each branching group is representative of a subfamily of channels.

32

Figure 1.2. High resolution structures for TRP channels initially resolved. A and B) Cryo-EM density maps of TRPV1 (Liao et al., 2013; Gao et al. 2016). C and

D) Cryo-EM density maps of TRPV2 (Huynh et al. 2016; Zubcevic et al. 2016). E)

X-ray crystal structure of TRPV6 (Saotome et al. 2016). F) Cryo-EM density map of TRPA1 (Paulsen et al. 2015). G and H) Cryo-EM density maps of TRPP2

(Grieben et al. 2016; Shen et al. 2016).

33

Figure 1.3. A schematic representation of the TRP channel pore showing the two regions of constriction (gates); The residues forming the two gates in various TRP proteins are listed in the table on the right

34

Figure 1.4. A schematic representation of the TRPP1 and TRPP2 interacting with each other through their C-terminal region and forming the receptor-channel complex.

35

Chapter 2

Understanding the molecular mechanism of the

TRPV2 channel gating during ligand activation by

cryo-EM

Portions of this chapter are under revision in:

Samanta A, Liu Y, Mayca Pozo F, Hughes TET, Dubyak GR, Han S, Lodowski DT,

Moiseenkova-Bell VY. Molecular mechanism of the TRPV2 channel pore dynamics during ligand activation. NSMB, Under revision.

36

2.1 Introduction

Transient receptor potential (TRP) channels play significant roles in human physiology and facilitate permeation of essential ions (Na+, Ca2+) through the plasma membrane[83, 96]. TRPV2, the second member of the Transient Receptor

Potential Vaniloid (TRPV) subfamily contributes to a broad range of physiological and pathophysiological events, yet remains the least studied TRPV channel at both cellular and molecular levels[97].

Cannabidiol (CBD), a natural product of the sativa plant, is a potent

TRPV2 agonist[98], which has been used to demonstrate the role of activated

TRPV2 in the inhibition of glioblastoma multiforme (GBM) cell proliferation[97, 99-

102]. It has also been shown that chemotherapeutic agents such as temozolomide

(TMZ, 194 Da) and doxorubicin (DOXO, 580 Da) permeate the TRPV2 channel pore after CBD activation[102], leading to increased TMZ and DOXO cytotoxicity in GBM cells[102]. These findings placed TRPV2 on the list of important anti-tumor drug targets[97, 99-102]. Nevertheless, the molecular details of TRPV2 activation by CBD or other agonists and the mechanistic basis for large organic molecules permeation of TRPV2 and other TRP channels have remained elusive.

Recently, cryo-electron microscopy (cryo-EM) structures of the truncated rat

TRPV1, truncated rabbit TRPV2, full-length rat TRPV2, and truncated xenopus tropicalis TRPV4 in the apo state revealed the atomic architecture of these channels. However, as the full-length TRPV1-TRPV4 structures in ligand activated states have yet to be determined, the ligand activation mechanism is not fully understood. Additionally, it has been suggested, that TRPV1-4 channels have two

37 gates (upper and lower) that facilitate channel opening and permeation[19, 20].

The upper gate or selectivity filter resides in the outer pore region of the channel which also contains an extracellular loop known as pore turret domain. In the truncated rat TRPV1[20] and the truncated rabbit TRPV2[23] structures this pore turret domain has been removed from the expression constructs, preventing us from understanding its role in channel opening and cation permeation.

To answer this outstanding question in TRP channel biology, we utilized the full-length rat TRPV2 channel and report a novel CBD-activated cryo-EM TRPV2 structure at 4.4Å resolution and an improved cryo-EM TRPV2 structure in apo- state at 4.6Å resolution. The apo-TRPV2 channel molecular model revealed architecture of the outer pore domain together with pore turrets that were not resolved in the previous full-length rat TRPV2 and truncated rabbit TRPV2 cryo-

EM structures. The CBD-activated TRPV2 structure uncovered conformational changes in the S5 helix, S6 helix and outer pore region of the channel that could potentially allow for large organic molecule permeation of the ion conducting pore and provided a framework for designing novel anticancer therapeutics.

2.2 Materials and Methods

2.2.1 Stable cell line generation and flux assay

Stable cell lines expressing 1D4 tagged TRPV2 (both wild type and Δ564-589 mutant) protein were generated by co-transfecting HEK293T cells with 3 µg

TRPV2 expressing plasmid constructs and 0.5 µg pCDNA3.1/hygro(+) plasmids using 3 µl Polyethylenimine (1 mg/ml) and incubated for 12 hrs at 37˚C in incubator with 5% CO2. Culture medium was replaced with new complete DMEM media

38 containing 10% FBS and supplemented with antibiotics. After 2 days 200 µg/ml hygromycin was added to start the selection for 3 days. Cells were re-plated in

10cm dishes at 1000 cells per dish and cultivated in DMEM containing 10% FBS,

1% antibiotics and 200 µg/ml hygromycin. Hygromycin-resistant clones were identified and TRPV2 expression (both wild type and Δ564-589 mutant) was monitored by western blot.

Wild type TRPV2 expressing, Δ564-589 mutant TRPV2 expressing and control HEK293T cells, which were previously placed in 24 well plates, were briefly washed with BSS containing Ca2+, 0.1% BSA, 5 mM glucose and 2.5 mM and then 0.5 ml of the same buffer supplemented with 1 μM fluo-4 AM was added to each well for the intracellular calcium flux assay. After incubation at

37°C for 30 min, a fresh 0.5 ml aliquot of BSS containing Ca2+ 0.1% BSA, 5 mM glucose and 2.5 mM probenecid was added to each well. The plate was placed into a BioTek Synergy HT reader preheated to 37°C and baseline fluorescence

(485 nm excitation → 528 nm emission at 30 sec intervals) was recorded for 5 min.

Cells were then stimulated with 30 µM CBD and changes in 485ex →

528emfluorescence were recorded at 30 sec intervals.

For the Yo-Pro uptake assay, wild type TRPV2 expressing, Δ564-589 mutant TRPV2 expressing and control HEK293T cells in 24 well plates were briefly washed with PBS prior to adding BSS supplemented with 1 μM YoPro2+ to each well. Baseline fluorescence (485 nm excitation–> 540 nm emission) for YoPro2+ at

30 sec intervals was first recorded for 10 min with a Bio Tek Synergy HT plate

39 reader preheated to 37°C. The cells were then stimulated with of 100 µM CBD and the change in fluorescence was recorded at 30 sec intervals.

2.2.2 Protein expression and purification

Full-length rat TRPV2 was expressed and purified as described previously[23].

Briefly, rat TRPV2 expressing plasma membranes were solubilized in a buffer containing 20 mM HEPES pH 8.0, 150 mM NaCl, 5% glycerol, 0.087% LMNG

(Anatrace), 2 mM Tris (2-carboxyethyl) phosphinehydrochloride (TCEP), 1 mM

Phenylmethylsulfonyl fluoride (PMSF) and supplemented with protease inhibitor cocktail tablet mini (Roche), for 1 hour. Detergent insoluble material was removed by ultra-centrifugation at 100,000 x g and the protein was purified from the supernatant by affinity chromatography by binding with 1D4 antibody coupled

CnBr-activated Sepharose beads. A column was packed with the above and washed with wash buffer containing 20 mM HEPES pH 8.0, 150 mM NaCl, 2 mM

TCEP and 0.006% DMNG. TRPV2 was eluted with wash buffer containing 3 mg/ml

1D4 (GenScript USA, Piscataway, NJ) and subjected to size-exclusion chromatography using a Superose 6 column (BioLogic DuoFlow chromatography system, Bio-Rad) with a buffer containing 20 mM HEPES pH 8.0, 150 mM NaCl, 2 mM TCEP and 0.006% DMNG.

2.2.3 Cryo-EM data collection

Prior to preparing cryo-EM grids, purified TRPV2 at a concentration of ~3 mg/ml was incubated with 30 µM cannabidiol (CBD) for 30 min. Fluorinated Fos- choline 8 was added to sample to a final concentration of 3 mM just before blotting to enhance particle distribution. This sample was double blotted (3.5 µl per blot)

40 onto 200 mesh Quantifoil 1.2/1.3 grids (Quantifoil Micro Tools) at 4°C temperature and 100% humidity and plunge frozen in liquid ethane cooled to the temperature of liquid nitrogen (FEI Vitrobot). Cryo-EM images were collected using a 300kV

FEI Titan Krios microscope equipped with a Gatan K2 Summit direct detector camera. Images were recorded using super resolution counting mode following the established protocol. Specifically, 38 frame movies were collected at 18,000 magnification with a physical pixel size of 0.69Å/pix and a dose rate of 6.85 electrons/pix/sec. Total exposure time was 11.4 seconds with one frame recorded every 0.3 seconds using the automated imaging software, Leginon[103]. Defocus values of the images ranged from -1.4 to - 3.0 µm.

2.2.4 Image processing

The movie frames were aligned using MotionCor2[104] to compensate for beam-induced motion. All subsequent data processing was conducted in RELION

2.0[105, 106] unless otherwise specified. Defocus values of the motion corrected micrographs were estimated using Gctf[107]. Initially, ~2000 particles were manually picked from the 3,970 micrographs and sorted into 2D classes to generate templates for auto-picking. Auto-picking with a loose threshold to ensure maximum picking resulted in ~821,000 auto-picked particles. These were then subjected to 2D classification to remove suboptimal particles and false positive hits. The best 268,221 particles were then auto-refined using the 3D auto- refinement option, followed by 3D classification into 10 classes. The resulting 3D structures showed heterogeneity in terms of the density distribution in the transmembrane region and the degree of opening of the central pore. The best two

41

3D classes that show a tighter pore, containing 31,525 particles, were used for 3D auto-refinement and post-processing yielding the 4.6 Å apo state map. The particles from the remaining eight classes were subjected to another round of 3D classification and from this reclassification the best two classes, showing wider pore opening, yielding 37,598 particles were used for the final 3D auto- refinement and post-processing resulting in a 4.8 Å overall resolution CBD- activated TRPV2 state map. These 37,598 particles were then processed using cisTEM software[108] and final resolution was 4.4 Å for CBD-activated TRPV2.

Local resolutions were estimated using the RESMAP software[109].

2.2.5 Model building

Our previously determined full-length TRPV2 structure (PDB: 5HI9) was employed as our starting model. We initiated model building by rigid body fitting the ankyrin repeat region and TM regions independently into new apo TRPV2 cryo-

EM map. This model was then manually adjusted to fit the density in COOT[110], and after this initial model fitting, we refined this model against the EM-derived maps using the phenix.real_space_refine program from the PHENIX software package[111], employing rigid body refinement, gradient minimization, local grid search and ADP refinement with secondary structure restraints and NCS constraints. The model was subjected to several additional rounds of manual model fitting followed by real space refinement (RSR), resulting in a final model to map cross-correlation coefficient of 0.675. We employed this improved model as the starting model for building the CBD-activated TRPV2 model, but due to the large scale differences we initiated model building by simply aligning the ankyrin

42 repeat region; following this, individual helices present in the apo structure were fit by hand as rigid bodies into the density of the CBD-activated map, followed by hand building and adjustment of the connecting regions. This model was again refined using multiple rounds of phenix.real_space_refine interspersed with manual model building and adjustment.

2.3 Results

2.3.1 Role of the pore turret domain in TRPV2 activation and large organic molecules uptake

To date, the rat TRPV2[112] and rabbit TRPV5[113] cryo-EM structures determined by our laboratory are the only full-length TRPV channel structures.

Constructs used to determine other TRPV channel structures by cryo-EM and X- ray crystallography were truncated or mutated for biochemical stability prior to structure determination[20, 23, 24, 113]. Previously, we have shown that deletion of the pore turret domain in the full-length rat TRPV2 at a comparable site, as truncated rat TRPV111 and rabbit TRPV218 used for cryo-EM analysis, produced a non-functional rat TRPV2 channel[112].

To further test the role pore turret region plays in rat TRPV2 channel activation by CBD and large organic molecules permeation upon CBD activation, we used previously characterized rat TRPV2 constructs[112] and created HEK293 stable cell lines expressing rat TRPV2 WT and rat TRPV2 564-589 (Figure 2.1A). These stable cell lines were loaded with fluo-4 fluorescent Ca2+ sensor dye and intracellular Ca2+ levels were monitored upon 30μM CBD application, which corresponds to ten times of the previously reported EC50[98] (Figure 2.1B).

43

Administration of 30μM CBD induced rapid Ca2+ influx in HEK293 stably expressing rat TRPV2 WT (Figure 2.1B), while no changes were observed in

HEK293 stably expressing rat TRPV2 564-589 or control HEK293 cells (Figure

2.1B).

Next, we tested if CBD could induce Yo-Pro 1 dye (376 Da) uptake through the rat TRPV2 channel and if red dye (RR) (786 Da) could block this effect.

We utilized 30μM and 100μM CBD and monitored 1μM Yo-Pro 1 dye uptake

(Figure 2.1C). The 30μM CBD induced negligible Yo-Pro 1 dye uptake in HEK293 stably expressing rat TRPV2 WT (Figure 2.1C), suggesting that the full-length

TRPV2 channel at 30μM CBD concentration gets activated but does not permeate large organic cations. Next, we used 100μM CBD, which initiated robust uptake of the Yo-Pro 1 dye after ~15 min CBD application (Figure 2.1C). These results suggest that higher concentration of CBD and prolonged application time of the activator promote large organic cations uptake through the full-length TRPV2 channel. Both HEK293 stably expressing rat TRPV2 564-589 or control HEK293 cells did not uptake Yo-Pro 1 dye (Figure 2.1C). Moreover, 10μM RR inhibited Yo-

Pro-1 dye uptake in HEK293 stably expressing rat TRPV2 WT (Figure 2.1C).

These results clearly indicate that the pore turret region is essential in TRPV2 channel gating and the full-length TRPV2 channel is necessary for uptake of Yo-

Pro 1 dye. Additionally, our data is consistent with previously reported results, which suggested that TRPV2 pore deletion abolishes CBD-induced Ca2+ influx and

DOXO uptake[102].

2.3.2 Architecture of the TRPV2 in apo- and CBD-activated states

44

To determine the TRPV2 structure in CBD-activated state, we used detergent solubilized full-length TRPV2 protein[112] and pre-incubated it with 30μM CBD for

30 min. We chose to incubate purified full-length TRPV2 for 30 min with CBD before freezing it in vitreous ice based on the reported incubation time for the

RTX/DkTx truncated TRPV1[18, 19].The cryo-EM images revealed a monodispersed distribution of TRPV2 protein in vitreous ice, allowing us to autopick more than 820,000 particles (Figure 2.2). After 2D classification, the best

268,221 particles were subjected to 3D auto-refine in RELION[105], which yielded a cryo-EM map of full-length TRPV2 channel at 5.2 Å (Figure 2.2B). To determine, if our TRPV2 data set had conformational heterogeneity, we subjected the 3D auto-refined particles to 3D classification with 10 classes in RELION[105] (Figure

2.2B). Inspection of central slices through the 10 refined 3D models in

RELION[105] and subsequent visual inspection of the corresponding 3D volumes in UCSF Chimera[114] identified two TRPV2 classes (31,525 total particles) that were very similar to previously determined apo full-length TRPV2[112] (Figure

2.2B). These particles were used to obtain the new apo state full-length TRPV2 cryo-EM map at 4.6Å resolution (Figure 2.2B, 2.3A, and 2.4A). While the overall resolution of the new apo state full-length TRPV2 structure is 4.6Å, the local resolution in the transmembrane (TM) region of the channel is between 3.5-4.0Å

(Figure 2.3B). This allowed us to build an atomic model of the new apo full-length

TRPV2 channel (Figure 2.5B). The new apo full-length TRPV2 structure (Figure

2.4A, 2.5A, and 2.5B) revealed all TM helices (S1–S6), the pore helix (P), the selectivity filter, the pre-S1 linker together with ankyrin repeat domain (ARD), TRP

45 domain and a portion of the C terminus (Figure 2.5A and 2.5B). Additionally, we were able to resolve previously undetermined TRPV2 channel structural elements[112], such as the S1–S2 linker (Ile422–Gly430) and the pore turret

(Glu561–Leu594) (Figure 2.5A and 2.5B). Even with the increased resolution of this structure, the first 71 residues of the N terminus (Met1–Glu71) and the last 36 residues of the C terminus (Asp725–Pro 761) were not resolved in this new cryo-

EM map (Figure 2.5A and 2.5B). Overall, the new apo full-length TRPV2 is very similar to the apo full-length TRPV2 determined at ~5 Å[112] (Figure 2.6).

The same 3D classification algorithm yielded two additional classes, which clearly represented a different conformational state of the channel. We assigned this state as CBD-activated TRPV2 (Figure 2.2B) and refined these representative classes (37,598 total particles) and obtained a full-length CBD-activated TRPV2 cryo-EM map at 4.9Å (Figure 2.2B). To further improve resolution of this full-length

CBD-activated TRPV2 cryo-EM map we used cisTEM software package and obtained new map of the channel at 4.4Å resolution (Figure 2.2B and 2.3E-F). At this resolution, with the guidance from the new apo state full-length TRPV2 model

(Figure 2.5B), we were able to build a CBD-activated TRPV2 structural model consistent with structural elements in the apo state and fit them to the CBD- activated maps (Figure 2.5C). Moreover, we observe a non-protein density positioned underneath the S5 helix, which we assigned with caution to CBD molecule (Figure 2.7). This density is located between two adjacent TRPV2 subunits and perhaps promoted the conformation change in the channel that we describe below.

46

2.3.3 Conformational differences between apo and CBD-activated TRPV2

Application of 30μM CBD for 30 minutes to the purified full-length TRPV2 channel had a profound effect on the channel architecture that is clearly visible in the CBD-activated cryo-EM map (Figure 2.4B) and the corresponding atomic model (Figure 2.5C). Comparison of the apo and CBD-activated cryo-EM maps revealed large conformational changes in the TM region of the channel (Figure

2.8A-C, Figure 2.9A, C, D), but minimal structural differences in ARD domain

(Figure 2.8D, Figure 2.9A) and in the S1-S4 domain of the channel (Figure 2.9A-

B), which are consistent with the observed static nature of these domains in the rat truncated RTX/DkTx-activated TRPV1 structure[18].

Upon first examination, an immediately obvious difference in the CBD-activated state compared to the apo state is the 40o rotation of the S5 helix in the CBD- activated channel (Figure 2.9C). Val532, which is located at the end of the S4-S5 linker, most likely acts as a hinge enabling this rotation of the S5 helix (Figure

2.9D). Subsequently, this S5 helix movement positions the pore helix of the channel between two adjacent S1-S4 domains (Figure 2.8A). This large conformational change is likely facilitated by movement of the pore turret (Figure

2.9C). These changes lead to the displacement of the S5 helix and the selectivity filter from the pore axis, promoting the large opening of the ion conducting pore that is observed in the CBD-activated structure (Figure 2.10).

The CBD-activated pore architecture (Figure 2.10A) is dramatically different from that of the apo TRPV2 pore[23] (Figure 2.10B). The apo TRPV2 pore is very similar to previously published truncated rabbit TRPV2[23] and truncated apo

47

TRPV1[18] structures (Figure 2.11). It is occluded at the selectivity filter (Gly606 and Met607) and at the lower gate (Met645 and His651) (Figure 2.11), which would prevent ion permeation through the channel (Figure 2.11). On the other hand, the

30μM CBD application induced rearrangements in the selectivity filter of the channel that moved the pore helix away from the pore axis, creating a large funnel- like opening in the channel (Figure 2.10A and 2.10C). These movements changed the diameter of the TRPV2 selectivity filter from 10Å in the apo state at Gly606

(Figure 2.12A) to 27Å in CBD-activated state at the Arg619 (Figure 2.12B), when measured Cα–Cα at the upper channel constrictions. Moreover, Gly606 moved away from the ion permeation pathway in the CBD-activated state structure (Figure

2.10A).

Additionally, the S6 helix of the CBD-activated TRPV2 at the lower gate is in different conformation compared to the RTX/DkTx-activated TRPV1[18] with the pore diameter 13Å at the Met645 (Figure 2.12B-C). The RTX/DkTx-activated

TRPV1[18] S6 helix is more tilted compared to the S6 helix of CBD-activated

TRPV2, which allowed for the expansion of the truncated TRPV1 lower gate to the

14Å at the Ile679 (Figure 2.12C). Thus, suggesting that CBD-activated TRPV2 channel is in a different conformation than the truncated RTX/DkTx-activated

TRPV1 channel (Figure 2.12B-C).

Among TRPV1-TRPV4 channels, TRPV2 and TRPV4 display high unitary conductance of 300 pS and 250 pS respectively and they both contain pore turret domains (Figure 2.13). The recently determined TRPV4 channel structure revealed that it has wide upper gate in the apo state (Figure 2.12D), suggesting

48 that it could conduct fully hydrated cations. The S6 helix of the TRPV2 channel in the CBD-activated state (Figure 2.12B) adopts a conformation different yet comparable to the TRPV4 in the apo state (Figure 2.12D), which suggests that

TRPV2 could permeate fully hydrated ions through the pore in the CBD-activated state.

Overall, it is clear from our study that the pore helix, pore turret and selectivity filter of the TRPV channels play a critical role in gating and plasticity of this region.

2.4 Discussion

TRPV1-TRPV4 channels share ~ 40% sequence homology and TRPV1,

TRPV2 and TRPV4 contain large pore turret domains that have been implicated in the dynamic process of ion selectivity in these channels (Figure 2.13). While TRP channels predominantly are non-selective cation channels, they have been shown to also permeate large organic cations[115-119]. The original findings suggested that the pore of the TRPV1 channel and other TRPs undergo conformational change after prolonged application of agonists[115-119], which leads to the widening of the channel pore to facilitate permeation of large organic cations [115-

119]. Recent studies have challenged this notion and provided evidence, which suggested that TRPV1 pore is wide enough for passage of these molecules and may not need to change size in order to permeate large organic cations[120, 121].

Like other TRP channels, TRPV2 has also been recently implicated in permeation of large organic molecule like DOXO (580 Da)[102] and in this study we were able to show that Yo-Pro 1 (376 Da) can also traverse the pore of TRPV2 at a higher concentration and prolonged application of CBD. We also saw that

49

TRPV2 Δ564-589, the pore turret truncated mutant, could not be activated by CBD and could not permeate Yo-Pro 1. This is in line with previously published results that TRPV2 Δ564-589 is insensitive to activators such as 2-APB[112]. These accumulative results suggest that the presence of the turret region in the full-length

TRPV2 channel facilitates conformational rearrangements in the channel that lead to channel opening and plays an essential role during TRPV2 channel gating.

Cryo-EM single particle analysis allowed us to visualize these structural changes in the TRPV2 channel and capture dynamic changes in the pore of the channel upon ligand application. In our studies, we were able to trap full-length

TRPV2 pore using the same incubation time as other TRPV channels in the presence of activators[18, 19], and solved a novel CBD-activated conformation that showed dramatic differences from the full-length apo TRPV2 structure. The

CBD-activated TRPV2 structure exhibited a large movement in the S5 helix, S6 helix and outer pore region of the channel that resulted in opening of the pore. The identified CBD binding site is well positioned underneath the S5 helix, which could foster such a conformation change in the channel.

Based on our results and previous scanning mutagenesis experiments, we propose that the rearrangement of the selectivity filter due to the movement of the

S5 helix is coordinated by the pore turret region of the channel that allowed for opening of the ion permeation pathway. The 27Å diameter of the upper part of the ion-conducting path in the CBD-activated state is also wide enough for NMDG+ with size 6Å x 6Å x 12.5Å[122] and Yo-Pro 1 with size 7Å x 8Å x 19Å[122] to potentially permeate the TRPV2 channel. However, since the lower gate diameter

50 is 13Å, the molecules would need to “snake” themselves through the pore as it has been recently proposed for P2X receptors[122]. Determination of the channel structure in the state of large organic cations permeation and visualizing how these molecules orient themselves in the pore of these channels will be the next challenge that cryo-EM could potentially reveal.

51

Figure 2.1. Functional characterization of wild type and truncated TRPV2 indicates the pore turret region is essential for passage of both Ca2+ and large cations. (A) Western blot analysis of TRPV2 WT and TRPV2 Δ564-589 mutant stably expressing in HEK293T cells. (B) Fluorescence based Ca2+ flux assay; application of 30µM CBD induces rapid Ca2+ influx into WT TRPV2 expressing HEK cells but not in control or TRPV2 Δ564-589 mutant expressing cells, monitored by Fluo-4 dye fluorescence. (C) Fluorescence based Yo-Pro flux assay; application of 30µM CBD induces negligible Yo-Pro uptake but 100µM CBD induces rapid Yo-Pro influx into WT TRPV2 expressing HEK cells. Yo-Pro uptake was not observed in control, TRPV2 Δ564-589 mutant expressing cells or in presence of (RR), monitored by Yo-Pro dye fluorescence.

52

Figure 2.2. Summary flowchart of TRPV2 data processing. (A). A representative micrograph of TRPV2 incubated with CBD frozen in vitreous ice

(left) and representative 2D class averages (right). (B) The workflow of data processing. The particles from the two green and two red boxed 3D classes were combined separately and leads to the refined structures of two TRPV2 states at

4.4 Å and 4.6 Å respectively.

53

Figure 2.3. Resolution data for TRPV2 refinement. The angular distribution of

2D views for the final particles used for the reconstructions of the (A) Apo and (D)

CBD-activated maps. High numbers of particles are represented as taller red cylinders while views with a low number of particles are shown as shorter blue cylinders. The final density maps are shown in salmon for apo and green for CBD- activated TRPV2. Local resolution map of (B) apo and (E) CBD-activated TRPV2.

Local resolution was determined using ResMap software. FSC curves for masked and unmasked reconstructions of (C) apo and (F) CBD-activated TRPV2. The dashed line represents an FSC of 0.143.

54

Figure 2.4. Cryo-EM analysis of apo and CBD-activated TRPV2 channel. Cryo-

EM density maps of (A) apo TRPV2 at 4.6Å and (B) CBD-activated TRPV2 at 4.4Å depicted as side views (left), top views (middle) and bottom views (right); one monomer in the tetrameric channel is colored salmon for apo and green for CBD- activated TRPV2.

55

Figure 2.5. Structural details of apo and CBD-activated TRPV2. (A) Linear representation of a monomer of TRPV2 depicting the various domains of the protein. Dashed lines denote regions that were not resolved in the apo state structure. PT: Pore turret. Atomic models of (B) apo TRPV2 and (C) CBD-activated

56

TRPV2 depicted as tetramers (left) and monomers (right). Coloring corresponds to the domains depicted in (A).

57

Figure 2.6. Structural comparison of 4.6Å apo TRPV2 and previously published ~5Å apo TRPV2 channel. (A) Overlaid cryo-EM density maps of 4.6Å apoTRPV2 (salmon) and previously published apo TRPV2 [EMD-6580] (grey) aligned by their ARDs depicting side view (left), top view (middle) and bottom view

(right). (B-D) Superimposed atomic models of 4.6 Å apo TRPV2 (salmon) and previously published apo TRPV2 [5HI9] (grey) aligned by their ARDs in tetrameric

(B) and dimer form (C) representations as well as a zoomed in view of the dimeric

TMD (D).

58

Figure 2.7. Putative CBD binding site. Zoomed in view of the CBD binding pocket (A) view from side of tetramer; (B) view from bottom of tetramer. CBD molecule (yellow sticks) is fitted in to the EM density (grey mesh) attributed to it.

The monomers are color coded to separate one from another.

59

Figure 2.8

60

Figure 2.8 Cryo-EM density map comparison of apo and CBD-activated

TRPV2 channel indicates large conformational change in the TMD but not in the ARD. Overlaid cryo-EM density maps of apo TRPV2 (salmon) and CBD- activated TRPV2 (green) aligned by their ARDs depicting differences in the TMD

(A-C) but not much difference in the ARD (D).

61

Figure 2.9.

62

Figure 2.9. Comparison of apo and CBD-activated TRPV2 models and TMD.

(A) Overlaid monomers of CBD-activated TRPV2 (multicolored) and apo TRPV2

(grey) with the helices depicted as cylinders. The regions of CBD-activated TRPV2 are labeled and colored based on the diagram in Fig. 3a. (B) Superposition of S1-

S4 of CBD-activated (blue) and apo (grey) states aligned by their ARDs with the helices depicted as cylinders. (C) Superposition of the pore domain of CBD- activated (red) and apo (grey) states aligned by their ARDs with the helices depicted as cylinders. (D) Cartoon diagram of superimposed S5 domain of CBD- activated (red) and apo (grey) showing the movement of this helix between the two states.

63

Figure 2.10. Architecture of the apo and CBD-activated TRPV2 pores. Pore profile (grey dotted surface) shown along with two diagonally opposed monomers of (a) CBD-activated (green) and (b) apo (salmon) TRPV2. (c) Pore radius of apo

TRPV2 (salmon) and CBD-activated TRPV2 (green) plotted along the ion conduction pathway. The grey dotted line indicates the radius of a Ca2+ ion.

64

Figure 2.11. Structural comparison of full length apo TRPV2, truncated apo

TRPV2 and apo TRPV1 pores. Pore profile (grey dotted surface) shown along with two diagonally opposed monomers of (a) full-length TRPV2 (salmon), (b) apo

TRPV1 in nanodiscs [5IRZ] (light blue) and (c) truncated TRPV2 [5AN8] (dark blue). Important residues lining the pore are shown as sticks. (c) Pore radius of full-length TRPV2 (salmon), closed apo TRPV1 in nanodiscs (light blue) and

65 truncated TRPV2 (dark blue) plotted along the ion conduction pathway. The grey dotted line indicates the radius of a Ca2+ ion.

66

Figure 2.12. Comparison of TRPV pore architectures. Pore profiles of (a) apo

TRPV2, (b) CBD activated TRPV2, (c) RTX/DkTx activated TRPV1 in nanodisc and (d) apo TRPV4 are shown between two diagonally opposite monomers of each channel. The measurements for the upper gate (widest part of the channel for CBD activated TRPV2) and the lower gate are between the Cα–Cα of the structures.

67

Figure 2.13. Comparison of the pore region of the TRPV channels. (Left)

Monomer of apo TRPV2 pore region. The turret is shown in green, the pore helix in red, the selectivity filter in blue and S5 and S6 in grey. PT: Pore turret. (Right)

Sequence alignment of a section of the pore region of the TRPV channels; the pore turret, pore helix and selectivity filter for TRPV2 sequence is color coded as in the helical model. TRPV2, TRPV1 and TRPV4 have more elaborate turret region compared to that of TRPV3, TRPV5 and TRPV6 as depicted from the sequence.

68

Figure 2.14. Electron density corresponding to regions of interest in the

TRPV2 structures validates model assignment. Various helices of (a-d) the apo

TRPV2 (salmon) and (e-f) CBD-activated TRPV2 (green) models shown as ribbons overlaid with their respective density maps (mesh) respectively. Select residues are shown as sticks to illustrate the accuracy of the model.

69

Chapter 3

Understanding the ligand induced conformational

changes of mouse TRPA1 channel during

activation and inhibition

Portions of this chapter were published in:

Samanta A, Kiselar J, Pumroy R.A., Han S, Moiseenkova-Bell VY. Structural insight into the molecular mechanism of mouse TRPA1 activation and inhibition. J

Gen Physiol. 2018 May 7;150(5):751-762. doi: 10.1085/jgp.201711876.

70

3.1 Introduction

TRPA1, the lone member of the mammalian TRPA subfamily, is a significant transducer of chemical, neuropathic and inflammatory pain signals [59, 61, 62,

123, 124]. It is expressed predominantly in small and medium sized peptidergic primary afferent neurons of the sensory ganglia [59, 125, 126]. TRPA1 is also expressed in various non-neuronal tissue types and organs, including epithelial cells, fibroblasts and cells [58, 127]. TRPA1 is a homo-tetrameric, non-selective cationic channel comprised of a transmembrane domain and a large cytosolic domain. Each monomer of the TRPA1 protein is made up of 6 transmembrane helices (S1 – S6) and a reentrant pore loop in the transmembrane domain, which is preceded by a large N-terminus and followed by a coiled coil structured C-terminus. Although TRPA1 has a structurally conserved transmembrane domain like other TRP channels, it is unique among mammalian

TRP channels in having a large number of ankyrin repeats (16 total) at its N- terminus.

TRPA1 is best known as a chemo-nociceptor in the body[63, 128, 129]. It can be activated by a multitude of structurally unrelated natural compounds like allyl isothiocyanate (in ), (in garlic), irritants like acrolein

(in cigarette smoke), vehicle exhaust, metabolic byproducts of chemotherapeutic drugs, and endogenous inflammatory molecules [61, 62, 64, 123, 130-133]. This wide range of TRPA1 ligands can be broadly classified into two groups: electrophilic modulators and non-electrophilic modulators. The electrophilic agonists activate TRPA1 via a cluster of cysteine residues present at the N-

71 terminus of the channel [134-136]. Binding of these electrophilic agonists to the channel leads to disulfide bond formation between these critical cysteine residues, which triggers conformational changes at the N-terminus and the opening of the

TRPA1 channel [134-137]. The activation mechanisms for non-electrophilic ligands are still unknown. Recently the structure of human TRPA1 was resolved at

4 - 4.5 Å resolution in the presence of either an electrophilic agonist (allyl isothiocyanate) or non-electrophilic antagonists (HC-030031 and A-967079).

While the potential binding site for one antagonist, A-967079, has been visualized, no conformational changes could be resolved among these cryo-EM structures in activated and inhibited states [138]. Therefore, the molecular mechanism of how

TRPA1 modulators affect the conformation of the tertiary structure of the protein to either open or close its gates is still elusive.

The N-terminus of TRPA1 contains 16 ankyrin repeats (AR1 – AR16), which are 30 – 34 amino acid long helix--helix motifs. The ankyrin repeats are arranged in tandem, forming an elongated ankyrin repeat domain (ARD) which is connected to the transmembrane domain via the pre-S1 region, and are usually involved in protein-protein and protein-ligand interactions [139, 140]. Chimeric and mutagenesis studies have suggested that modulations in the ARD can be translated to the pore, leading to opening or closure of the channel [141, 142].

Chimeric studies have also shown that the ARD can be divided into two parts: a primary module comprised of AR10 – AR15 and an enhancer module comprised of AR3 – AR8 [141]. Existing literatures of mutagenesis studies have suggested that thiol reactive activators of TRPA1 interact with the sulfhydryl groups of

72 specific, conserved cysteine residues (C415, C422, C622, C642, C666, C174,

C193, C634 and C859; numbering is for mouse TRPA1) [133-135] and it has been shown that these critical undergo disulphide bond formation or rearrangements, leading to N-terminal conformational changes, channel activation, and/or desensitization [137, 143]. Furthermore, the C-terminus of

TRPA1, which is linked to the transmembrane region by unstructured loops, β- sheets and a TRP like domain, has also been suggested to play a role in channel gating [138].

To determine the structural basis of TRPA1 channel modulation by ligands, we employed a classic technique: limited proteolysis. Limited proteolysis is a well- established, simple biochemical technique used to probe information regarding protein structure and conformational changes [144-147]. The theory underlying limited proteolysis is that a protein is incubated with a relatively low concentration of a protease of choice, which induces cuts at exposed recognition sites throughout the protein, mostly at loops and other flexible regions. Substrate binding, or activation and inhibition by other kinds of stimuli induce conformational changes in the protein, changing the regions that are exposed and thus the pattern of protease cleavage. Subsequent analysis of the cleaved fragments by mass spectrometry results in an extremely robust technique for the evaluation of ligand-induced structural changes in various proteins [148]. These changes are sometimes so minor that they are difficult to determine by conventional biophysical methods, short of resolving a structure at atomic resolution. In these cases, biochemical assays like limited proteolysis can provide useful and accurate information about

73 the change in the topology of the protein. Therefore, in this study we used limited proteolysis coupled with in-solution mass spectrometry to identify the regions of

TRPA1 that undergo conformational changes during activation or inhibition by both electrophilic and non-electrophilic ligands.

3.2 Materials and Methods

3.2.1 Expression and purification of TRPA1

Mouse TRPA1 was expressed in the S. cerevisiae yeast strain BJ5457 and plasma membranes were isolated as described previously (Cvetkov et al 2011).

Briefly, mouse TRPA1 expressing yeast cells were lysed using a microfluidizer (M-

110Y, Microfluidics, Newton, MA), and the lysate was cleared of cell debris by centrifuging first at 3,000g for 10 mins, and the supernatant further centrifuged at

14,000g for 35 mins. Plasma membranes were obtained by using an ultracentrifuge to spin down the supernatant at 100,000g for 45 mins and storing the pelleted membranes at -80˚C for future use. For protein purification all the following steps were performed at 4˚C. Membranes were solubilized in a buffer containing 20mM HEPES at pH 8, 500mM NaCl, 10% glycerol, 8mM FC-12

(Anatrace), 0.4mg/ml Soybean polar lipids, 1mM Phenylmethylsulfonyl fluoride

(PMSF) and10mM Tris(2-carboxyethyl) phosphinehydrochloride (TCEP) supplemented with a protease inhibitor cocktail tablet mini (Roche) for 1 hour.

Detergent insoluble material was removed by centrifuging at 100,000X g for 45 mins. 1D4 antibody coupled CnBr-activated Sepharose beads were added to the supernatant and incubated for 2 hrs. A column was packed with the above beads and washed with buffer containing 20mM HEPES, pH 8.0, 150mM NaCl, 10 mM

74

TCEP, 10% glycerol, and 0.25% A8-35 (amphipol). TRPA1 was eluted with the same buffer supplemented with 10 mg/ml 1D4 peptide (GenScript USA,

Piscataway, NJ). The protein was concentrated by spin concentrator (Millipore

Amicon Ultra 50K Ultracel) and subjected to size-exclusion chromatography on a

Superose 6 column (GE Healthcare) with a buffer containing 20mM HEPES, pH

8.0 and 150mM NaCl.

3.2.2 Limited proteolysis of TRPA1

Purified TRPA1 was concentrated to ~ 1mg/ml in a buffer suitable for limited proteolysis (20 mM HEPES, pH 8.0 and 150 mM NaCl). The protein was then pretreated with either 2mM NMM, 333µM A-967079 (Pfizer), 1mM menthol, 333µM

PF-4840154 (Pfizer) or buffer for the apo state. Freshly prepared mass spectrometry-grade trypsin was added at a protease:TRPA1 mol/mol ratio of 1:300 at room temperature for 15 mins, and the reaction was quenched by adding 10 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF), 5-fold excess of Soybean

Trypsin Inhibitor II-S (Sigma) and placing on ice. All samples were precipitated with 10% trichloroacetic acid (TCA)/acetone overnight at -20 oC, washed three times with acetone, and air dried. The precipitation procedure was repeated twice.

Air dried samples were reconstituted in 10 µL of 50 mM Tris, pH 8.0, and then reduced and alkylated with 10 mM DTT and 25 mM iodoacetamide, respectively.

Subsequently, all samples were fully digested with Asp-N an enzyme to protein ratio of 1:10 at 37 oC overnight, followed by liquid chromatography coupled with high-resolution mass spectrometry (LC-MS).

3.2.3 LC-MS Analysis

75

LC-MS analysis of digested samples was carried out on the Orbitrap Elite mass spectrometer (Thermo Electron, San Jose, CA) interfaced with a Waters nanoAcquity UPLC system (Waters, Taunton, MA). Approximately 300 ng of proteolytic peptides for each sample were loaded on a trap column (180 μm × 20 mm packed with C18 Symmetry, 5 μm, 100 Å (Waters, Taunton, MA)) for desalting, and then separated on a reverse phase column (75 μm x 250 mm nano column, packed with C18 BEH130, 1.7 μm, 130 Å (Waters, Taunton, MA)) using a gradient of 2 to 42% mobile phase B (0.1% formic acid in acetonitrile (ACN)) versus mobile phase A (0.1 % formic acid in water) over a period of 60 minutes with a flow rate of 300 nl/min. Peptides eluting from the column were introduced into the nano- electrospray source using a capillary voltage of 2.5 kV. For MS analysis, a full scan was recorded for eluted peptides (m/z range of 380–1700) in the Fourier Transform

(FT) mass analyzer with resolution of 120,000 followed by MS/MS of the 20 most intense peptide ions scanned in the ion trap (IT) mass analyzer. All MS data were acquired in the positive ion mode. The resulting MS and MS/MS data were searched against a TRPA1 protein sequence database using Mass Matrix software

[149] to identify peptides sequence and overall TRPA1 sequence coverage. In particular, MS and MS/MS spectra were searched for peptides derived from the dual digestion of TRPA1 with trypsin and Asp-N enzymes using mass accuracy values of 10 ppm and 0.8 Daltons respectively, with allowed variable modifications including carbamidomethylation for cysteines and oxidative modifications for methionines. The total number of MS/MS ion scans (spectral counting) for each identified peptides were used to compare peptides abundances [150].

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3.3 Results

3.3.1 Limited proteolysis and in-solution mass spectrometry to determine ligand induced conformational changes in TRPA1

To identify ligand induced conformational changes of the full-length mouse

TRPA1 ion channel, we used Saccharomyces cerevisiae as the heterologous expression system to express the full length protein [70, 151]. Although inositol hexakisphosphate (IP6) was required for the purification of human TRPA1 in a previous cryo-EM study [138], the purification method employed here has been proven to produce functional mouse TRPA1 and other TRP channels that can be used for structural, biochemical and biophysical studies [70, 151]. The protein was purified using detergent and amphipol A8-35 and the peak corresponding to tetrameric TRPA1 (Figure 3.1A) was collected and used for all described studies.

For limited proteolysis, the first digestion is usually performed using a protease with broad specificity. Complete digestion of a denatured protein with such an enzyme will thus yield relatively small peptides. However, when used for a short period of time at a low substrate to enzyme ratio, digestion of a protein in its native state will result in the formation of larger, stable peptides due to the inaccessibility of buried or protected sites by the protease. Here we used the serine protease trypsin at 1:300 trypsin:TRPA1 ratio and checked three time points over

30 minutes by SDS-PAGE, which showed large, stable peptides appearing quickly and surviving for the entire time course. Based on this time course, we decided to use this ratio of trypsin and an intermediate digestion time of 15 minutes for all experiments. Using the above ratio of trypsin to protein and digesting for 15

77 minutes yielded a distinct cleavage pattern for apoTRPA1 and drug treated TRPA1

(Figure 3.1B). Therefore, these conditions were used for all experiments.

Electrophilic and non-electrophilic agonists and antagonists that have been used in this study are outlined in Table 1.

Instead of using bands cut out from SDS-PAGE gels for mass spectrometric analysis, we employed in-solution mass spectrometry to get better overall coverage. Upon full digestion with endoproteinase Asp-N and subsequent analysis with in-solution mass spectrometry, the sequence coverage for all the experiments was ≥ 90%, thus enabling us to identify the regions of conformational changes with confidence. To achieve this coverage and reliable peptide quantification, we used spectral counting approach, which relies on comparing the number of identified tandem mass spectrometry (MS/MS) spectra from the same protein in each of the multiple liquid chromatography tandem mass spectrometry (LC-MS/MS) datasets to quantify the number of identified nonredundant peptides [152]. The increase in protein abundance correlates with the increase in the number of its proteolytic peptides, which subsequently results in an increase in the number of identified unique peptides, and the number of identified total MS/MS spectral count for a specific protein [150, 152, 153]. Thus, spectral count can be used as a simple but reliable index for relative peptide/protein quantification.

To illustrate TRPA1 conformational changes in the presence of various modulators, we created a TRPA1 channel dimer model representation (Figure 1).

For this we downloaded the published 3J9P atomic model from the (Figure 3.2A) [138] and used ITASSER software [154, 155] to build a

78 homology model for AR1 – AR11 (Figure 3.2B). Next, we overlaid these two parts of the structure in accordance to the proposed “propeller and independent wings” model by ’s group [138] to get the complete structural representation

(Figure 3.2C).

3.3.2 Analysis of NMM (electrophilic agonist) induced conformational change in TRPA1

NMM (N-methyl-maleimide) is an electrophilic agonist that is commonly used to study TRPA1 ion channel activation [134, 137]. It has been proposed that NMM and other electrophilic agonists covalently modify cysteine residues C415, C422,

C622, C642, C666 on the N-terminus of the channel, which leads to channel opening [134, 135]. Purified full-length TRPA1 was incubated with 2mM NMM for

10 mins and then the reaction was quenched with 10mM DTT before performing limited proteolysis with trypsin. Partially digested apo-TRPA1 and NMM-activated

TRPA1 were precipitated, reconstituted, alkylated, reduced and fully digested with endoproteinase Asp-N before MS analysis. Apo and NMM treated TRPA1 showed different cleavage patterns in the MS analysis (Figure 3.3): peptide DTNLKCT (536

– 542) was not present in apo-TRPA1, but appeared in the NMM-activated TRPA1 sample with a peptide count of 2 & 4 in two separate experiments (Figure 3.3A and

3.3B). Region D536 – T542 is located at the N-terminus and is part of AR14 and the loop connecting AR14 and AR15 (Figure 3.3C). This region is positioned between two clusters of cysteines residues: a) C415 and C422; b) C622, C642 and C666; which have been implicated in channel activation by NMM and other electrophilic agonists (Figure 2C). Also, the mass spectrometric analysis revealed

79 that a number of these critical cysteines, like C415, C622, C634, C666, etc, were modified by NMM, indicating that these cysteine residues are available for interaction with NMM in the full length TRPA1 protein. A few of these modifications are shown in Fig. 3.4.

Additionally, there were also a couple of regions of missed trypsin cleavages in the NMM-TRPA1 sample at residues R1004 and R1014, located in the linker domain (Figure 3.7A). Interestingly, residue R423 was protected from being cleaved by trypsin in apo TRPA1, but was exposed for cleavage in the presence of NMM (Figure 3.7A).

Taken together, our data shows that TRPA1 modification by NMM leads to

N-terminal conformational changes in the ARD11-ARD15 region of the protein. As

NMM is an agonist and mediates channel opening, it seems likely that the conformational changes we observe in ARD11-ARD15 are related to the opening of the channel.

3.3.3 Analysis of PF-4840154 (non-electrophilic agonist) induced conformational change in TRPA1

TRPA1 is a genuine polymodal channel as it can be activated not only by structurally unrelated electrophilic ligands but also by a large number of non- electrophilic ligands [60-62, 124, 156]. Recently, a potent non-electrophilic agonist

(PF-4840154) was developed by Pfizer that has been shown to selectively activate

TRPA1 and elicit a pain sensation [157]. Purified TRPA1 was incubated with

333µM PF-4840154 for 10 mins before performing limited proteolysis with trypsin.

Partially digested apo-TRPA1 and PF-4840154-activated TRPA1 were

80 precipitated, reconstituted, alkylated, reduced and then fully digested with endoproteinase Asp-N and subjected to LC-MS analysis. Analysis of the peptide products by MS revealed that peptide EYLLMK (707 – 712), located in the pre-S1 helix, was detected in apo-TRPA1 with a peptide count of 2 and 13 in two separate experiments, but was not present in the PF-4840154 treated sample (Figure 3.5A and 3.5B). Intriguingly, the E707 – K712 peptide was also present in the NMM- activated TRPA1 samples (Figure 3.5B), suggesting that electrophilic agonist activation of the channel differs profoundly from activation by non-electrophilic agonists such as PF-4840154.

The cryo-EM structure revealed that the pre-S1 region of the channel resides in close proximity to the TRP-like domain at the C-terminus of the channel and harbors critical cysteine and lysine residues that are involved in channel activation (Figure 3.5D). The E707 – K712 peptide belongs to the pre-S1 helix

(Figure 3.5C and 3.5D), which has been implicated in allosteric modulation of the channel by electrophilic ligands through its interaction with the TRP-like domain and transmission of the signal to the pore of the channel [138]. Additionally, it has been suggested that the TRP-like domain interacts with the pre-S1 helix and the

S4-S5 linker via hydrophobic interactions that would be affected upon ligand binding [138]. Recently, the S4-S5 linker has been shown to play a critical role in activation or inhibition of another “pain” sensor, TRPV1 [18]. It has been suggested that TRPV1 modulators (activator capsaicin and inhibitor ) interact with the S4-S5 linker and induce conformational changes in this region that open or close the channel.

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Based on these recent discoveries, we speculate that the absence of E707

– K712 peptide in the PF-4840154 treated sample could be due to disruption of the hydrophobic interactions between the TRP-like domain and the pre-S1 helix upon PF-4840154 binding to the S4-S5 linker in TRPA1 (Figure 3.5C), thus triggering channel activation.

3.3.4 Analysis of menthol (non-electrophilic modulator) induced conformational change in TRPA1

Menthol is a widely used naturally occurring non-electrophilic modulator of

TRPA1 which has been shown to have a bimodal effect on rodent TRPA1 channels

[65]. Specifically, it has been shown that 1mM of menthol reversibly blocks the channel, while a sub-micromolar to low-micromolar range of menthol concentrations activates the channel. Purified TRPA1 was incubated with 2mM of menthol for 10 mins before performing limited proteolysis with trypsin. Partially digested apo-TRPA1 and menthol-blocked TRPA1 were precipitated, reconstituted, alkylated, reduced and then fully digested with endoproteinase Asp-

N and subjected to MS analysis. With 2mM menthol we had expected to capture the channel in an inhibited state, but surprisingly we found a cleavage pattern very similar to PF-4840154-activated TRPA1, with peptide E707 – K712 absent in menthol treated TRPA1 (Figure 3.5A and 3.5B). Analysis of missed cleavages revealed that although majority of the same residues (K109, R465, R621, R706,

K712 and R978) were shielded from cleavage by trypsin in both menthol treated and PF-4840154-activated TRPA1, there were a few exceptions. Residue K662, which missed cleavage in the menthol treated sample, was not affected by

82 treatment with PF-4840154, while residues K213, K239 and K636 were not cleaved in PF-4840154 bound TRPA1 but were trypsinized in both apo and menthol bound TRPA1 (Figure 3.7C). The majority of these differences were observed in the N-terminal linker domain and in the flexible N-terminal ARs.

Given these similarities to the PF-4840154-activated state and that menthol is an agonist at low concentrations, we suggest that with 2mM menthol the channel was first activated and then desensitized and that we have captured the desensitized state of TRPA1. However, it is entirely possible that we captured

TRPA1 in an inhibited state and that there is a very similar global mechanism of

TRPA1 activation, desensitization and inhibition by non-electrophilic ligands which could possibly be conserved among other TRP channels.

3.3.5 Analysis of A-967079 (non-electrophilic antagonist) induced conformational change in TRPA1

To determine whether a non-electrophilic antagonist causes conformational changes in similar regions of the full-length TRPA1 we used A-967079, a commonly used inhibitor of TRPA1 activity [158-160]. Purified TRPA1 was pretreated with 333M A-967079 for 10 mins before performing limited proteolysis with trypsin. Partially digested apo-TRPA1 and A-967079-inactivated TRPA1 were precipitated, reconstituted, alkylated, reduced and then fully digested with endoproteinase Asp-N and subjected to MS analysis. In this case we detected two different peptides that were present in the apo-TRPA1 state but absent in the A-

967079 treated sample: INTCQRLLQ (460 – 468) with a peptide count of 1 and 2 and STIVYPNRPR (1005 – 1014) with a peptide count of 3 and 3 in two separate

83 experiments (Figure 3.6A-C). Structurally, I460 – Q468 forms one of the helices of the helix-turn-helix of AR12 and S1005 – R1014 is a part of the flexible linker that connects the C-terminus to the transmembrane domain (Figure 3.6D). The published cryo-EM structure of human TRPA1 revealed the binding pocket of A-

967079 to be surrounded by transmembrane helices S5 and S6 and pore helix 1.

Since this is quite far from the regions of missed peptide cleavages, this suggests that channel inhibition by A-967079 occurs through a global conformational change. Additionally, as AR1-11 were too flexible to be resolved in the cryo-EM

TRPA1 structure, AR12 is the first of them visible in that structure and so is at the transition point between the stable and dynamic portions of TRPA1. As one of the missed cleavage sites is found in AR11 (Figure 3.7D), this suggests the intriguing possibility that A-967079 inactivation of TRPA1 may involve modulating the interaction between the relatively stationary main channel and the flexible N- terminal AR region.

3.4 Discussion

The mechanism for how a multitude of structurally diverse and unrelated compounds can modulate the polymodal TRPA1 has remained elusive. In this study we reveal the critical regions of TRPA1 that undergo conformational rearrangement upon ligand interaction to accomplish the opening and closing of the ion conducting pore. We examined the topological changes of TRPA1 upon the addition of a diverse group of modulators encompassing both electrophilic and non-electrophilic ligands by limited proteolysis and mass spectrometry. It has been demonstrated before that certain cysteine and lysine residues in the cytosolic

84 domain of the protein are critical for activation of the channel by electrophilic compounds [134, 135], however, the process of how modification of these residues gets translated to the pore is still unclear. The molecular mechanism by which the non-electrophilic ligands control the gating mechanism is also unknown. In this study we illustrated that across all of the modulators tested, TRPA1 saw topological rearrangements stretching from the primary module of the ARD (AR10-

15) [141] through the linker region comprising the loops, β-sheets, pre-S1 helix and TRP like domain, suggesting that these are the critical regions involved in the opening and closing of the pore.

The primary module of the ARD (see star 1 in Fig. 3.8) undergoes changes for all tested modulators. Of particular interest are the changes seen on treatment with A-967079 and NMM. On A-967079 treatment, peptide I460 – Q468, which forms the second helix of AR12, becomes protected from cleavage. This is far away from the binding site of A-967079 and the pore, indicating that binding of this ligand induces allosteric conformational changes in the protein. From recent cryo-

EM data of the TRP channel NOMPC [161] and the different subpopulations resolved, it was proposed that the force experienced by the channel in the N- terminus could be transduced to the linker region via the spring like ARs. Our limited proteolysis data corroborates these structural observations. In the NMM treated TRPA1 sample, peptide D536 – T542, found in AR14 and the AR14-AR15 linker, becomes accessible for cleavage, indicating that NMM induces conformational rearrangement in this region. It is interesting to note that this region lies in between the two clusters of cysteine residues that were implicated by two

85 separate groups [134, 135] to be necessary for channel activation by electrophilic ligands. Moreover, mass spectrometric analysis of the NMM treated TRPA1 sample depicted that a lot of these critical cysteines (Cys-193, Cys-415, Cys-463,

Cys-622, Cys-634, Cys-666,) along with some other cysteines (Cys-31, Cys-45,

Cys-66, Cys-89, Cys-105, Cys-214, Cys-259, Cys-274, Cys-541, Cys-609, Cys-

1087) were modified by NMM, indicating that NMM binding to TRPA1 could lead to disruption of some existing disulfide bonds and formation of new disulfide bonds

[137], making the 536-542 region accessible to trypsin cleavage.

For all of the modulators, the data shows that the linker regions between the transmembrane domain (TMD) and the two cytosolic domains play a major role in channel gating and experience conformational changes. On the N-terminal side

(see star 2 in Fig. 3.8), the pre-S1 helix and a linker domain made of two helix- turn-helix motifs connected by two anti-parallel β sheets harbor some of the cysteine and lysine residues (C622, C642, C666 and K710) (Figure 3.5D) that are required for activation by electrophilic ligands [134, 135]. The non-electrophilic modulators also modify this region, as menthol, PF-4840154, and A-967079 treatment all cause a conformational change in the pre-S1 helix, as peptide E707

– K712, comprising the majority of the pre-S1 helix, is protected from the proteolytic effect of trypsin in all three cases. On the C-terminal side, the linker regions and TRP-like domain form the connection to the TMD. Peptide S1005 –

R1014, which is found in the linker connecting the C-terminal coiled coil to the

TRP-like domain, is protected from cleavage in the presence of both NMM and A-

967079 despite being highly flexible. This is intriguing, since the binding site of A-

86

967079 is very close to the pore in the TMD, but it somehow causes structural rearrangement in the linker region (see star 3 in Fig. 3.8). These behaviors of

TRPA1 can potentially be explained by the structural proximity of the linkers connecting the N-terminus and the C-terminus to the TMD, as seen in the cryo-EM structure. The TRP like domain, which lies parallel and close to the inner leaflet of the membrane, can interact with the pre-S1 helix and the S4-S5 linker.

Furthermore, in case of TRPV1 it has been proposed that the area encompassing

S4, S4-S5 linker, S6 and the TRP domain is critical for opening or closure of the channel. Both agonist (RTX/DkTx) and antagonist (Capsazepine) binds to the same pocket but trigger different structural rearrangements, one leading to channel opening and the other to its closing [18]. It is possible that a very similar mechanism also exists for TRPA1 modulation by different ligands and our data validates this by revealing that across different classes of ligand, the entire linker region is vital in governing the ion conducting path. It is likely that this mechanism is conserved throughout the TRP channel family.

While our study highlights the conformational changes specific to different gating states of the mouse TRPA1 ion channel, it has some limitations due to sample preparation. The mouse TRPA1 channel has been shown to require Ca2+ ions and polyphosphates for its activation [162], but in our study, we used purified full-length mouse TRPA1 that has been reconstituted in amphipol A8-35 without addition of IP6 and only trace amounts of Ca2+ were present in our buffers. The human TRPA1 channel purification conditions in the cryo-EM study were very similar to ours with the exception of the presence of IP6 in the buffers. Based on

87 our previous work [70, 137], the presence of IP6 is not necessary for channel purification, but it would be important in our future experiments to test how IP6 together with the Ca2+ influence the conformational changes in the channel in the presence of different modulators. Like other TRP channel structural biology groups, we also utilized amphipol A8-35 to stabilize purified protein for our analysis, however we could not resolve conformational differences in the transmembrane region of the channel using in-solution mass spectrometry. This is likely due to presence of A8-35 in the mouse TRPA1 transmembrane region, shielding it from proteolysis.

It is also very important to note that the methods used in this current study do not fully allow us to interpret how local conformational changes in the mouse

TRPA1 result in the overall conformational changes of the channel. Although further cryo-EM and mass spectrometry studies are required to visualize the molecular mechanism of TRPA1 gating, our data suggest that for all ligand classes, the primary module of the ARD, the pre-S1 helix, the TRP-like domain and the linker regions of the channel are vital for conformation changes in the channel that lead to channel opening.

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Table 3.1

Table 3.1. List of TRPA1 ligands used here.

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Figure 3.1. TRPA1 purification and limited proteolysis of pure TRPA1. A, Gel filtration profile of purified mouse TRPA1; the peak corresponding to tetrameric, amphipol stabilized TRPA1 is shown by the dotted black lines. B, Commassie stained SDS-PAGE gel showing purified TRPA1, purified TRPA1 cleaved with trypsin for 15 mins, purified TRPA1 treated with A-967079 for 10 mins and then cleaved with trypsin for 15 mins.

90

Figure 3.2. Construction of a representation of the full-length TRPA1 channel. A, Model 3J9P downloaded from the RCSB protein data bank and depicted as a dimer. B, Homology model of residues M1 – P450 generated by I-

TASSER software. C, The two models were aligned to generate a dimeric representation of the full channel.

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Figure 3.3 Effect of NMM on TRPA1 conformation. A, MS/MS spectra of doubly protonated ions for peptide 536-DTNLKCT-542 containing cysteine modified with

N-methyl-maleimide. B, Graph of the occurrence of peptide 536-DTNLKCT-542 in apo and NMM treated TRPA1. C, Peptide 536-DTNLKCT-542 (shown in green)

92 mapped onto the dimeric representation of TRPA1; the cysteines necessary for channel activation by electrophilic ligands are shown as spheres in yellow.

93

Figure 3.4.

94

Figure 3.4. Nano- LC-MS/MS spectrum of the modified peptides after Asp-N digestion of NMM-activated TRPA1. A, MS/MS spectrum of the doubly protonated ion (m/z 453.20) of the 536-542 peptide has mass shift of 111.032 Da at Cys-540 residues corresponding to cysteine modification by N-methylmaleimide

(NMM). The y*- and b*- fragment ions correspond to the y- and b- fragment ions that were modified by NMM. B, MS/MS spectrum of the doubly protonated ion (m/z

729.30) of the 413-423 peptide has mass shift of 222.064 Da that corresponds to modification by N-methylmaleimide (NMM) at both Cys-415 and Cys-422 residues.

The y*, b*- and y**, b**- fragment ions correspond to the y- and b- fragment ions that were modified by one and two NMM respectively. C, MS/MS spectrum of the doubly protonated ion (m/z 523.76) of the 192-199 peptide has mass shift of

111.032 Da at Cys-193 residues corresponding to cysteine modification by N- methylmaleimide (NMM). The y*- and b*- fragment ions correspond to the y- and b- fragment ions that were modified by NMM.

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Figure 3.5. Effect of non-electrophilic modulators on TRPA1 conformation.

A, MS/MS spectra of doubly protonated ions for peptide 707-EYLLMK-712 with oxidized methionine for PF-4840154 treated TRPA1. B, Graph of the occurrence of peptide 707-EYLLMK-712 in apo, menthol and PF-4840154 treated TRPA1. C,

Peptide 707-EYLLMK-712 (shown in red) mapped onto the dimeric representation of TRPA1. D, Magnified view of the linker region with cysteine and lysine residue highlighted.

96

Figure 3.6.

97

Figure 3.6. Effect of A-967079 on TRPA1 conformation. MS/MS spectra of doubly protonated ions for A, peptide 460-INTCQRLLQ-468 with carbamidomethylated cysteine and for B, peptide 1005-STIVYPNRPR-1014. C,

Graph of the occurrence of peptide 460-INTCQRLLQ-468 and peptide 1005-

STIVYPNRPR-1014 in apo and A-967079 treated TRPA1. D, The two peptides

(shown in red) are mapped onto the dimeric representation of TRPA1; the binding pocket for A-967079 is shown in blue.

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Figure 3.7. Dimer representations of TRPA1 showing additional regions of miscleavage upon ligand interaction. Miscleaved regions are mapped onto the dimeric representation of TRPA1. A, Residue R423 (highlighted in dark green) was protected and residues R1004 and R1014 (highlighted in pink) were exposed for miscleavage upon treatment with NMM. Residues that were exposed for miscleavage upon treatment with B, PF-4840154 or C, menthol are highlighted in

99 pink. D, Residue R423 (highlighted in dark green) was protected from miscleavage and several other residues were exposed for miscleavage (highlighted in pink) upon treatment with A-967079.

100

Figure 3.8. Cartoon representation of TRPA1 showing the regions involved in channel gating. The regions critical for channel gating are highlighted with numbered yellow stars.

101

Chapter 4

Discussion and Future Directions

102

4.1 Summary

In the first part of the dissertation we had aimed to understand the molecular events that lead to opening of the TRPV2 channel’s ion conducting pore. The data revealed that in TRPV2 the pore turret is an important region that is necessary for channel opening and also aids in channel plasticity that leads to permeation of cations and large organic molecules through the pore. Here we have determined the cryo EM structures of apo- and CBD- activated TRPV2. This apo state structure at 4.6Å resolution allowed us to build a more accurate model of the pore helix and the pore turret region compared to previously published structures. This, therefore, gave us a more detailed understanding of the architecture of this important region.

Moreover, by comparing the apo state with the CBD-activated structure, we could reveal a potential molecular mechanism of channel activation and identify the conformational change endured by the outer pore region during this activation. The

CBD-activated 4.4Å structure revealed a distinct gating mechanism and architecture that involved rearrangement of the S5 and S6 helices, pore turret and the selectivity filter. These results provide a foundation towards further understanding TRP channel gating properties and the divergent physiological functions of channels in this family.

In the second part of the study we intended to understand the effect of both electrophilic and non-electrophilic ligands on TRPA1 channel architecture. TRPA1 is localized in “”, which transduce chemical, inflammatory and . TRPA1 is known to be modulated by a wide range of structurally unrelated electrophilic and non-electrophilic compounds. It was known that

103 electrophilic ligands activate TRPA1 channels by interacting with certain critical cysteine residues present on the N-terminus of the channel via their covalent modification and/or disulfide bonds formation/rearrangement [134, 135]. This explained the structural diversity of this group of ligand to some extent. However, the structural diversity of the non-electrophilic ligands was still elusive since they did not require interaction with these critical cysteine residues. Moreover, there was no structural information available on the gating mechanism of such a polymodal channel, even though the cryo-EM structure of this channel has been resolved in presence of ligands [70]. Our data revealed that irrespective of the ligand, the N-terminal “primary module” ankyrin repeats (AR10 – AR15), the pre-

S1 helix, the TRP-like domain, and the linker regions of the TRPA1 channel experiences conformational rearrangements during both activation and inhibition.

Taken together, the major findings from this dissertation revealed structural nuances of TRP channel gating. We found that although TRPV2 shares ~50% sequence homology with TRPV1, unlike TRPV1, in TRPV2 the pore turret is absolutely necessary for a functional channel. Truncation of this region (TRPV2

564-589) rendered the channel non-functional. In the presence of CBD, the S5 helix makes an approximately 40˚ rotational movement. This rearranges the pore turret and subsequently moves the selectivity filter out of the pore entrance leading to the funnel like opening of the ion channel (Figure 4.1). This is the first time that the open state structure of a full length TRPV channel has been resolved. In the case of TRPA1, we found that a section of the ankyrin repeat domain and the linker region that connects the TMD to the cytosolic part of the channel sustain

104 conformational rearrangement during the activation and/or inhibition of the channel. Thus, these two regions are critical in regulating the opening and closing of the TRPA1 channel irrespective of the ligands.

4.2 Impact of this study

4.2.1 Application for structural studies of other membrane proteins

Previously, little was known regarding the structural details and the architecture of the TRP channels due to the unavailability of high resolution structure. However, in the past couple of years, with the advancement in the field of cryo-EM, high resolution structures of many TRP channels have been resolved

(Figure 4.2) [163-167]. Some have also been resolved in presence of ligands [18,

19, 113, 164, 168, 169]. Our study further establishes that cryo-EM is a technique that can be used to study the structural architecture and biophysical properties of membrane proteins which are either resistant to crystallization or are too big for

NMR studies. Moreover, cryo-EM allows us to capture the protein in a more native environment since the protein is frozen in a buffer solution.

However, like most other techniques, cryo-EM cannot be the answer for all problems. There are some proteins which are so flexible that cryo-EM is not able to resolve high resolution structures, for example ~ 50% of the TRPA1 protein was not resolved in the cryo-EM structures. Additionally, often proteins are mutated or truncated to achieve stable and homogeneous sample suitable for cryo-EM imaging. Mutation or truncation can sometimes give an incomplete answer about the role different regions of the protein play in achieving various physiological

105 outcomes. In such cases well-established biochemical techniques like limited proteolysis can be used to look at protein stability under various conditions like addition of agonist, antagonist, change in temperature, etc (Figure 4.3). This can then be combined with mass spectrometry to determine the regions that are exposed to or shielded from proteolysis under these conditions, providing insight to the regions that experience topological rearrangements.

4.2.2 Application on health and disease

TRPV2 protein is ubiquitously expressed throughout the human body and has been implicated in various pathological conditions including cancer [97, 99,

100]. Among the various types of cancer, Glioblastoma multiforme (GBM) is considered to be the deadliest type and accounts for ~ 50% of all primary brain tumors (ref). One of the major reasons for the devastating prognosis of GBM patients is the resistance to standard chemotherapeutic drugs. Recently, the compound CBD, a non-psychotropic component of the Cannabis sativa plant, was shown to be a potent agonist for TRPV2 channel [99-102]. It was also shown that

TRPV2 plays critical role in GBM cell proliferation. Co-application of CBD and temozolomide (TMZ), a chemotherapeutic, increased TMZ uptake subsequently potentiating its cytotoxicity in GBM cells [101, 102]. This placed TRPV2 on the list of important anti-tumor drug targets [99-102]. This study revealed the molecular mechanism of TRPV2 channel activation by CBD consequently allowing the passage of cations and other molecules through the pore. This knowledge lays the foundation for the development of other small molecule compounds that can be

106 used for activation of TRPV2 and act as chemotherapeutics to treat various other types of cancer.

TRPA1 plays a key role in sensory transduction of chemical, neuropathic and inflammatory pain [61, 62, 128, 170-174] . Therefore, TRPA1 has often been a target for development of analgesics and anti-inflammatory agents [160, 175, 176].

Our study along with the cryo-EM structure [138] gives an in-depth understanding of the molecular mechanism of activation/inhibition/desensitization of the TRPA1 channel. Our results highlight the regions that are either shielded or exposed to the aqueous environment in presence of various ligands. This knowledge will help the pharmaceutical companies to manufacture small molecule drugs that can target a similar conformational change of these regions of TRPA1 to either activate/desensitize or inhibit the channel and alleviate pain.

4.3 Future directions

4.3.1 Permeation of large organic cations through TRP channels

Along with cations, large organic molecules such as N-methyl-d-glucamine

(NMDG+, 195 Da), QX-314 (343 Da), Po-Pro 3 dye (351 Da), FM 1-43 dye (611

Da) and Yo-Pro 1 dye (630 Da) have been also shown to permeate TRPV1-TRPV4 and other TRP channels [115, 117, 120, 177-179]. Based on extensive scanning mutagenesis experiments, it has been proposed that the pore turret, pore helix, and selectivity filter domains play significant role in large organic cation permeation through the TRPV1 channel upon ligand activation. Additionally, it has also been shown that chemotherapeutic agents like temozolomide (TMZ, 194 Da) and

107 doxorubicin (DOXO, 580 Da) permeate the TRPV2 channel pore after CBD activation. However, the structural basis of this uptake is unknown due to unavailability of the full-length TRPV1-TRPV4 structures in ligand-activated states[18-20]. Here we have resolved the structure of CBD-activated full length

TRPV2, that could provide the groundwork for understanding the potential mechanism of permeation of these large organic cations through TRP channels.

Resolving the structure of TRPV2 in presence of both CBD and Yo-Pro 1 dye could help us in understanding this phenomenon. Also, since our study showed that for Yo-Pro 1 uptake higher CBD concentration along with prolonged incubation time is required, in future, we can resolve the structure of TRPV2 at a higher concentration of CBD or at various CBD exposure times. This will give a more in depth understanding of the pore dilation phenomenon and the mechanism of large molecule uptake.

4.3.2 Characterization of TRPV2 specific blocker and understanding the mechanism of TRPV2 inhibition

TRPV2 has long been termed as an “orphan channel” due to lack of specific and proper pharmacological tools to study this channel. Therefore, it has remained as the least studied TRPV channel at the molecular level [97]. However, lately

TRPV2 has been proposed to play integral role in proliferation and migration of cancer cells [97]. Unlike in GBM, in some cancer types it has been reported that upregulation of TRPV2 leads to disease progression and malignancy [97]. Thus, identification and characterization of a TRPV2 specific inhibitor and resolving the inhibited state structure of full length TRPV2 is essential.

108

Recently, one of our collaborators have found that piperlongumine (PL), an natural compound of the plant Piper longum and has been shown to selectively kill cancer cells [180], is an inhibitor of TRPV2 activity. We ran some preliminary test to confirm this and found that PL indeed inhibits TRPV2 activity to some extent

(Figure 4.4). We also did some preliminary data collection of TRPV2 incubated with PL and could resolve a structure to ~5Å resolution (Figure 4.5). At this resolution we observed a very strong density in the pore at the lower gate region of the channel. A better resolution structure of TRPV2 with PL will be able to clarify the mechanism of TRPV2 inhibition by PL.

4.3.3 Resolving the structure of full length TRPA1 reconstituted into nanodiscs using cryo EM

Although full length TRPA1 was used for determining the structure of TRPA1 by cryo-EM, only 50% of the structure could be resolved [138]. In this paper the authors had used amphipol, PMAL-C8 to stabilize their TRPA1 sample. In our study also we have used detergent solubilized and amphipol (A8-35) stabilized pure TRPA1. However, there is some debate in the field about whether amphipols allow for extensive conformational changes to occur. Additionally, neither David

Julius’s group nor our group saw any changes in the transmembrane region of the

TRPA1 channel. In contrast, nanodiscs provide a more natural environment for the protein molecules. Amphipols might have also caused hindrance to detection of conformational changes in transmembrane regions by limited proteolysis and mass spectrometry. Furthermore, the structure of NOMPC, another TRP channel protein with extensive ankyrin repeats, was recently resolved in nanodiscs [161]

109 with various ligands and the authors had observed changes in the gates of the protein. Therefore, it is necessary to try a similar approach for resolving the full length structure of TRPA1 in its apo state and in presence of various ligands. It will be also beneficial to perform limited proteolysis and mass spectrometry of nanodisc reconstituted TRPA1 in its apo state and in the presence of various ligands to get an overall understanding of how the protein functions at a molecular level.

4.3.4 Regions of TRPA1 molecule affected by other electrophilic and non- electrophilic ligands

Being a polymodal channel, TRPA1 is modulated by a wide variety of structurally unrelated compounds. Here, we studied the effect of one electrophilic agonist, one non-electrophilic antagonist, one non-electrophilic agonist and one non-electrophilic modulator. In the future, it will be interesting to see the effects on

TRPA1 architecture of more electrophilic agonists like AITC, DADS; non- electrophilic agonists like cinnamaldehyde, allicin; non-electrophilic antagonists like HC-030031 and many other drugs that are in clinical trials. This will enhance our understanding of molecular mechanism of functioning of such an intriguing and multimodal channel. This knowledge will help in development of better analgesics that do not target the opioid receptors.

4.4 Concluding remarks

Both TRPV2 and TRPA1 play important roles in various physiological functions and are implicated in various disease conditions and hence, both are

110 primary drug targets of interest. Therefore, studying them at a cellular and molecular level to better understand their biophysical properties is important for the development of therapeutics. Our study here lays the ground work for understanding some of the basic molecular mechanisms of these two TRP channels belonging to two different subfamilies. Further studies are needed to understand how TRPV2 and TRPA1 are modulated by other ligands. Additionally, revealing the structure of the full length TRPA1 is necessary to better understand how the various ankyrin repeats are oriented and how they communicate between themselves and other intracellular components.

111

Figure 4.1. Cartoon representation of gating mechanism of TRPV2 in presence of CBD. Interaction with CBD initiates rotation and large scale displacement of the S5 helix which in turn leads to movement of the pore helix and pore turret away from the pore thereby causing a funnel like opening of the channel.

112

TRPML3 TRPP2 TRPML2 TRPM8 TRPP3

TRPM2 TRPP5 TRPP1 TRPML1 TRPM4 TRPP4

TRPM5

TRPM7 TRPA1 TRPM6 TRPM3 TRPM1 TRPC3 TRPC7 TRPV6 TRPC6 TRPV5 TRPV1 TRPV4 TRPC4 TRPV3 TRPV2 TRPC2 TRPC5 TRPC1

Figure 4.2. A tree representing the mammalian TRP superfamily of ion channels with most of the high resolution structures. Recently resolved most of the high resolution structures of TRP superfamily are shown. In the past few years the field has resolved structures from all the subgroups except TRPC.

113

Limited Proteolysis Mass Spectrometric Analysis

Protease

Protein

Ligand Protease

Protein

Figure 4.3. Schematic representation of the principle of limited proteolysis followed by mass spectrometry. Limited proteolysis of a protein in apo state and in ligand bound state by a favorable protease will yield different peptides due to ligand induced conformational changes in the protein. These different peptides can then be identified and analyzed by mass spectrometry.

114

Figure 4.4. Inhibition of TRPV2 activity by PL and preliminary cryo-EM data of PL-inhibited TRPV2. (A) Fluorescence based Ca2+ flux assay; application of

30µM CBD induces rapid Ca2+ influx into WT TRPV2 expressing HEK cells but not in presence of 50µM PL, monitored by Fluo-4 dye fluorescence. (B) Representative micrograph of TRPV2 incubated with PL. (C) Representative 2D classes of TRPV2 incubated with PL.

115

Figure 4.5. Cryo-EM analysis of CBD-activated and PL-inhibited TRPV2 channel. Cryo-EM density maps of (A) CBD-activated TRPV2 and (B) PL-inhibited

TRPV2 depicted as top views. C) Sliced view through the cryo-EM density map of

PL-inhibited TRPV2. Putative PL density in the lower gate of the channel pore is shown by arrow.

116

Appendix

1) Portions of Samanta et al, Subcell Biochem. 2018;87:141-165. doi:

10.1007/978-981-10-7757-9_6, were reproduced in Chapter 1.

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Copyright Clearance Center’s RightsLink® service makes it faster and easier to secure permission for the reuse of Springer Nature content to be published, for example, in a journal/magazine, book/textbook, coursepack, thesis/dissertation, annual report, newspaper, training materials, presentation/slide kit, promotional material, etc.” https://www.springer.com/us/rights-permissions/obtaining-permissions/882

2) Portions of Samanta et al, Nature structural biology, under revision, were

reproduced in Chapter 2.

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Ownership of copyright in in original research articles remains with the Author, and provided that, when reproducing the contribution or extracts from it or from the

117

Supplementary Information, the Author acknowledges first and reference publication in the Journal, the Author retains the following non-exclusive rights:

To reproduce the contribution in whole or in part in any printed volume (book or thesis) of which they are the author(s).” https://www.nature.com/reprints/permission-requests.html

3) Portions of Samanta et al, J Gen Physiol. 2018 May 7;150(5):751-762. doi:

10.1085/jgp.201711876, were published in Chapter 3.

“It is the mission of Rockefeller University Press to promote widespread reuse and distribution of the articles and data we publish. In this spirit, authors retain copyright to their own work and can reuse it for any purpose as long as proper attribution is provided.” http://www.rupress.org/content/permissions-and-licensing

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