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Structure and function of adenylyl , key

in cellular signaling

1,2,3 2,3 1,2,3

Basavraj Khannpnavar , Ved Mehta , Chao Qi and Volodymyr

Korkhov1,2

The adenylyl cyclases (ACs) catalyze the production of the prokaryotes, the mammalian genomes encode only the

ubiquitous second messenger, cAMP, which in turns acts on a class III ACs. Despite the profound differences in the

number of effectors and thus regulates a plethora of cellular structures and domain organization, the six AC classes are

functions. As the key enzymes in the highly evolutionarily functionally very similar, catalyzing the conversion of a

conserved cAMP pathway, the ACs control the physiology of molecule of ATP into cAMP (Figure 1a).

the cells, tissues, organs and organisms in health and disease.

A comprehensive understanding of the specific role of the ACs

Bacterial cAMP systems

in these processes of life requires a deep mechanistic

In bacteria, one of the most extensively studied roles of

understanding of structure and mechanisms of action of these

cAMP is in regulation of the Escherichia coli glucose

enzymes. Here we highlight the exciting recent reports on the

metabolism via the cAMP receptor protein CRP (or

biochemistry and structure and higher order organization of the

catabolite activator protein, CAP) [1]. In addition to its

ACs and their signaling complexes. These studies have

role in regulation of glucose metabolism, CRP can exert

provided the glimpses into the principles of the AC-mediated

profound influence on global expression in E. coli via

homeostatic control of cellular physiology.

its influence on more than 380 promoters and 70 transcrip-

Addresses tion factors [2], contributing to the multiple roles of

1

Institute of Biochemistry, ETH Zurich, Switzerland cAMP in processes ranging from carbon metabolism to

2

Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen regulation of virulence phenotypes in pathogenic bacteria

5232, Switzerland

[3–5]. The bacterial cAMP systems also play key roles in

regulation of the cellular homeostasis, phototaxis, protein

Corresponding author: Korkhov, Volodymyr ([email protected])

3 secretion and virulence [6]. For example, in Pseudomonas

Contributed equally.

aeruginosa, cAMP pool generated by adenylyl cyclases

Current Opinion in Structural Biology 2020, 63:34–41 CyaA and CyaB regulates the expression of the type-III

secretion system (T3SS) and other important virulence

This review comes from a themed issue on Membranes

determinants via the cAMP-binding protein Vfr [7].

Edited by Beili Wu and Fei Sun

Many of the bacterial species have evolved the ACs (or

more broadly, nucleotidyl cyclases, NCs) as virulence

https://doi.org/10.1016/j.sbi.2020.03.003 effectors or toxins [8]. These toxins are secreted by the

pathogenic bacteria inside the host cell, hijacking the host’s

0959-440X/ã 2020 The Author(s). Published by Elsevier Ltd. This is an

open access article under the CC BY-NC-ND license (http://creative- cAMP system and aiding the pathogenic bacteria in invad-

commons.org/licenses/by-nc-nd/4.0/). ing the host immune system. The anthrax edema factor

(EF) from Bacillus anthracis is a prototypical and a very

extensively studied AC toxin (REF). Other well-studied

Introduction AC toxins include the Hemolysin-AC CyaA from Bordetella

0 0

pertussis, ExoY from P. aeruginosa, and MARTX ExoY-like

Adenosine 3 ,5 -monophosphate (cAMP) is a ubiquitous

edema factor from Vibrio species [9–12]. These nucleotidyl

signaling molecule across all the domains of life. The

cyclases toxins are active inside the host organisms and

cAMP signaling pathway is a highly conserved regulatory

depend on the host-specific binding partners such as cal-

mechanism that plays a pivotal role in a wide range of

modulin or actin for maximal catalytic activity (REF). The

fundamental cellular process. Adenylyl cyclases (ACs) are

cAMP pool generated by these ACs suppresses the host

the enzymes that generate cAMP, and thus the key

immune system, triggers the reorganization of the actin

components of the cAMP signaling and regulation path-

cytoskeleton causing bleb-niche formation, inter-endothe-

ways. In this review we provide a brief general overview

lial cell gap formation and increased vascular permeability.

of the field, focusing on the recent breakthroughs in our

This ultimately leads to tissue edema and injury, prevent-

understanding of the structure and function of the ACs as

ing the wound healing [8,13].

the key enzymes in cellular signaling.

The ACs in all kingdoms of life include six distinct classes The most abundant class of the ACs, the class III

(class I–VI). Although the six classes are present in enzymes are often coupled to domains that can be used

Current Opinion in Structural Biology 2020, 63:34–41 www.sciencedirect.com

Structure and function of adenylyl cyclases Khannpnavar et al. 35

Figure 1

(a)

Class II CyaA: 1XFV

(b)

Class III AC10: 4CM0

(c)

Class IV YpAC-IV: 3N0Y

Current Opinion in Structural Biology

Representative structures of ACs.

(a) A crystal structure of a class II AC anthrax edema factor showing the key residues involved in binding and catalysis of ATP into cAMP. (b)

Active site architecture of human soluble ACs (AC10) in the presence of an ATP analogue and divalent metal ions. (c) X-ray structure of a class IV

AC from Yersinia pestis, bound to an ATP analogue. Despite the significant differences in structural folds, these ACs are able to maintain similar

configurations involving acidic residues for coordination of divalent metal ions and basic residues for stabilization of negatively charged

ATP in the catalytic pocket.

to sense and respond to a variety of stimuli. Because of metabolites, and so on [18]. Some of these proteins have

this feature, the class III ACs can directly receive, amplify been assigned a role in pathogenicity of the mycobacte-

and transmit the information [14]. For example, the rium. This is the case, for example, for Rv0386, which has

adenylate CyaB responsible for major cAMP pool been shown to be critical for macrophage infection [19].

in P. aeruginosa, is a class III AC coupled to a membrane-

anchored MASE2 sensor domain, which enables the Mammalian ACs

direct regulation of the expression of T3SS and other All mammalian ACs are class III enzymes, with nine

virulence determinants by the environmental stimuli membrane-integral isoforms participating in the GPCR

(CO2/HCO3 ) [15–17]. The genome of Mycobacterium and G protein-mediated signaling pathway (AC1-9), and

tuberculosis encodes 16 ACs which facilitate regulation one soluble AC (AC10 or sAC) that is not directly linked

of gene expression, metabolism, and virulence in to the GPCR signaling. The most conserved regions of

response to extracellular stimuli such as pH, bicarbonate, these enzymes are the class III catalytic domains. The

www.sciencedirect.com Current Opinion in Structural Biology 2020, 63:34–41 36 Membranes

membrane ACs also share the architectural similarity light [30 ,35]. The structure of the catalytic domain of a

˚

(detailed in section 3 below): the 12 transmembrane light-sensitive rhodopsin-cyclase was reported at 2.3 A

(TM) domains, and two ‘helical domains’ that link resolution and depicted similar homodimeric structural

TM6 and TM12 to the catalytic domains 1 and 2 (C1a arrangement seen in Class III ACs with relatively

and C2a, respectively). The less conserved C1b and C2b conserved catalytic residues [30 ]. Although it remains

domains are predicted to play an isoform-specific role in to be determined how the different domains interact

regulating the activity or the assembly of ACs and their with and regulate the light-sensitive ACs (and GCs),

complexes. The role played by the TM region in the understanding how these enzymes operate and how

membrane ACs, apart from membrane anchoring and cyclic nucleotide production can be controlled by light

trafficking [20] or oligomerization of these enzymes under various conditions will enable new optogenetic

[21], is currently not clear. applications in biology and medicine.

Membrane bound cyclases have been shown to play Structure of the soluble domain of Rv1625c, the

crucial role in mammalian physiology. AC1 and AC8 have evolutionary ancestor of the membrane ACs

been shown to be involved in learning and memory [22]. One of the M. tuberculosis ACs, the membrane-integral

AC1 has been implicated in neuropathic and inflamma- Rv1625c (Cya), has been suggested to be an evolutionary

tory pain, playing a critical role in nerve injury-induced ancestortothemammalianmembraneACs[36].Theprecise

plasticity in regions of the brain responsible for pain role of Rv1625c in mycobacteria is currently unknown,

perception and has been considered as a potential target although the protein has been shown to be involved in

for pain [23]. Both AC1 and AC8 knock out mice showed mycobacterial cholesterol metabolism [37,38 ] as well as in

reduced inflammatory, neuropathic and muscle pain [23]. response to heat stress [39]. The sequence of Rv1625c

AC3 has been linked to obesity and diabetes [24,25]. The includes six TM helices and a catalytic soluble domain

Gbg and Gas subunits released upon receptor activation [36]. The functional unit of the protein is a dimer. Whereas

are responsible for activating AC2 and AC4; this contrib- mammalian ACs are regulated by G proteins, the factors that

utes to the excitatory effects of chronic opioid use and to regulate the Rv1625c/Cya in mycobacteria are unknown. A

the development of tolerance [26]. The role of AC5 and recent study suggested that the TM domain of Rv1625c may

6 has been extensively studied in cardiac function [27,28]. be a receptor for a yet unknown signal, however this remains

Mutations in AC5 have been linked to a movement to be experimentally verified [40].

disorder, familial dyskinesia and facial myokymia

(FDFM) [29]. The X-ray structure of the soluble domain of the

M. intracellulare Cya (a homologue of Rv1625c) provided

Insights into the structure and function of the the first structure of a complete soluble domain of a

ACs membrane AC (Figure 2c) [41 ]. The structure revealed

Light-sensitive ACs a canonical type III catalytic domain, fused to the helical

Some of the bacterial genomes feature ACs fused to domain. The helical domain was determined to be very

light-sensing domains. These types of enzymes have important for the catalytic activity of Cya. Grafting of the

attracted a considerable amount of attention due to inactivating mutation P228S, which mimicks the similar

their potential use in optogenetics [30 ,31]. In 2016, the mutation in the helical domain of the human retinal GC

X-ray structure of the photoactivated AC (PAC) retGC1, produced a protein with a severe defect in its

from the photosynthetic cyanobacterium Oscillatoria ability to catalyze cAMP production. Despite this, the

acuminata was solved, revealing the key role of central mutated soluble domain was capable of binding MANT-

coiled-coil domain in the transduction of the signal GTP, a fluorescent nucleotide analogue, and was able to

received by the blue-light sensing BLUF domain dimerize in a MANT-GTP-dependent manner. Remark-

[32] (Figure 2a and b). A number of crystal structures ably, the helical domain of Cya clearly resembles the

of PACs from Oscillatoria and Beggiatoa were solved in similar structural element in the BLUF domains of the

dark and light-activated states, highlighting-specific PACs (Figure 2e). The observations of the minor changes

structural elements involved in the relay between induced by light in the PAC upon its activation, together

the light-induced changes in the flavin chromophore with the severe effects on enzymatic activity induced by

of the BLUF domain and the active site of the cyclase the disrupting mutation in Cya, suggest that the helical

catalytic domain [33 ,34 ] (Figure 2b). More recently, domain plays a key role in controlling the function of

a novel class of light-sensitive membrane bound ACs, the ACs.

also known as the two-component cyclase opsins

(2c-cyclops) belonging to rhodopsin family of proteins Cryo-EM structures of the mammalian AC9-Gas

have been discovered [35]. These proteins include the complex

8-TM helical bundle, similar to the rhodopsin- The early structures of a mammalian AC were those of

phosphodiesterases RhoPDE), show ATP-dependent the homodimeric C2a catalytic domain of the rat AC2

guanylyl cyclase (GC) activity, and are inhibited by [42], and the pseudo-dimer of the C1a domain of canine

Current Opinion in Structural Biology 2020, 63:34–41 www.sciencedirect.com

Structure and function of adenylyl cyclases Khannpnavar et al. 37

Figure 2

(a) (c) hν

FMN FMN

ATPcAMP ATP cAMP

(b) (d)

Fmn HD

BLUF Oscillatoria acuminata PAC : Mycobacterium intracellulare Cya: 5X4T 5O5L (e)

Fmn BLUF

Beggiatoa sp. PS AC, β-subunit: 5NBY

Current Opinion in Structural Biology

X-ray structures of the photoactivated ACs (PACs) and the soluble domain of Rv1625c/Cya.

(a) A schematic representation of the bacterial photoactivated ACs. The catalytic domains of the homodimeric enzymes are fused to the light

sensing BLUF domains, which bind a flavin mononucleotide. (b) Examples of the BLUF domain containing PACs from Oscillatoria and Beggiatoa.

(c) A schematic of the mycobacterial membrane AC, Rv1625c/Cya. Each monomer in the homodimeric Cya is composed of 6 TM domains, linked

to the catalytic domain (C) by a helical domain. The bracket indicates the region for which a structure is available. (d) X-ray structure of the M.

intracellulare Cya, revealing the arrangement of the helical domain (HD). (e) The helical domains in PACs and Cya are structurally conserved.

AC5 and C2a domain of the rat AC2, bound to the [44–47]. These structures provided important insights

bovine Gas subunit [43]. Subsequently, number of into the asymmetric catalytic and allosteric pockets at

crystal structures of the latter complex in a variety of the interface between the AC catalytic domains, as well

substrate and co-substrate-bound states were solved into the details of the two-metal ion-dependent

www.sciencedirect.com Current Opinion in Structural Biology 2020, 63:34–41 38 Membranes

Figure 3

(a) (c) C1a Gαs GTPγS

ATP cAMP

Effectors (PKA, CNG, EPAC, etc.)

(b) C2a C2b bAC9-bGαs: 6R4P out

(d) MANT-GTP in

HD Gαs

Forskolin

C2a

C1a α bAC91250-bG s-MANT-GTP-Forskolin: 6R4O bAC9-bGαs: 6R3Q

Current Opinion in Structural Biology

Cryo-EM structures of the bovine AC9-Gas complex.

(a) A schematic illustration of the G protein-coupled receptor/cAMP pathway. A hormone binds and activates a GPCR, which activates the

heterotrimeric G protein; the G protein a subunit dissociates and binds to the membrane AC, leading to cAMP production. The generated cAMP

˚

acts on a number of downstream effector proteins to regulate the cellular physiology. (b) A 3.4 A cryo-EM structure of bovine AC9, bound to the

Gas protein subunit. The helical domain (HD) and the two catalytic domains C1a and C2a are indicated in the panel. The G protein is bound to the

non-hydrolysable GTP analogue (GTPgS). (c) A view of the catalytic site from the cytosolic side of the membrane shows the location of the

occluding peptide, the C2b domain, which binds to the active and allosteric sites of the AC9. (d) Removal of the C2b domain enabled structure

determination of the MANT-GTP-bound and forskolin-bound state of the AC9-Gas complex.

reaction [44], inhibition by calcium ions [47] and phar- purified AC9-Gas complex in detergent micelles

macological modulation of the ACs using P-site inhibi- (digitonin).

tors [45].

The structure revealed the overall architecture of AC9,

The first insights into the structure of the full-length including the 12-TM region linked to the C1a and C2a

membrane AC were provided by the cryo-EM structure domains by the helical domain (Figure 3b). The 12-TM

of the bovine AC9-Gas complex [48 ] (Figure 3). The portion shows a pseudo-two-fold symmetrical arrangement

˚

3.4 A resolution structure was determined using single of TM1-6 and TM7-12, which can be superimposed with a

particle analysis using the recombinantly expressed and relatively low RMSD [48 ]. Although the membrane

Current Opinion in Structural Biology 2020, 63:34–41 www.sciencedirect.com

Structure and function of adenylyl cyclases Khannpnavar et al. 39

portions of the ACs have been proposed to potentially act as Acknowledgement

channels or transporters [49], there is currently no biochemi- The research of VMK’s group is supported by the Swiss National Science

Foundation (grants 150665, 176992, 184951).

cal evidence for this. The cryo-EM 3D reconstruction of the

AC9-G protein complex does not feature a membrane per-

meation pathway within the TM region of AC9. Thus, References and recommended reading

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