View Showing the Overlay of C Trace of Gankyrin WT (Black) and Modeled I79D Mutant (Green)

Study of the Structure and Function Relationship of Oncoprotein Gankyrin

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Yi Guo, B.S.

Ohio State Biochemistry Graduate Program

The Ohio State University 2009

Dissertation Committee:

Professor Ming-Daw Tsai, Advisor

Professor Ross Dalbey, Co-advisor

Professor Shang-Tian Yang

Yi Guo, B.S.

2009

ABSTRACT

Gankyrin, a newly defined oncoprotein also known as PSMD10 and P28, functions as a dual-negative regulator of the two most prominent tumor suppressor pathways: the CDK/pRb and MDM2/P53 pathways. Its aberrant expression has been prevalently found in human hepatocellular carcinomas (HCC) and esophagus squamous cell carcinomas (ESCC), which indicates gankyrin is a potential diagnostic and therapeutic target in cancers. Gankyrin is an ankyrin repeat (AR) protein which is composed of seven ankyrin repeats. Each repeat of gankyrin exhibits a canonical helix- turn-helix conformation and these seven repeats are stacked together near linearly to form a helix bundle, in which the neighboring ankyrin repeats are linked by loops of varied size which orientate perpendicularly to the axes of the helices of ankyrin repeats.

Our previous studies showed that while both specific CDK4 inhibitor p16INK4A

(P16, exclusively consists of four AR repeats) and gankyrin bind to cyclin-dependent kinase 4 (CDK4) in similar fashion, only P16 inhibits the kinase activity of CDK4. While this could explain why P16 is a tumor suppressor and gankyrin is oncogenic, the structural basis of these contrasting properties was unknown. In this study we show that a double mutant of gankyrin, L62H/I79D, inhibits the kinase activity of CDK4, similar to

P16, and such CDK4-inhibtory activity is associated with the I79D but not L62H mutation. In addition, mutations at I79 and L62 bring about a moderate decrease in the ii

stability of gankyrin. Further structural and biophysical analyses suggest that the substitution of Ile79 with Asp leads to local conformational changes in loops I–III of gankyrin. Taken together, our results allow the dissection of the “protein–protein binding” and “CDK4 inhibition” functions of P16, show that the difference between tumor suppressing and oncogenic functions of P16 and gankyrin, respectively, mainly resides in a single residue, and provide structural insight to the contrasting biological functions of the two AR proteins.

The second part of the research on gankyrin involves an important structural characteristic of AR proteins: the presence of TPLH tetrapeptide or a variant at the beginning of canonical helix-turn-helix motif. Hydrogen bonding involving the Thr and

His residues in the same and between adjacent tetrapeptide motifs presumably contributes to the formation of a hydrogen-bonding network and consequently the stability of the molecule. Thus, we investigated the structural role of this TPLH network in AR proteins by studying gankyrin, an oncogenic protein composed of seven ARs and six TPLH tetrapeptides, and p16INK4a, a tumor suppressor with four ARs and no TPLH tetrapeptides. Our results show that disrupting the TPLH network in the middle by removing Thr or both Thr and His from AR4 and AR5 of gankyrin significantly decreases its stability in both chemical- and heat-induced unfolding. On the other hand, introducing the TPLH network in p16INK4a in the middle (on AR3) increases its conformational stability. Our results suggest that the hydrogen bonding between neighboring TPLHs stabilize the structure of AR proteins when the TPLH motifs are in the middle of a long stretch of ankyrin repeats.

iii

Dedicated to my family

ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Ming-Daw Tsai, for his guidance during my

Ph.D studies. Dr. Tsai has always been encouraging his students to think as a scientist, which has truly helped me in my scientific career.

I would also like to thank Dr. Junan Li and my dissertation committee members,

Dr. Ross Dalbey and Dr. Shang-Tian Yang, for their time and vested interest in my research. Dr. Junan Li has been guiding me with my research during the past two years.

With his help on research, I was able to tackle problems and finish research projects.

I would like to acknowledge my past lab members, including Hongyuan Li,

Shngjiang Tu, Anjali Mahajan, Haiyan Song, Yu Wang, Sandeep Kumar, Hyun Lee,

Brandon Lamarche, and Marina Bakhtina. This group of incredibly talented scientists has taught me so much not only about science, but also about being human. It is an honor to have worked with each of these individuals.

Finally, I thank my family for the unconditional love and support. My mother has been extremely supportive of my scientific career even though she is thousands of miles away. My lovely wife, Jie, has been my most solid support during my Ph.D career.

VITA

November 29, 1980...... Born – Wuhan, Hubei, China.

June 2002...... Bachelor of Science in Biochemistry, Wuhan University.

2004 – 2005...... Ohio State Biochemistry Program Fellow, The Ohio State University.

2005 – present...... Graduate Teaching and Research Associate, The Ohio State University.

PUBLICATIONS

1. Guo, Y. , Yuan, C., Weghorst, C. M., Tsai, M.-D., and Li, J. (2009) Diversified Contributions of Conserved TPLH Tetrapeptides to the Conformational Stability of Ankyrin Repeat Proteins. J Biol Chem (submitted).

2. Li, J. and Guo, Y. (2009) Gankyrin Oncoprotein: Structure, Function, and Involvement in Cancer. Current Chemical Biology (In Press).

3. Guo, Y. , Mahajan, A., Yuan, C., Joo, S. H., Weghorst, C. M., Tsai, M.-D., and Li, J. (2009) Comparisons of the Conformational Stability of Cyclin-Dependent Kinase (CDK) 4-Interacting Ankyrin Repeat (AR) Proteins. Biochemistry 48, 4050-4062.

4. Mahajan, A. †, Guo, Y.† , Yuan, C., Weghorst, C. M., Tsai, M.-D., and Li, J. (2007) Dissection of protein-protein interaction and CDK4 inhibition in the oncogenic versus tumor suppressing functions of gankyrin and p16. J. Mol. Biol. 373, 990-1005. ( †A.M. and Y.G. contributed equally to this work)

FIELDS OF STUDY

Major Field: Ohio State Biochemistry Program vi

TABLE OF CONTENTS

Page

Abstract ...... ii

Dedication ...... iv

Acknowledgments ...... v

Vita ...... vi

List of Tables...... xi

List of Figures ...... xii

List of Abbreviations ...... xiv

Chapters:

1. Introduction ...... 1

1.1 The discovery of oncoprotein gankyrin ...... 1

1.2 The structure of gankyrin ...... 3

1.3 Diverse functions of gankyrin ...... 5

1.3.1. Gankyrin and Rb, p53 ...... 6

1.3.2. Gankyrin and the proteasome ...... 8

1.3.3. Gankyrin and MAGE A4, RelA ...... 8

1.3.4. The structure basis for the function of gankyrin ...... 9 vii 1.4 Gankyrin and cancer ...... 12

1.4.1. Aberrant expression of gankyrin is prevalent in human cancers . . 12

1.4.2. Aberrant expression of gankyrin could be an early event in the development of human cancers ...... 13

1.4.3. Potential mechanisms underlying the involvement of gankyrin in cancer ...... 14

1.5 Summary ...... 16

2. Dissection of protein-protein interaction and cdk4 inhibition in the oncogenic versus tumor suppressing functions of gankyrin and p16 ...... 18

2.1 Introduction ...... 18

2.2 Results ...... 23

2.2.1. Structure-based protein engineering ...... 23

2.2.2. I79D and L62H/I79D of gankyrin bind and inhibit CDK4 ...... 24

2.2.3. I79D and L62H/I79D mutations bring about substantial perturbation to the local conformation of gankyrin ...... 27

2.2.4. I79D and L62H/I79D destabilize the global structure of gankyrin 33

2.3 Discussion ...... 35

2.3.1. Possible conformational adjustments of local loops upon I79D mutation of gankyrin ...... 35

2.3.2. Structural basis for the decreased stability of the gankyrin mutants ...... 39

2.3.3. Structural basis for the functional diversity of the AR proteins . . 40

2.3.4. Potential biological significance ...... 42

2.4 Materials and Methods ...... 43

viii 2.4.1. Cloning, expression, and purification of human gankyrin and its mutants ...... 43

2.4.2. Pull-down assay ...... 44

2.4.3. In Vitro CDK4 kinase assay ...... 45

2.4.4. Circular dichroism (CD) analyses of gankyrin proteins ...... 46

2.4.5. NMR analyses ...... 47

2.4.6. Bioinformatics analysis ...... 47

3. Contributions of conserved tplh tetrapeptides to the conformational stability of ankyrin repeat proteins ...... 48

3.1 Introduction ...... 48

3.2 Results ...... 54

3.2.1. Mutagenic effect on gankyrin-CDK4 association ...... 55

3.2.2. Mutagenic effect on the conformational stability of gankyrin . . . . 57

3.2.3. Mutagenic effect on the structure of gankyrin ...... 61

3.2.4 Mutagenic effect on P16 ...... 68

3.3 Discussion ...... 70

3.4 Materials and Methods ...... 76

3.4.1. Database analysis...... 76

3.4.2. Cloning, expression, and purification of human gankyrin, p16 INK4a and their mutants...... 77

3.4.3. Pull-down assay ...... 78

3.4.4. In vitro CDK4 kinase assay ...... 78

3.4.5. Circular dichroism (CD) analyses ...... 79

ix 3.4.5. NMR analyses ...... 80

Bibliography ...... 81

LIST OF TABLES

Table Page

2.1 Biochemical and biophysical parameters of gankyrin and its mutant proteins ...... 35

3.1 Conformational stability of TPLH mutants of gankyrin and P16 . . . . . 54

LIST OF FIGURES

Figure Page

1.1 Sequence alignments of human, wolf, mouse, rat, hamster, zebrafish, and yeast gankryin proteins ...... 3

1.2 Solution structure of human gankyrin ...... 4

1.3 The dual-negative regulatory roles of gankyrin in the pRb and P53 pathways ...... 7

1.4 Proposed mechanisms of gankyrin-mediated deregulation of cell cycle control ...... 16

2.1 Structural Basis of p16/CDK6 (or CDK4) Interaction ...... 20

2.2 Structure-based sequence homology between P16 and gankyrin ...... 22

2.3 Interactions between gankyrin proteins and CDK4 as evaluated using pull-down assays and in vitro kinase assays ...... 25

2.4 Quantitative measurement of the CDK4-inhibitory activities of gankyrin proteins ...... 27

2.5 Comparison of 15 N-1H HSQC spectra of wild type gankyrin and its mutants ...... 29

2.6 Mapping of chemical shift changes on the gankyrin structure ...... 30

2.7 2D NOESY spectra on WT (a), I79D (b), and L62H (c) showing the proton downfield region ...... 32

2.8 Chemical- and heat-induced unfolding of gankyrin mutants monitored by far-UV CD ...... 34

xii

2.9 Docking model showing the interactions of I79 of gankyrin with the active site of CDK6 ...... 38

α 2.10 Stereoview showing the overlay of C trace of gankyrin WT (black) and modeled I79D mutant (green) ...... 39

3.1 The consensus sequence of an AR motif and the TPLH-mediated hydrogen bonding network ...... 50

3.2 Probability of T/SxxH appearing on each AR of proteins containg three to twlve ARs ...... 51

3.3 The TPLH network in Gankyrin and P16 ...... 53

3.4 Functional analyses of gankyrin and P16 mutants ...... 56

3.5 Chemical- and heat-induced unfolding of representative gankyrin and p16 INK4a mutants monitored by far-UV CD ...... 59

3.6 2D 1H-15 N HSQC spectra of gankyrin WT showing the all-or-none unfolding ...... 60

3.7 Residue pairwise chemical shift difference between gankyrin mutant and WT ...... 63

3.8 Selected spectral regions from 2D NOESY spectra on gankyrin ...... 65

3.9 2D 1H-15 N HSQC of gankyrin proteins after 25 hour H/D exchange at room temperature ...... 66

3.10 Ribbon diagram showing the slow exchanging amide proton detected in gankyrin T42A (GankTPLH1A) ...... 67

3.11 Selected spectral regions from 2D NOESY spectra on (A) P16 WT, (B) mtant R80T (P16TPLH2) ...... 70

xiii

LIST OF ABBREVIATIONS

AAA ATPases Associated with a variety of cellular Activities

AEBSF 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride

AR Ankyrin Repeat

BSA bovine serum albumin

°C degrees Celsius cal calorie

CD Circular Dichroism

CDK Cyclin-dependent kinases

D Dalton

DTT dithiothreitol

EDTA ethylenediaminetetraacetic acid

EGTA ethylene glycol tetraacetic acid g gram(s)

GdnHCl guanidinium hydrochloride

GST glutathione-S-transferase h hour(s)

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HMBC heteronuclear multiple-bond correlation

xiv

HPLC high performance liquid chromatography

HSQC Heteronuclear Single Quantum Coherence

IC inhibition concentration

IPTG isopropyl-beta-D-thiogalactopyranoside

κ kappa k kilo

µ micro l liter m milli

M moles per liter

MAGE melanoma antigen gene min minute(s) mol mole(s) n nano

NOESY Nuclear Overhauser Enhancement Spectroscopy

NMR Nuclear magnetic resonance

PCR polymerase chain reaction

PDB Protein Data Bank

Rb retinoblastoma protein

SCC Small Cell Carcinoma

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

WT wild-type

CHAPTER 1

INTRODUCTION

1.1 The discovery of oncoprotein gankyrin.

Recently, a novel oncogenic protein was discovered by Higashitsuji et al and it was shown to deregulate the pRb pathway of cell cycle control. The novel gene was initially isolated from a subtraction cDNA library which contained overexpressed genes in hepatocellular carcinomas compared with non-cancerous liver tissues. After cloning the full length cDNA from human placenta (Genbank accession no. NM_002814), it was found that the clone encoded a 25 kDa ankyrin repeat protein with six ankyrin repeats (1).

Higashitsuji et al discovered that in all 34 hepatocellular carcinomas studied, the mRNA level of this oncoprotein was increased by 360% on average, compared with levels in the corresponding non-cancerous portions of the liver. In the meantime, the expression level of this protein was also increased significantly in these 34 samples. Therefore, this oncoprotein was named gankyrin (gann ankyrin-repeat protein; gann means “cancer” in

Japanese).

Gankyrin, originally named p28, was first purified and characterized by Hori et al . It was isolated from the purified bovine erythrocyte PA700 (19S) complex by HPLC and SDS–PAGE, and reported as a novel regulatory subunit of the 19S complex, a

regulatory complex of the human 26S proteasome (2). Thus, gankyrin was thought to function in the proteasome-mediated degradation. Later on, genes encoding fission yeast, mouse, rat, Golden hamster, wolf, and zebrafish have been identified. As shown in

Figure 1.1, the sequence identities of gankyrin vary among species. The sequence

homologies between human and other mammals are higher than 90%, which is consistent

with the fact that the proteasome system is highly conserved among mammals. While

there is a homology of 72% between human and zebrafish gankyrin, the sequence

homology between human and fission yeast is relatively low, only 29%. However, most

of the key residues important for the sketch structure of ankyrin repeats are conserved,

implying that human and fission yeast gankyrin proteins could have a higher structural

similarity.

Human MEGC VSN LMV CN LA YSG KLEE LKESILA DKSLA TRTDQDSRTAL HWA CS AGHT EIV EFLL 60 wolf MEGC VSN LMV CN LA YGG KLEE LKERIL ED KSLA TRTDQDSRTAL HWA CS AGHT EIV EFLL 60 mouse MEGC VSN IMI CN LA YSG KLDE LKERILA DKSLA TRTDQDSRTAL HWA CS AGHT EIV EFLL 60 rat MEGC VSN LMV CN LA YNG KLDE LKESILA DKSLA TRTDQDSRTAL HWA CS AGHT EIV EFLL 60 hamster MEGC VS—KVCN LA YTG KLDE LKESILA DRTLA TRTDQDNRTAL HWA CS AGHT EIV EFLL 58 zebrafish MEARVSN VEVCN LA YGG KFEE LKK CVL SDNS LAA KTDQDSRTAL HWA CS AGH VNIA QFLL 60 yeast --- MV YASLGKAI EE NC PEE YVEQAI QN DPNS LNAV DDD KR TPL HWA CS VGKVNT IYFLL 57 * : : ::: : * . .* :*.**.******.* :.: ***

human QLG-VPV NDKDD AGWSPL HIAA SAG—RDE IV KALL GKG-AQVNAV NQNGCT PL HY AA SK 116 wolf QLG-VPV NDKDD AGWSPL HIAA SAG—RDE IV KALL GKG-AQVNAV NQNGCT PL HY AA SK 116 mouse QLG-VPV NDKDD AGWSPLHIAA SAG—RDE IV KALLV KG-AHVNS VNQNGCT PL HY AA SK 116 rat QLG-VPV NEKDD AGWSPL HIAA SAG—RDE IV KALLI KG-AQVNAV NQNGCT AL HY AA SK 116 hamster QLG-VPV NDKDD AGWSPL HIAA SAG—RDE IV KALLI KG-AQVNAV NQNGCT PL HY AA SK 114 zebrafish DLG-VEVDLKDD ACWTPL HIAA SAG—REE IVRSLI SKG-AQLNS VNQNGCT PL HY AA SK 116 yeast KQPNIKPDE KDE AGWTPLMI SINN RSVP DNVI EE LI NRSDVDPTITT RGGQTC LHY AA GK 117 . : : ** :* *:** *: . :::: . *: : ...... :.* * *****.*

human NRHEIAVMLL EGG ANPDAKDHY E-ATAM HRAAA KGN LKMI HILLYY KASTN IQDTEGNT P 175 wolf NRHEIAVMLL EGG ANPDAKDHY E-ATAM HRAAA KGN LKMI HILL YY KASTN IQDTEGNT P 175 mouse NRHEISVMLL EGG ANPDAKDHY D-ATAM HRAAA KGN LKMV HILLF YKASTN IQDTEGNT P 175 rat NRHEIAVMLL EGG ANPDAKNHY D-ATAM HRAAA KGN LKMV HILLF YKASTN IQDTEGNT P 175 hamster NRHEIAVMLL EGG ANPDAKDHY E-ATAM HRAAA KGN LKMV HILLF YKASTN IQDTEGNT P 173 zebrafish NLYEIA QILL ENG ADPNATDKLQ-ST PL HRASAKGNY RLI QLLL KESASTN IQDSEGNT P 175 yeast GRLSIV QLL CDKAP ELI RKK DLQGQT PL HRAAAV GKIQVV KYLI SQ RAPL NTS DSYG FTP 177 . .* :* : .. : ... : *. :*** :* *: :::: *: *. * .* : * **

human LHLA CDEE RVEE AKLLV SQG ASIYIENKEE KTPL QVA KGG LGL—IL KR MV EG----- 226 wolf LHLA CDEE RVEE AKLLV SQG ASIYIENKEE KTPL QVA KGG LGL—IL KR MV EG----- 226 mouse LHLA CDEE RVEE AKFLV TQG ASIYIENKEE KTPL QVA KGG LGL—IL KR LA ESEE ASM 231 rat LHLA CDEE RVEE AKLLV TQG ASIYIENKEE KTPL QVA KGG LGL—IL KR IA ESEE ASM 231 hamster LHLA CDEE RVEE AKLLV TKGASIYIENKEE KTPL QVA KGG LG------215 zebrafish LHLA CDEE RAEAA KLLVEHG ASIYIENKEKMTPL QVA KGG LGS —VL KR IV EG----- 226 yeast LHFALA EGH PDVGVELV RAGADTLRK DSENHT AL EVCPDRIV CN EFL EACKEQN LEI- 234 ** :* * : : . ** **. :: .* : *.* :*. . :

Figure 1.1. Sequence alignments of human, wolf, mouse, rat, hamster, zebrafish, and yeast gankryin proteins. The identification codes for these gankyrin proteins are NM_002814, NCBI XP_538135.1, GenBank BAA36869.1, NCBI NP_446377.1, GenBank AAN76708.1, GenBank ABD43170.1, and NCBI NP_593722.1, respectively. The alignment was performed using Clustal W2 provided by EMBL_EBI (www.expasy.ch ). Rectangles represent α helices.

1.2 The structure of gankyrin.

The structure of human gankyrin has been determined by X-ray chromatography

(3) and NMR spectroscopy (4) independently. As shown in Figure 1.2 , gankyrin is exclusively composed of seven ankyrin repeats, each of which exhibits a canonical helix-

turn-helix conformation with the two helices in an antiparallel fashion (5). These seven ankyrin repeats are stacked together near linearly to form a helix bundle, and neighboring ankyrin repeats are linked by loops of varied size which orientate perpendicularly to the axes of the helices of ankyrin repeats. While it is generally described as “linearly stacked”, a slight bending of the repeat stack toward the α-hairpin loop can be clearly discerned for gankyrin (5). The N- and C-terminal repeats are capping repeats and are less similar to the consensus sequence; the five internal repeats of gankyrin are 33.1% identical and 61.8% homologous in terms of pairwise sequence.

A B

Figure 1.2. Solution structure of human gankyrin. A , Structure of gankyrin with identified target-binding domains; B, Tertiary structures of CDK4-interacting AR proteins, gankyrin, P16, P18, and I κBα67-302. The first four Ars of P18, I κBα67-302, and gankyrin are superimposed with the corresponding Ars in P16. While the coordinates of I κBα67-302 (yellow) are derived from the crystal structure of I κBα/NF κB complex, the structures of P16 (green), P18 (blue), and gankyrin (magenta) are solution

structures solved by NMR in our laboratory. In both A and B, N and C represent the N- and C- termini, respectively.

Like most of ankyrin repeat proteins, there is no disulfide bond or long-range intramolecular interaction present in gankyrin. The elongated gankyrin structure is mainly stabilized by inter- and intra-ankyrin repeat hydrophobic interactions predominantly associated with conserved nonpolar residues in the helical regions, as well as hydrogen bonding interactions between polar residues and the main chain atoms from adjacent ankyrin repeats (6,7).

The crystal structures of mouse gankyrin (8) and yeast Nas6 (gankyrin homolog in fission yeast) (9,10) have been reported. No significant differences have been found in the sketch structures of these proteins, especially at the helical regions, even though the sequence homology between human gankyrin and yeast Nas6 is only about 29%.

Moreover, the solution structure of gankyrin is almost superimposable to that of gankyrin in complex with the C-terminal domain of S6 ATPase (11), implying that free gankyrin is in a biologically active conformation.

1.3 Diverse functions of gankyrin.

Gankyrin belongs to the ankyrin repeat protein group, which is one of the largest

repeat protein groups. Proteins in this group are involved in various physiological

processes exclusively through mediating protein-protein interactions (6,7). A number of

important proteins have been identified as physiological targets for gankyrin binding and

modulating. Some of these targets, such as MDM2 (12), pRb (1) and CDK4 (13,14), play pivotal roles in cell cycle progression, apoptosis, and tumorigenesis.

1.3.1 Gankyrin and Rb, p53.

In their previous study (1), Higashitsuji and his colleagues demonstrated that gankyrin binds to pRb through a conserved pRb-binding motif LxCxE at its C-terminus, and such binding is essential for gankyrin-induced transformation of NIH 3T3 fibroblasts.

More importantly, they showed overexpression of gankyrin led to increased pRb hyperphosphorylation (loss of suppressor activity), activation of the E2F transcription factor (expression of DNA synthesis genes) and accelerated the degradation of pRb, which suggests that increased expression of gankyrin could promote tumorigenicity by targeting pRb to the proteasome ( Figure 1.3) (1). Interestingly, it has also been shown that gankyrin is able to modulate the pRb pathway through an alternative mechanism.

Briefly, gankyrin competes with P16 as well as other INK4 proteins for binding to

CDK4, which prevents the INK4 proteins from inhibiting the kinase activity of CDK4.

This results in enhanced pRb phosphorylation and concomitant deregulation of E2F1- mediated transcription and cell cycle progression (14). Evidently, these studies indicate that gankyrin deactivates the pRb pathway at multiple levels: it directly binds to pRb and facilitates its degradation, and it also ensures the inactivation of pRb through inhibition of

CDK4 kinase activity.

More recently, the role of gankyrin in tumorigenesis has been expanded with the evidence that it disrupts the P53 tumor suppressor pathway (12,15). It has been shown that gankyrin binds to MDM2, an E3 ubiquitin ligase, and enhances the ability of MDM2

to ubiquitinate P53 (12,16,17). Gankyrin recruits the MDM2 and P53 complex to the protease and encourages the turnover of P53 in an MDM2-dependent manner. Moreover, silencing gankyrin expression by RNA interference led to increased P53 protein level and activity which in turn promotes apoptosis. In addition, it has been reported that pRb inhibits MDM2-mediated P53 ubiquitination in a gankyrin-dependent manner and the pRb-gankyrin interaction is critical for pRb-induced P53 stabilization (18). Furthermore, acute ablation of pRb facilitates gankyrin-mediated P53 destablization and desensitizes cancer cells for chemotherapy-induced apoptosis, indicating that pRb antagonizes gankyrin to inhibit MDM2-mediated P53 ubiquitination in cancer cells and the status of both P53 and Rb are important for efficacy of cancer chemotherapy (18). Taken together, gankyrin functions as a dual-negative regulator in both pRb and P53 pathways ( Figure

1.3).

Figure 1.3. The dual-negative regulatory roles of gankyrin in the pRb and P53 pathways. Arrows represent positive modulation, and bars represent negative regulation.

1.3.2 Gankyrin and the proteasome.

The 26S proteasome is the primary component of a major non-lysosomal

proteolytic system which is responsible for the degradation of a wide variety of

intracellular proteins including tumor suppressors, transcription factors, and proteins that

regulate the cell cycle (2,19-21). The 26S proteasome is a large, multi-subunit, multi-

catalytic protease found in the nucleus and cytosol of all eukaryotic cells. It has a 700 kD,

28 subunit protease called the 20S proteasome that is capped at one or both ends by the

900 kD 19S activator. The 19S activator consists of six AAA ATPase subunits that may

form a ring structure to unfold proteins targeted for proteasome degradation and

translocate them into the 20S proteasome’s central proteolytic chamber. Other 19S

subunits include enzymes with processing or editing roles and structural proteins that

serve as docking platforms for binding partners. Gankyrin is a subunit of the 19S

activator by virtue of its interaction with the C-terminal domain of the 19S S6b ATPases

(2,11) and appears to be a shuttle protein for transporting ubiquitinated proteins, such as

P53 to the proteasome for degradation (17). Disruption of the possible gankyrin homolog

in yeast (NAS6) resulted in non-disruption of the 26S proteasome, further implicating its

supportive role in protein degradation (2).

1.3.3 Gankyrin and MAGE A4, RelA.

In addition to the aforementioned roles in the P53 and pRb pathways, gankyrin

has also been found to interact with melanoma antigen (MAGE)-A4, a tumor specific

antigen with potentials in cancer immunotherapy (22). This interaction is mediated by

the C-terminal half of MAGE-A4 and is very specific, other MAGE family proteins

structurally similar to MAGE-A4, i.e. MAGE-A1, MAGE-A2, and MAGE-A12, do not bind to gankyrin. While it remains unknown how the interaction with gankyrin influences the function of MAGE-A4, it has been shown that the expression of MAGE-

A4 partially suppressed both anchorage-independent growth in vitro and tumor formation in athymic mice incells where gankyrin was stably overexpressed. This indicates that

MAGE-A4 could counteract the oncogenic activity of gankyrin. Moreover, gankyrin is involved in the regulation of the I κB/NF-κB pathway (23,24). On one hand, gankyrin

directly binds to NF-κB/RelA and exports RelA from nucleus through a chromosomal region maintanence-1 (CRM-1) dependent pathway, thus suppressing the nuclear translocation of NF-κB/RelA as well as its activity (23). On the other hand, gankyrin binds to NF-κB and negatively regulates its activity at the transcription level through

modulating acetylation via SIRT1, a class III histone deacetylase (24). Finally, it has

been shown that overepxression of gankyrin in human hepatocellular cell line Huh-7 up-

regulated expression of insulin-like growth factor binding protein 5 (IGFBP-5), which

subsequently promoted cell proliferation (25).

1.3.4 The structure basis for the function of gankyrin.

In general, the ankyrin repeat is a conserved motif with a consistent pattern of key

residues which maintain the characteristic helix-turn-helix topology. There exists a

certain sequence homology as well as a structural similarity among most ankyrin repeat

proteins (6,7). From this point of view, gankyrin has a simple, repetitive sketch structure

of seven helix-turn-helix units. Thus, it is interesting to find out how this “small and

simple” protein can bind to multiple targets that are significantly different in structure

and function. Previous structural and biochemical studies performed by our laboratory and other groups have provided insights about the structural basis for the functional diversity of gankyrin (1,4,6,11,13,14).

The pRb-binding motif has been mapped to 178 LxCxE 182 in the fifth ankyrin repeat of gankyrin ( Figure 1.2A ). E182 has been shown to be critical for binding with pRb since mutations in E182 completely disrupt pRb/gankyrin association (1,14). As revealed in the crystal structure of the gankyrin/S6-C terminal domain complex (11),

E182 is located at the edge of the S6-C terminal domain interface, hence it is very likely that gankyrin binds to pRb and form a pRb/gankyrin/S6-C terminal domain complex, which locates pRb to the vicinity of the ATPases for processing and degradation.

Furthermore, it has been demonstrated that for gankyrin, the binding of CDK4 is distinct from the binding of pRb. The evidence is that, an Rb-binding peptide,

176 LHLACDEERN 185 , which could eliminate the binding of gankyrin to pRb but does not influence the gankyrin/CDK4 association (14). On the other hand, truncated gankyrin protein with the first four ankyrin repeats remaining is substantially potent in binding to

CDK4 and conteracting the inhibitory function of p16 (14). Interestingly, the structure of first four ankyrin repeats of gankyrin is almost superimposable with the structure of P16

(as well as the structures of the first four ankyrin repeats in another two CDK4- interacting ankyrin repeat proteins, P18 and I κBα), especially in the helical regions where most contacts between CDK4 and P16 are located ( Figure 1.2B ) (26). Such high structural resemblance between P16 and gankyrin provides the basis for their potential similarity in binding to CDK4. Indeed, besides residues universally conserved for the formation of the helix-turn-helix conformation of an ankyrin repeat, residues mainly

contributing to CDK4 binding, such as E26, D74, and D92 of P16, are also conserved in gankyrin, indicating that gankyrin interacts with CDK4 in a way similar to that P16 does

(26,27). As revealed in the crystal structures of P16/CDK6 (28), P19/CDK6 (29), and

P18/CDK6/viral cyclin complexes (30), most of CDK4-intercating residues are located within the second and third ankyrin repeats while the first and fourth ankyrin repeats are required to form a stable structure, which is consistent with our previous observation that the first four ankyrin repeats of gankyrin are responsible for CDK4 binding. Similarly, the structural resemblance between gankyrin and I κBα in the first four ankyrin repeats enables gankyrin to compete with I κBα in binding to NF-κB/RelA which modulates NF-

κB-mediated transactivation (23,24,26). In addition, deletion of the last ankyrin repeat of

gankyrin abolishes its binding with MDM2, implying that the last ankyrin repeat is

critical for MDM2 binding ( Figure 1.2B ) (12). While the domain responsible for

MAGE-A4 binding remains to be discovered (22), the association with the C-terminal

domain of the S6 ATPase involves a number of residues discontinuously dispersed in all

of the seven ankyrin repeats of gankyrin, including residues at the tips (defined as the

first residue of an ankyrin repeat and the last residue of the preceding ankyrin repeat),

adjacent to the tips, and in the helical regions (11). Such discontinuous, multiple-residue-

interacting patterns have been found in all crystal structures of complexes containing

ankyrin repeat proteins, such as the 53BP2-P53 complex (31) and the I κBα/NF-κB

complex (32).

1.4 Gankyrin and cancer.

1.4.1 Aberrant expression of gankyrin is prevalent in human cancers.

Gankyrin appears to be one of the few oncogenic proteins which negatively modulate both pRb and P53 tumor suppressive pathways. Considering the fact that in more than 90% of cancer cells, the pRB and P53 pathways were inactivated directly or indirectly (15), the status of gankyrin in cells could be highly related to the development of human cancers. In an endeavor to explore the potential involvement of gankyrin in human cancer, Higashitsuji and his colleagues evaluated the expression of gankyrin mRNA in primary hepatocellular carcinomas (1). Increased expression of gankyrin mRNA was found in 34 of 34 cases (100%) compared with corresponding histologically normal tissues. In another independent study (33), the expression of gankyrin mRNA was increased in 57 of 64 HCC specimens and moderately increased in another 5 HCC specimens in the same cohort. In comparison with liver cirrhosis and para-carcinomas liver tissues, the average expression of gankyrin mRNA in HCC was increased by 3.6- and 5.2-fold, respectively. In chemically-induced rodent HCC (34), gankyrin mRNA was overexpressed in all tested HCC specimens in comparison with the matched

“histologically normal” samples. Taken together, these results demonstrate that overexpression of gankyrin plays an important role in the development of HCC.

Additionally, a recent study on human esophageal SCC (ESCC) (35) showed that all 30 tested tumor specimens and 11 ESCC cell lines exhibited high levels of gankyrin expression, and gankyrin overexpression was positively correlated with lower survival rate, extent of the primary tumor, lymph node metastasis, distant lymph node metastasis

and stage, indicating that gankyrin might be important during the development of malignancy potential in ESCC, and may play an important role in its progression.

1.4.2 Aberrant expression of gankyrin could be an early event in the development of human cancers.

With regard to cancer diagnosis and prevention, the ability to identify the molecular events occurring at the earliest stage of cancer development is essential.

Recently, emerging evidence demonstrates that the overexpression of gankyrin could be one of such events in the development of hepatocellular carcinoma. In the aforementioned chemically-induced rodent hepatocarcinogenesis model, Lim et al . (36) discovered that several different aberrant molecular events occurred sequentially in association with the different stages of HCC development. Hypermethylation of the p16 gene and mutations in the p53 appear at the late stage (the HCC stage), whereas the

overexpression of gankyrin occurred early just after carcinogen treatment (the liver

fibrosis stage), preceding the loss of pRb (the cirrhosis stage) and hepatocellular adenoma

formation (the HCA stage). Then, Tan et al . reported that the frequencies of gankyrin overexpression in Edmondson’s grade I to II, III, and IV human HCCs were 82%, 63%, and 22%, respectively (37). Similarly, Fujita et al . observed positivity in 81% and 35% of low and high TNM stage of human HCCs (38). These results indicate that the overexpression of gankyrin is associated with the early clinical stage of human HCCs.

Hence, gankyrin may play an important role in the early stage of liver and oral cancer progression, and serve as a biomarker with potentials in cancer diagnosis and therapeutics. Moreover, it has been reported recently that overexpression of gankyrin

confers multi-drug resistance of gastric cancer cells (39), indicating that down-regulation of gankyrin could provide a novel approach to combat against drug resistance, a challenge frequently found in cancer chemotherapy.

1.4.3 Potential mechanisms underlying the involvement of gankyrin in cancer.

The detailed molecular mechanisms underlying the contribution of aberrant gankyrin expression to cancer progression remain to be further investigated. However, three distinct possible mechanisms are now emerging in which gankyrin is incorporated into the well-known cell cycle control machinery (Figure 1.4) . First of all, the overexpression of gankyrin may deregulate the pRb pathway of cell cycle control by a mechanism similar to the one associated with the human papilloma virus-16 (HPV-16)

E7 protein (1,40,41). Basically over abundant gankyrin would bind to hypo- and hyper- phosphorylated pRb, targeting both for proteasomal degradation. Secondly, P21 functions as a universal inhibitor of CDKs, such as CDK2 and CDK4, and its inhibition function is modulated by P53 (42). Hence, the overexpression of gankyrin may indirectly deregulate the cell cycle progression through facilitating and enhancing the ubiquitination and degradation of P53. Finally, in normal cells, there exists a dynamic balance between gankyrin and P16 in controlling the activity of CDK4 as well as cell cycle progression

(14). The overexpression of gankyrin could drive this balance in the direction favoring gankyrin/CDK4 interaction thus compromising the tumor suppressor activity of P16.

Since several molecular events, including the activation of proto-oncogenes and the inactivation of tumor suppressor genes such as P53 and P16, are involved in tumor development, the question is whether it is necessary to have two functionally-related

molecular events, gankyrin overexpression and P16 inactivation as the control at the same time. Especially when gankyrin overexpression or P16 inactivation alone is sufficient to “functionally” inactivate the P16/CDK/pRb pathway. The reasons lie in the following facts. First of all, as mentioned earlier (36), the overexpression of gankyrin most likely occurs in an earlier stage of cancer progression than the inactivation of P16.

Secondly, not all p16 gene alterations result in functionally inactivated P16 proteins.

Some P16 missense mutations led to P16 proteins with only moderately reduced CDK4-

inhibitory activities (6). Thirdly, the loss of P16 activity via p16 alterations could be

rescued by the gene redundancy and the upregulation of other INK family members (e.g.,

p15, p18 and p19) (43,44), whereas gankyrin counteracts against the CDK4-inhibitory

activities of all INK4 proteins. Finally, gankyrin can always de-regulate the cell cycle

progression in mechanisms other than competition with P16 ( Figure 1.3) (1,11), while

P16’s cell cycle deregulating properties could be manifest through its inactivation in

other critical non-CDK/pRB regulatory pathways (e.g. cell senescence, anoikis, cell

spreading, and angiogenesis) (45-47).

Figure 1.4. Proposed mechanisms of gankyrin-mediated deregulation of cell cycle control. Arrows represent positive regulation, and bars represent negative regulation. In B, two red crosses indicate that the inhibition of P16 and P21 to CDK4 can be “eliminated” by overexpressed gankyrin.

1.5 Summary.

In a relatively conserved AR motif, residues forming its helix-turn-helix framework are strictly conserved, whereas residues directly involved in target binding are less conserved and usually are located on the surfaces of the helices as well as in the flexible loops connecting neighboring ARs (6,7). Additionally, some residues in the loops, such as the TPLH tetrapeptide, are well conserved, but they are not directly involved in target binding or the formation of the sketch structure (4). Instead, these residues mainly function to stabilize the global structure of an AR protein, thus indirectly impacting its function. These structural features enable us to use chemical biology approaches to modify the functioning of gankyrin or even generate novel gankyrin-like molecules with potentials in therapeutics. First of all, as described earlier, the four N- terminal ARs of gankyrin and P16 exhibit high structural similarities, and both proteins 16

compete with each other for binding to CDK4 (14,26). Nevertheless, the impacts of gankyrin and P16 are totally different: P16 inhibits CDK4-mediated phosphorylation of pRb and acts as a tumor suppressor, while gankyrin enhances CDK4-mediated phosphorylation of pRb (14). In chapter two, the structral basis of these contrasting properties is studied. Secondly, there are six TPLH tetrapeptides in the loop regions of gankyrin ( Figure 1.1 and Figure 1.2) (4). Introducing or eliminating TPLH tetrapeptides from different locations of gankyrin may change the conformational stability of the global structure. In chapter three, we explore the roles of the TPLH tetrapeptide in gankyrin and AR proteins. Thirdly, information from consensus analyses of AR proteins including gankyrin is used to generate a novel AR motif, in which residuals other than those essential for forming the helix-turn-helix framework are randomly chosen (6,7).

This novel AR motif is used as a building block, and different copies of this AR motif are linked together to form a combinatorial library, whose members can form a simple, rod- like structure as gankyrin but retain potentials to bind to any chosen target protein. To date, a number of such “novel” AR proteins have been reported, including those specifically binding to aminoglycoside phosphotransferase (3’)-IIIa (APH) (48), maltose binding protein (MBP) (49), p38 AMP kinase (50), and JNK-2 (50). From this perspective, consensus-based AR combinatorial libraries could be a very attractive approach in chemical biology to generate novel molecules with potentials in clinic.

CHAPTER 2

DISSECTION OF PROTEIN-PROTEIN INTERACTION AND CDK4 INHIBITION IN THE ONCOGENIC VERSUS TUMOR SUPPRESSING FUNCTIONS OF GANKYRIN AND P16

2.1 Introduction.

P16 and gankyrin are two AR proteins (consisting of 4 and 7 ARs, respectively) with opposite biological functions. P16 specifically binds to CDK4 and inhibits the latter’s Rb-phosphorylating activity (42,51). Such inhibition precludes the release of transcription factors E2Fs from incompetent Rb/E2F complexes, thus blocking the transactivation of downstream genes required for entry into the S phase. It has been well established that P16 is a tumor suppressor whose genetic inactivation through deletion, methylation, or mutation has been found in almost every type of human cancers (42). In contrast, binding of gankyrin to CDK4 renders the kinase resistant to P16 inhibition

(13,14), which results in enhanced Rb-phosphorylation and stimulation of E2F transcription activity, thus effecting cell cycle progression. Gankyrin is also able to directly bind Rb and facilitate Rb phosphorylation and degradation (1). Furthermore, gankyrin interacts with MDM2 and promotes the ubiquitination and targeting of P53 to the proteasome for degradation (12). Therefore, gankyrin functions as a negative regulator of three prominent tumor suppressors, P16, Rb, and P53 (15,52), and overexpression of gankyrin leads to cell transformation (1). 18

The goal of this study was to understand the structural basis of the contrasting properties of P16 and gankyrin, and to use these studies to further understand how AR proteins control their biological specificity. As revealed in the crystal structure of

P16/CDK615 (a close homolog of CDK4), and other biochemical studies (5,53,54)

(Figure 2.1), contacts between P16 (or other INK4 proteins, in general) and CDK4 occur in discontinuous patches, and a number of residues located in both loop and helical regions of P16 contribute to CDK4 binding through electrostatic, hydrogen bonding, and van der Waals interactions (7). While mutations of most of CDK4-interacting P16 residues only lead to mild decrease in its inhibitory activity, the D84H mutant of P16 (a mutant found in human cancers), or the corresponding mutant of P18, D76A, loses all inhibitory activity even though these two mutants are still able to bind to CDK4 (5,54).

On the other hand, it has been reported that CDK4 R24C, a mutant frequently found in human cancers, retains its kinase activity but is resistant to P16 inhibition (55,56).

Further analysis of the crystal structure of the P16/CDK6 complex indicates that there is a strong electrostatic interaction between the side chains of D84 of P16 (D76 of P18) and

R24 of CDK4 (R26 of CDK6), and mutation in either D84 of P16 or R24 of CDK4 disrupts this interaction, thus abolishing P16 inhibition.

Figure 2.1. Structural Basis of p16/CDK6 (or CDK4) Interaction. (A) Structural positioning of the functionally important residues of P16 in contact with CDK6 is shown using the crystal structure of the P16/CDK6 complex (PDB code: 1BI7). (B) Quantitative contributions of functionally important residues of P16. Residues are presented in different colors based on changes in the values of IC 50 when mutated. Residues with >20 fold increase in IC 50 when mutated are indicated in red (L78 and D84); 10-20 fold, orange (W15, D92 and R124); 5-10 fold, green (H66 and E69), and 3-5 fold, purple (E26, N71, P76, A77, T80, H83, F90, W110, and L121).

Gankyrin competes with P16 for CDK4 binding (14), but it does not influence the kinase activity of CDK4, suggesting that gankyrin acts as a P16 blocker rather than a

CDK4 inhibitor or activator. The structure of gankyrin in complex with CDK4 is not available, although the structure of free gankyrin has been solved by both NMR (4) and

X-ray (3,8,9). Our structure-based sequence homology analysis showed that the residue corresponding to D84 of P16 is I79 in gankyrin ( Figure 2.2). Using site-directed mutagenesis, we show that I79D mutant of gankyrin, as well as a double mutant

L62H/I79D, binds to CDK4 and inhibits its kinase activity similar to P16. Further structural analyses suggest that these substitutions bring about notable local conformational changes in gankyrin, most likely facilitating the positioning of a negatively-charged residue into the proximity of positively-charged R24 of CDK4.

Analysis of conformational stability also suggests that the CDK4-inhibitory activity likely comes at the expense of stability. Overall, our studies provide insights into the molecular mechanisms underlying interactions between AR proteins and their partners.

Leu6 His66 Ile79

Asp84

His66/ Leu62

Asp84/ Ile79

Figure 2.2. Structure-based sequence homology between P16 and gankyrin. (A) Surface charge distribution of P16 (PDB ID: 1DC2; left) and gankyrin (PDB ID: 1TR4; right) indicating the location of the two residues of interest. (B) Structure-based sequence alignment of human P16 and the first four ARs of gankyrin. Two residues of interest, H66 and D84 of P16, and corresponding gankyrin residues L62 and I79, are highlighted in red. (C) Surface-fitted overlay of P16 and gankyrin (left) and the ribbon drawing of best fit superimposition of the backbone (N, C α, and C) atoms of the NMR structures of P16 (gold) and the first four ankyrin repeats of gankyrin (green). Two residues of interest, H66 and D84 of P16 (gold) as well as their corresponding residues, L62 and I79 (green) in gankyrin are highlighted.

2.2 Results.

2.2.1 Structure-based protein engineering.

Previous studies in our as well as other laboratories showed that gankyrin binds to

CDK4 in a way similar to that of P16 in vitro and in vivo, and this binding counteracts

the CDK4-inhibiting activity of P16 (13,14). Further fragmentation experiments

demonstrated that the first four ARs of gankyrin are required and sufficient for CDK4

binding and P16 counteraction (14). To understand the differences between the functions

of gankyrin and P16, structural and sequence alignments of the two proteins were

performed as shown in Figure 2.2 (A and B, respectively). As shown in Figure 2.2B ,

four negatively-charged CDK4-binding residues, E26, E27, D74, and D92 of P16 align

perfectly with E20, E21, D70, and E87, respectively, of gankyrin thus justifying this

structure-based sequence homology analysis ( Figure 2.2C ). These residues are very

likely involved in the binding of gankyrin with CDK4 (4).

One interesting notion of this analysis is that D84 of P16 corresponds to I79 in

gankyrin. Apparently, the hydrophobic side chain of I79 is incapable of interacting via a

salt bridge or a hydrogen bond with the positively charged side chain of R24 at the active

site of CDK4, although a much weaker nonpolar contact could not be ruled out.

Therefore, this residue is an interesting candidate to explore the functional role and to see

if introducing a negatively charged Asp residue in place of I79 of gankyrin could rescue

its CDK4-inhibiting activity. H66 is another important residue in P16 which is not

conserved between P16 and gankyrin. The crystal structure of the P16/CDK6 complex

showed that this residue does not directly contact CDK4, however, mutations of this

residue led to the loss of 80% of the CDK4-inhibitory activity of P16 (5), suggesting that

H66 contributes indirectly to inhibition, likely through stabilizing the structure of P16 and facilitating the positioning of D84. The residue corresponding to H66 in P16 is L62 in gankyrin, and it can be reasoned that introducing a L62H mutation in gankyrin may influence the stability of gankyrin but not its inhibitory ability, as long as the critical Asp is absent at position 79 of gankyrin. To confirm the above notion, two single mutants,

L62H and I79D, and one double mutant, L62H/I79D of gankyrin were generated by site- directed mutagenesis, and their biochemical and biophysical properties were subsequently evaluated.

2.2.2 I79D and L62H/I79D of gankyrin bind and inhibit CDK4.

The binding of these gankyrin mutants to CDK4 was first investigated using pull- down assays with glutathione-S-transferase (GST)-tagged gankyrin proteins and the

CDK4/cyclin D2 holoenzyme. Instead of CDK4, the CDK4/cyclin D2 holoenzyme was used in this assay mainly due to the fact that the holoenzyme is the biological active form and there is no interaction between gankyrin and cyclin D2 (14). As shown in Figure

2.3A , CDK4 was detected in the reaction mixtures containing GST-gankyrin wild type

(lane 3) and mutant proteins (lanes 4-6) as well as in the reaction mixture containing

GST-P16 (lane 2), which was used as positive control in this assay, suggesting that all three gankyrin mutants bind to CDK4. Even though this assay should not be used to quantitatively evaluate protein/protein interaction, the amounts of CDK4 in the pull-down products were comparable to each other, indicating that there are no significant differences among the wild type and the mutant gankyrin proteins in their CDK4-binding affinity.

Figure 2.3. Interactions between gankyrin proteins and CDK4 as evaluated using pull-down assays and in vitro kinase assays. (A) Pull-down assays. The reaction mixtures containing GST-tagged proteins and the CDK4-cyclin D2 holoenzyme were incubated with reduced glutathione-agarose, and after elution with reduced glutathione the bound proteins were separated by SDS-PAGE, and blotted against anti-human CDK4 antibody (Santa Cruz, C-22). Lanes: 1, the input only containing 5% of the amount of purified CDK4-cyclin D2 in other lanes; 2, positive control, GST-P16 (0.5 M)/CDK4- cyclin D2 (0.1 M); 3, GST-gankyrin wild type (0.5 M)/CDK4-cyclin D2 (0.1 M); 4, GST-gankyrin L62H (0.5 M)/CDK4-cyclin D2 (0.1 M); 5, GST-gankyrin I79D (0.5 M)/CDK4-cyclin D2 (0.1 M); 6, GST-gankyrin L62H/I79D (0.5 M)/CDK4-cyclin D2 (0.1 M); 7, mock lane, GST (0.5 M)/CDK4-cyclin D2 (0.1 M); 8, BSA, as a negative control in Western Blot. (B) in vitro kinase assays. Each reaction mixture included 3 units of CDK4-cyclin D2 holoenzyme (about 0.3 g), 50 ng GST-Rb791-928, 5 Ci [ γ- 32 P] ATP, and varying amounts of effector proteins. After incubation at 30 °C for 15 minutes, the reaction mixtures were separated by 10% SDS-PAGE, dried, and analyzed by autoradiography.

The CDK4-modulating activities of gankyrin and the three mutants were then assessed using an in vitro kinase assay as previously described (14). As shown in Figure

2.3B , neither gankyrin nor L62H mutant brought about any significant change in CDK4-

mediated phosphorylation of Rb, suggesting that wild type gankyrin and L62H mutant do

not influence the activity of CDK4. However, in the reaction mixtures containing mutant

I79D or L62H/I79D, as the concentration of I79D or L62H/I79D increased, CDK4-

mediated phosphorylation of Rb decreased, indicating that unlike wild type gankyrin,

both I79D and L62H/I79D mutants inhibit the kinase activity of CDK4. As a positive

control, increasing amounts of P16 in the reaction mixtures also led to a decrease in the

kinase activity of CDK4. Further quantitative analyses yielded IC 50 values of 170 ± 32 nM and 120 ± 27 nM for I79D and L62H/I79D mutants, respectively ( Figure 2.4), only slightly higher than that for P16 (72 ± 15 nM), suggesting that the CDK4-inhibitory activities of I79D and L62H/I79D are comparable to that of P16. In contrast, the IC 50 values for wild type gankyrin and L62H mutant were higher than 3.0 M, indicating that gankyrin wild type and L62H mutant do not inhibit CDK4 under the experimental conditions used. Interestingly, our previous studies showed that while mutations at D84 of P16 completely abolished its CDK4-inhibitory activity, mutations at H66 of P16 also led to an 8-fold decrease in the CDK4-inhibitory activity suggesting that H66 of P16 is able to affect the CDK4-inhibitory activity through certain ways (5). In contrast, gankyrin L62H mutation does not exhibit any detectable CDK4-inhibitory activity, and the IC 50 values for gankyrin I79D and L62H/I79D are almost identical. Taken together,

an Ile Asp substitution at position 79 of gankyrin does not significantly affect the binding of gankyrin to CDK4 but enables gankyrin to inhibit the kinase activity of 26

CDK4. In comparison, a Leu His mutation at position 62 apparently affects neither

the binding nor the inhibition.

120

100

60 Gankyrin Gankyrin L62H Gankyrin I79D 40 Gankyrin L62H/I79D P16

0 Relative CDK4-Inhibitory Activity (%) 0 500 1000 1500 2000 2500 Concentration of Modulator (nM)

Figure 2.4. Quantitative measurement of the CDK4-inhibitory activities of gankyrin proteins. The in vitro kinase assays were performed as described in Figure 3B, and the incorporation of 32 P into the Rb substrate was quantitated using ImageQuant (Molecular Dynamics). IC 50 was defined as the inhibitor concentration at which half of the maximum inhibition can be obtained. The experimental error for this assay was estimated to be ± 30%. All assays were performed in triplicate, and P16 was used as a positive control.

2.2.3 I79D and L62H/I79D mutations bring about substantial perturbation to the local conformation of gankyrin. 2D 1H-15 N heteronuclear single-quantum coherence (HSQC) NMR spectroscopy

was subsequently used to investigate the possible structural perturbation caused by the

above gankyrin mutations. As shown in Figure 2.5A , the spectrum of L62H is almost

identical to that of wild type gankyrin. The few notable chemical shift changes are

mapped to residues Lys30, Lys35, Thr34, Thr42, Phe58, and Val64, all of which are

located in the first two ARs, and thus in close vicinity of L62H mutation ( Figure 2.6A ).

Hence, L62H mutation brings about only minor changes in local environments rather 27

than any significant change in the global structure of gankyrin. In contrast, there are considerable and extensive changes in the spectra of I79D and L62H/I79D in comparison to the spectrum of wild type gankyrin ( Figures 2.5B and 2.5C ). In addition, the spectra of I79D and L62H/I79D are almost superimposable, further supporting our previous observation that L62H mutation does not cause any significant change in the global structure of gankyrin.

A C

B D

Figure 2.5. Comparison of 15 N-1H HSQC spectra of wild type gankyrin and its mutants. The HSQC spectra were obtained in D 2O solution using a 600 MHz spectrometer. (A) L62H (red) and wild type (black); (B) I79D (blue) and wild type (black); (C) L62H/I79D (green) and wild type (black); (D) superimposition of spectra in A, B, and C. L62 and I79 peaks in the spectra of wild type gankyrin are in overlapping regions, hence, cannot be precisely pointed at in the respective mutant data. The peaks that show larger chemical shifts are labeled with arrows.

Figure 2.6. Mapping of chemical shift changes on the gankyrin structure. (A) L62H (highlighted in red) mutation and the residues affected as a consequence are localized in AR1 and AR2. (B) I79D (highlighted in red) mutation and the residues affected as a consequence are localized in loop regions between AR1-2, AR2-3 and AR3-4.

On the basis of 3D 15 N-edited NOESY recorded on a 15 N-labeled I79D sample together with the previous assignments on wild type (4), a total of 168 backbone amides have been assigned, among which the residues in the AR2 and AR3 are least assigned due to the largest chemical shift perturbations. It can be concluded that while the 30

residues in AR5-AR7 retain virtually the same chemical shifts, the residues perturbed as a result of the Ile Asp substitution are located in the flexible loop regions within the first four ARs, including Val10, Arg35, Arg37, Ser40, Thr42, Ala43 (the first loop), Gly73,

Trp74, Ser75, Ser82 (the second loop), Ala101, Val102, Gly106, and Thr108 (the third loop) ( Figure 6B ). Interestingly, all these residues are part of the TPLH interaction network of central ankyrin repeats of gankyrin (4). These results suggest that the conformations of loops I to III are perturbed in the I79D mutant, and raise the possibility that the observed functional change upon this mutation may not be a simple effect of a side chain substitution. Instead, the I79D substitution possibly leads to local structural adjustments that help to position the side chain for the inhibitory function.

This concept of full integration of structure and function is further supported by some preliminary NOE analysis on the aforementioned 3D 15 N-edited NOESY as well as

parallel 2D 1H homonuclear NOESY recorded on WT, I79D, and L62H. First of all, in

3D NOESY data set relative NOE intensity changes have been observed for the limited number of residues that have been assigned in the second and third loop. For example,

γ2 the NOE between H /T37 and H N/S40 is becoming much stronger whereas the sequential

α NOE between H /N67 and H N/D68 is weaker in I79D mutant. Secondly, in the

downfield region of 2D NOESY recorded in H 2O ( Figure 2.7), I79D mutant shows

ε2 contrasting result compared with L62H mutant concerning the H of the histidine residue in a TPLH motif. The significant change in I79D is highlighted by the large perturbation

ε2 ε2 on H /H78 resonance and more importantly, the disappearance of the H /H45 signal

despite extensive search aided by 2D 1H-15 N HSQC and 2D 1H-15 N heteronuclear

ε2 multiple-bond correlation (HMBC) experiments on a 15 N-labeled I79D. Since H /Η45 is 31

the bridging proton of the inter-AR hydrogen bond between H45 imidazole ring and the backbone oxygen of W74, its disappearance could be indicative of the disruption of the inter-AR interaction and the local conformational changes on both AR2 and AR3.

Figure 2.7. 2D NOESY spectra on WT (a), I79D (b), and L62H (c) showing the ε2 proton downfield region. The cross peaks designated A-C are NOEs from H /His in a ε1 δ2 α TPLH motif to H and H of the same histidine residue, and H /(His+30) residue α (H /T207 for H177), respectively. The latter is a supporting evidence of inter-ankyrin ε2 ε2 repeat hydrogen bond between N /His and O/(His+29) (e.g. N /H177 and O/K206). It is clear from the comparisons that L62H mutant retains the TPLH features from AR2 to AR6, while I79D has large perturbation in TPLH of AR3 (H78) and ANK4 (H111), and ε2 likely disruption in 42 TALH 45 evidenced by the disappearance of H /H45 signal. 32

2.2.4 I79D and L62H/I79D destabilize the global structure of gankyrin.

The effect of the above gankyrin mutations on the conformational stability of gankyrin was evaluated using far-UV Circular Dichroism (CD) spectroscopy. In guanidinium hydrochloride (GdnHCl)-induced unfolding, changes in the ellipticity at 222 nm, indicative of changes in the α-helical content, were monitored. The unfolded

fraction was derived from the raw data and plotted against the concentration of

denaturant ( Figure 2.8A ). The unfolding curves of all three mutants as well as the wild

type gankyrin can be fitted well to a model with a two-state transition between native and

water unfolded proteins (57); the resultant values of ∆Gd (the denaturation free energy in water), D 1/2 (the denaturant concentration at the midpoint of transition), and the slope m

are listed in Table 2.1. Since the m values of the mutants and the wild type gankyrin are

water almost identical, differences in the values of ∆Gd can be directly interpreted as their

differences in conformation stability (57). Compared to wild type gankyrin, both L62H

and I79D mutations have moderately destabilized the conformation by 0.51 and 0.85

kcal*mol -1, respectively. The double mutant, L62H/I79D, which possesses the highest

CDK4-inhibitory activity, has additional 0.34 kcal*mol -1 loss of conformational stability from I79D. The relative stability has been further confirmed in a heat-induced unfolding experiment ( Figure 2.8B ), in which the temperatures at the midpoint of two-state

unfolding transition (58,59), Tm values, were extracted ( Table 2.1). Compared to

gankyrin WT, all three mutants have decreased Tm values, and the double mutant has the

lowest Tm. In conclusion, it appears that the relative conformation stability of the three

mutants coincides with the relative perturbations in HSQC, and the CDK4-inhibitory

activity via mutation likely comes at the expense of stability.

1.2

1.0

0.8

0.6

0.4 Gankyrin Gankyrin L62H

Fraction Fraction Unfolded 0.2 Gankyrin I79D Gankyrin L62H/I79D

0.0 0 2 4 6 8 Concentration of GdnHCl [M] B

1.2

Gankyrin 1.0 Gankyrin L62H Gankyrin I79D 0.8 Gankyrin L62H/I79D

0.6

0.4

Fraction Unfolded 0.2

0.0 0 10 20 30 40 50 60 70 Temperature ( °C)

Figure 2.8. Chemical- and heat-induced unfolding of gankyrin mutants monitored by far-UV CD. (A) GdnHCl-induced unfolding. Samples containing 7.5-10.0 M proteins were incubated with different amounts of GdnHCl on ice overnight and the ellipticity at 222 nm was monitored by far-UV CD (190-260nm) at 25 ºC. The fraction unfolded, defined as (the ellipticity at 222 nm at a denaturant concentration-the ellipticity at 222 nm at the native state) / (the ellipticity at 222 nm at the fully unfolded state-the ellipticity at 222 nm at the native state), was plotted against the GdnHCl concentration (60). The unfolding curve was fitted to a model of two-state approximation (57). (B) Thermal melting spectra were recorded at 222nm by heating from 3 °C to 65 °C with a rate of 1 °C per minute and a 1 °C interval. Tm, the temperature at the midpoint of transition, was obtained through fitting the melting curve to a two-state transition model.

In vitro Chemical-induced unfolding b Heat- CDK4 induced a water c Protein inhibition Gd D1/2 m unfolding

(kcal*mol -1) (M) (kcal*mol -1 *M - IC 50 (nM) Tm (°C) 1) gankyrin > 3000 2.77 1.54 1.80 51.5 gankyrin L62H > 3000 2.26 1.38 1.64 44.5 gankyrin I79D 170 ± 32 1.92 1.14 1.68 42.0 gankyrin L62H/I79D 120 ± 27 1.58 1.01 1.57 38.5

Table 2.1. Biochemical and biophysical parameters of gankyrin and its mutant proteins . a IC 50 was defined as the concentration of gankyrin proteins required for 50% of the maximum inhibition. The highest concentration of gankyrin proteins tested in such in vitro kinase assay was 3.0 M, and an IC 50 value higher than 3.0 M indicated that at the tested concentration range of gankyrin proteins, the CDK4 kinase activity was higher than 50% of that in the absence of any gankyrin protein in the reaction mixture. The estimated error in the determination of IC 50 is ± 20%, and the experiments were performed in triplicate (61). b water Gd , D 1/2 , and m values were calculated according to a two-state model, and the error water limit in Gd is estimated to be ± 0.5 kcal/mol (62). cTm was defined as the temperature at the midpoint of transition (59), and the error is estimated to be ± 0.5 °C.

2.3 Discussion

2.3.1 Possible conformational adjustments of local loops upon I79D mutation of gankyrin. In spite of the striking similarity between the skeletal structures of P16 and the first

four ARs of gankyrin, available structures indicate that the microenvironments around the

“effector” residues, i.e. D84 and I79 for P16 and gankyrin, respectively, are different

from each other. In P16, D84 is located at the beginning of the first helix of AR3 and

structurally, it has more freedom than the residues in the middle of the helix (5). Hence,

even though D84 is only slightly exposed on the surface of the free protein ( Figure

2.2A ), the conformationally flexible loop (loop 2) preceding D84 could influence the

microenvironment around D84 and make this negatively charged residue accessible to the

solvent or the positively-charged side chain of R24 at the active site of CDK4. In the

crystal structure of the P16/CDK6 complex, negatively charged side chain of D84 is in

the vicinity of a positively charged environment ( Figure 9 ). In gankyrin, I79 structurally

and sequentially corresponds to D84 of P16. While I79 is located at the beginning of the

first helix of AR3, its bulky, aliphatic side chain presumably makes it less accessible to

the aqueous surrounding. As shown in the docking model of gankyrin and CDK6

(Figure 2.9), nonpolar I79 is positioned in an unfavorable electrostatic surrounding at the

active site of CDK6. Additionally, the preceding H78 or other nearby residues are unable

to provide any assistance in stabilizing the aliphatic side chain of I79. Therefore, after

introduction of Asp residue at position 79, the microenvironments surrounding this

residue need to be adjusted in order to position the negatively-charged side chain of Asp

in the proximity of the active site of CDK4 and facilitate the kinase inhibition. Such

structural adjustment in gankyrin I79D mutant is supported by the substantial chemical

shift changes in the HSQC spectra of gankyrin I79D and L62H/I79D mutants and the

discernable changes in NOE pattern. While large chemical shift changes were observed

mostly for residues located in the flexible loops connecting AR2, AR3, and AR4,

significant changes were also observed for the residues constituting the TPLH network.

The residues involved in the TPLH network include, for example, T42 and A43 of

42 TALH 45 (AR2), S75 of 75 SPLH 78 (AR3) and T108 of 108 TPLH 111 (AR4), as well as the residues preceding the TPLH sequence, such as S40, G74, W75, and G106. Most of

these residues are located on the concave surface of gankyrin facing CDK4 ( Figure 2.6).

The 2D NOESY have provided more insights about the structural perturbation in TPLH

motifs, 42 TALH 45 in particular, induced by I79 mutation. The observation is not

surprising, considering that the side chain of I79 is pointing to AR2 and directly interacts

δ1 δ2 with H45 evidenced by a NOE assigned previously between H /I79 and H /H45 21 . It is expected that such interaction between these two side chains helps to orient the histidine

δ2 imidazole ring and restrict its flexibility, enabling H45 to adopt an appropriate N -H tautomeric form and engage in several hydrogen bonds. A modeling structure of I79D

(Figure 2.10 ) shows that the Asp side chain is incapable of such an interaction without

significant structural adjustment. Since P16 does not have the TPLH motif or its close

variant, and specifically, 46 RPIQ 49 of P16 is aligned with 42 TALH 45 of gankyrin, the

destabilization in TPLH motifs induced by I79 mutation likely makes the local

conformation and dynamics at the binding site mimic the counterpart in P16.

In conclusion, our results suggest a structural adjustment in I79D, especially in

the loop regions, and that such adjustment may facilitate the positioning of negatively-

charged D79 for an electrostatic interaction with R24 of CDK4. Moreover, this

conformational adjustment was observed for free gankyrin I79D mutant under

physiological condition. Since structural analyses show that there is no significant

structural change on P16 or P18 upon binding to CDK4 (data not shown), i.e. no induced-

fit event happens on the AR protein part after binding to CDK4, like P16 or P18, free

gankyrin I79D mutant is present in a functional conformation (in regard to CDK4 binding

and inhibition).

Figure 2.9. Docking model showing the interactions of I79 of gankyrin with the active site of CDK6. (A) Docking model of the putative gankyrin/CDK6 complex. This model was constructed by superimposing the first four ARs of gankyrin (ribbon diagram, green) onto P16 (ribbon diagram, golden) in the crystal structure of the P16/CDK6 complex 15 (PDB ID: 1BI7; CDK6 is shown in solid-filled diagram). Since no structure of CDK4 is available, crystal structure of CDK6, a close homologue of CDK4, is used in this modeling. H66 and D84 of P16, as well as L62 and I79 of gankyrin are highlighted. Surface residues of CDK6 with positive and negative charges are shown in red and blue, respectively, in the solid-filled diagram. The active site of CDK6 is framed and analyzed in (B). (B) Interactions between D84 of P16 (corresponding to I79) and the active site of CDK6. P16 is in golden, and gankyrin is in green. Apparently, interactions with the charged surface of R26 in the active site of CDK6 (R24 in CDK4) depend on the negatively charged Asp of P16.

α Figure 2.10. Stereoview showing the overlay of C αα trace of gankyrin WT (black) and modeled I79D mutant (green). Only the AR1-AR3 repeats are shown. The side chains of the following residues are highlighted: H45, L62 and I79 of gankyrin WT (in blue), and D79 of I79D mutant (in red). In previous NMR studies an NOE between δ2 δ1 H /H45 and H /I79 was observed (dashed line in magenta) (4). Apparently the modeling I79D structure indicates that such side-chain interactions would be impossible without major structural adjustment. Thus the local structures around H45 including the binding residues may be destabilized by this mutation.

2.3.2 Structural basis for the decreased stability of the gankyrin mutants.

As demonstrated by our GdnHCl- and heat-induced unfolding experiments, the conformation of gankyrin mutants was destabilized in the order L62H < I79D <

L62H/I79D. One structural difference between P16 and gankyrin is that there is a TPLH stretch, or its variant, present in at least six out of the seven ankyrin repeats of gankyrin but none as highly matched TPLH variant is present in any of the four P16 ankyrin repeats. These TPLH stretches are located within neighboring loops and form an intra- and inter-AR interaction network that stabilizes the global structure of gankyrin (4,6,53).

Besides the potential electrostatic and steric disturbance caused by the above mutations, impairment to the TPLH interaction network could be the major cause for structural destabilization of gankyrin mutants. As discussed earlier, an Ile Asp substitution at

position 79 brings about considerable conformational changes in residues within/around these TPLH stretches which perturb the TPLH interaction network and subsequently destabilize the global structure of gankyin. As for L62H, such mutation only caused

“minor” conformational changes in residues within the first two ARs (including the first

TPLH stretch), and the “impairment” to the TPLH network is minor and local ( Figure

2.6A and Figure 2.7C ). This can explain why L62H mutation destabilizes the

conformation to a less extent and does not affect the function in CDK4 binding and

modulation. In contrast, due to the absence of the TPLH interaction network, the

corresponding H66Y mutation in P16 with marginal stability could strongly destabilize

the structure, which consequently can impair its CDK4-inhibitory activity as evidenced

by a loss of 80% of CDK4-inhibitory activity of this mutant (5).

2.3.3 Structural basis for the functional diversity of the AR proteins.

The residues in P16 could play three major roles: (1) maintaining the skeletal

structure of P16, e.g. L63 and L64, which are conserved in almost all AR motifs (61,63);

(2) CDK4 binding (but not inhibition), e.g. E28 and E29 (5); and (3) inhibition (may also

contribute to binding), e.g. D84. Our studies suggest that these three functions are inter-

related. For example, residues in groups 1 and 2 that are responsible for generating the

skeletal structure, may facilitate the positioning of D84 to R24 at the active site of CDK4,

thus inhibiting its kinase activity ( Figure 2.9). Therefore, mutations in group 1 and 2 residues could affect the inhibition by destabilizing the global structure and/or impairing

CDK4 binding, whereas mutation of D84 directly abolishes the CDK4-inhibtory activity.

On the other hand, binding of gankyrin to CDK4 brings the extremely hydrophobic I79

residue in close proximity of R24 of CDK4 ( Figure 2.9) which precludes any

electrostatic interaction between the side chains of these two residues and as a result

gankyrin just acts as a P16 blocker rather than a CDK4 inhibitor. However, when a

negatively-charged Asp residue is introduced at position 79 of gankyrin, following

certain adjustments in the local environment, its electrostatic interaction with R24 of

CDK4 resumes, and so does the inhibition.

In regard to the modular and repetitive nature of AR proteins, one might assume

that the AR proteins are structurally very rigid. However, the results presented in this

and previous reports suggest that AR proteins are highly tolerant of structural variations,

which could be the basis for its functional diversity since structural and functional

properties are intimately related. Some of these properties, relating to the structural

pliancy of AR proteins, are summarized here: First, there are significant structural

variations in the loops linking neighboring ARs among AR proteins, even though the

global topology, including the small curvature, is highly conserved (6,63). Second, some

AR proteins can tolerate addition or deletion of AR repeats in the middle or at the end of

AR proteins. For example, our previous biochemical studies demonstrated that the

removal of up to three ARs at the C-terminus of gankyrin did not affect its structural

topology and CDK4-binding ability (14). It has also been reported that deletion of

individual ARs from Notch AR domain, or insertion of various copies of ARs from

Notch AR domain or from consensus-designed ARs, did not affect chemical- and heat-

induced unfolding of Notch AR domain (64-66). Last but not least, our results from the

gankyrin mutants showed that when negatively-charged Asp was introduced to the site

originally occupied by Ile, the local conformation of the loops adjusted accordingly

(accompanied by partial loss of stability), likely to help position the Asp residue for the

CDK4-inhibiting function. These properties suggest that the AR proteins are structurally pliant and are capable of conformational adjustments as a result of relatively minor changes in the sequence. This, in turn, can introduce the functional diversity seen in many of the AR proteins.

2.3.4 Potential biological significance.

In its natural form, gankyrin is bi-functional in regulating the CDK4-Rb pathway: it counteracts P16 inhibition of CDK4 (14), and facilitates the ubiquitin-mediated degradation of Rb, both of which result in forcing the cells into cell cycle progression

(1,52). Through structure-based protein engineering, we have transformed gankyrin from a simple competitor of P16 in CDK4 binding into a potent CDK4 inhibitor. In addition, it is plausible that for the gankyrin I79D mutant its Rb-binding and promoting function may remain intact since the conformational adjustments upon this mutation are limited to loops I-III, and results from our previous studies showed that the three C-terminal ARs are responsible for Rb binding, and this binding is independent of CDK4 binding.

Therefore, gankyrin I79D mutant likely remains bi-functional, but the outcomes of these two functions are now in conflict: while its Rb-binding and promoting role assists cell cycle progression, its CDK4-inhibitory role acts against cell cycle progression. It will be very interesting to see which of these two opposing functions is dominant when gankyrin

I79D is overexpressed in cells. Results from such studies could further our understanding of how nature balances the two functions of gankyrin, or the contrasting functions between gankyrin and P16 in relation to cell cycle and cancer.

Recently, the crystal structure of the complex between mouse gankyrin and the C- terminal domain of S6 ATPase of the 26S proteasome was reported (11). In this complex, most of S6 ATPase-interacting residues are located in the concave surface of mouse gankyrin, especially in loops II and III, which also contribute to binding to CDK4 as demonstrated in previous studies (reviewed in Reference 7) as well as in our current study. Furthermore, mouse gankyrin binds to S6 ATPase in the presence and absence of

Rb, indicating that like CDK4 binding, S6 ATPase binding is independent of Rb binding.

These findings suggest that CDK4 and S6 ATPase may compete with each other for gankyrin binding in cells. From this perspective, the similarities between CDK4 and S6

ATPase in binding to gankyrin make our question about the molecular mechanisms in which gankyrin balances and coordinates its versatile functions in cells more interesting and challenging. The significance of our current study, however, goes beyond binding – it dissects the binding and inhibition functions of gankyrin toward CDK4.

2.4 Materials and Methods.

2.4.1 Cloning, expression, and purification of human gankyrin and its mutants.

Human gankyrin cDNA was cloned into pGEX-6p-2 vector (Amersham) and expressed in Escherichia coli BL21 (DE3) Codon plus cells (Novagen) as glutathione-S- transferase-fusion proteins (GST) upon IPTG induction. GST-fusion gankyrin protein was purified from the cell lysate using a reduced glutathione-agarose column (about 15 mL of G beads, Sigma). After washing with TES buffer (10 mM Tris-HCl-1 mM EDTA-

1 mM β-mercaptoethanol-0.15 M NaCl, pH 7.5), 2.0 mL of protein-bound G beads were washed with 10 mL of TES-50 mg/mL reduced glutathione (pH 7.5) and the elute was

further purified using a Q Fastflow column (Pharmacia) to get pure GST-gankyrin. To obtain gankyrin without the GST tag, the remaining protein-bound G beads (about 13 mL) were suspended in 30 mL of TES in a 50 mL tube, and 100 units of PreScission protease (2 units / L, Amersham) were added into the tube. After incubation at 4 °C for

24 hours, the G beads were re-packed on a column and the flowthrough was further

purified by an S100 column (Pharmacia) equilibrated with 5 mM HEPES, 1 M EDTA, and 1 mM DTT (pH7.5). After SDS-PAGE analysis, fractions containing free gankyrin were pooled, concentrated, and lyophilized for further analyses.

All gankyrin mutants were generated through PCR-based Quickchange site- directed mutagenesis (Stratagene), and were expressed and purified as wild type.

2.4.2 Pull-down assay.

To investigate the binding of gankyrin proteins to CDK4, 25 g of GST-gankyrin

proteins were incubated with 10 g of recombinant CDK4-cyclin D2 complex (see below) and were incubated at 4 °C in 250 L of TES (pH 7.5) for 2 hours (14). The concentrations of the holoenzyme and GST-gankyrin proteins were 0.4 and 2.0 M, respectively. Subsequently, 250 L of fresh G beads were added into the reaction mixture, and after incubation at 4 °C for one hour, the reaction mixture was loaded onto a spin column (Fisher Scientific) and centrifuged at 4 °C, 1500 rpm for 3 minutes. After washing the G beads with TES 5 times, 1.0 mL each time, the beads were eluted with 200

L of TES-50 mg/mL reduced glutathione, and the elute was further analyzed with

Western Blot using rabbit polyclonal anti-human CDK4 antibody (Santa Cruz, C-22). In this assay, GST-P16 was used as positive control, and GST was used as negative control.

2.4.3 In vitro CDK4 kinase assay.

The in vitro CDK4 activity assay was performed as previously described (5).

Briefly, each reaction mixture contained 0.3 g of recombinant CDK4/cyclin D2 holoenzyme and varying concentrations of AR proteins in 15 L of the kinase buffer, 50 mM HEPES, 10 mM MgCl 2, 2.5 mM EGTA, 0.1 mM Na 3VO 4, 1 mM NaF, 10 mM β–

glycerolphosphate, 1 mM DTT, 0.2 mM AEBSF, 2.5 mg/mL leupeptin, and 2.5 mg/mL

aprotinin. After incubation at 30 ºC for 30 minutes, 50 ng of GST-Rb791-928 and 5 Ci

[γ-32 P] ATP were added in the reaction mixture which was then incubated at 30 ºC for another 15 minutes. Proteins in the reaction mixture were separated by SDS-PAGE, and the incorporation of 32 P into GST-Rb791-928 33 was quantitatively evaluated using a

PhosphorImager (Molecular Dynamics). The concentrations of AR proteins were

determined using absorbance at 280 nm in the kinase buffer and the molar extinction

coefficients from ProtParam at ExPASY. The IC 50 value was defined as the concentration of kinase inhibitor required for 50% of the maximal inhibition of CDK4, and measurements were repeated in triplicate.

Recombinant CDK4/cyclin D2 holoenzyme was expressed and purified as previously described (5,14). Briefly, human CDK4 and cyclin D2 cDNAs were cloned into pBacBAK8 and pBacBAK6 vectors, respectively. Of note, a Hisx6 tag was fused to the C-terminus of CDK4 to facilitate the following purification of the CDK4/cyclin D2 holoenzyme. Each construct was co-transfected into Spodoptera frugiperda SF-9 cells with Autographa California nuclear polyiruhedrosis virus BacPAK6/Bsu-361 DNA (BD

Clontech) to generate baculovirus particles. Both baculovirus particles were co- transfected into HighFive insect cells (Invitrogen), and the CDK4/cyclin D2 holoenzyme

was purified through affinity chromatography using Talon resin (BD Clontech). The final product was concentrated to approximately 0.3 mg/mL in the above kinase buffer, and aliquots were stored at -80 ºC.

2.4.4 Circular dichroism (CD) analyses of gankyrin proteins.

In GdnHCl-induced unfolding, recombinant gankyrin proteins were dissolved in

20 mM sodium borate buffer (pH 7.4) containing 40 M DTT and dialyzed against this

borate buffer at 4 ºC overnight. Samples containing 7.5-10.0 M proteins were incubated with different amounts of GdnHCl (in a stock solution of 8.5 M) on ice overnight and then equilibrated at 25 ºC just prior to CD analysis. The rotation at 222 nm was measured on an AVIV far-UV spectropolarimeter using a quartz microcell (Helma) of 0.1 cm light pass length, and the exact concentrations of GdnHCl were determined using its refractive index, and three scans were averaged. In this study, the ellipticity at 222 nm, an indicator of the existence of α–helical secondary structure was taken as the measure of

the degree of structure present in the protein at each GdnHCl concentration, and the free

energy of protein denaturation in aqueous condition was obtained on the basis of two-

state approximation (57). To determine the protein concentration, absorbance at 280 nm

was measured in the borate buffer, and the molar extinction coefficient for each protein

was determined using ProtParam at ExPASY.

Heat-induced unfolding experiments were performed using 10 M proteins in the borate buffer (pH 7.4) with 1 nm bandwidth and 10 second response time. Thermal melting spectra were recorded at 222nm by heating from 3 °C to 65 °C at the rate of 1 °C per minute and a 1 °C interval and then cooling down to 3 °C at the same rate. Tm was

defined as the temperature at the midpoint of transition (58). The heat-induced unfolding of these gankyrin proteins was highly reversible, and for each protein, more than 90% of the CD signal was recovered after returning to the initial temperature.

2.4.5 NMR analyses.

Unlabeled WT, I79D, and L62H, and uniformly 15 N-labeled I79D proteins were

prepared as previously described (4). The NMR samples contained 4 mM HEPES, 1 mM

2 DTT, and 5 µM EDTA in 95% H 2O/5% H2O at pH 7.5, and about 0.4 mM protein. All

NMR experiments, including 2D 1H-15 N HSQC, 2D 1H-15 N HMBC, 2D 1H homonuclear

NOESY, and 3D 15 N-edited NOESY, were performed at 27 °C, on a Bruker DRX-600 or

Bruker DRX-800 spectrometer equipped with cryoprobe. The NOE mixing time was

150ms. Data were processed with NMRPipe (67) and analyzed with NMRView.

2.4.6 Bioinformatics analysis.

All structural modeling and comparisons were performed using DS VISUALIZER

(ACCELRYS) and MOLMOL. The molar extinction coefficient of gankyrin was calculated using ProtParam from ExPASY.

CHAPTER 3

CONTRIBUTIONS OF CONSERVED TPLH TETRAPEPTIDES TO THE CONFORMATIONAL STABILITY OF ANKYRIN REPEAT PROTEINS

3.1 Introduction.

Ankyrin repeat proteins are one of the most abundant classes of repeat proteins that have been identified in the expanding protein sequence data bank (7). Through mediating protein-protein interactions, ankyrin repeat proteins are involved in numerous physiological processes across most of life forms, such as apoptosis, cell cycle control, cell signaling, cytoskeleton integrity, development and differentiation, inflammatory response, transcriptional regulation, and transport. These proteins are composed of various numbers of structural units, namely, ankyrin repeat motifs (hereafter, abbreviated as AR), which are stacked in a nearly linear fashion to form an elongated architecture that provides an extended and solvent-exposed binding surface highly versatile in protein binding (6,7,63).

One structural characteristic of AR proteins is the prevalence of the TPLH motif or its close variants, T/SxxH. This TPLH tetrapeptide is located at the 4 th – 7th positions

of an AR that consists of 30-34 amino acid residues ( Figure 3.1A ), and it initiates the

helix-turn-helix conformation of an AR while generating a tight turn from the preceding

AR (4). Out of 5785 AR sequences included in the Swiss-Prot ANK database

(http://www.expasy.org/sprot/ ), 2359 of them contain the T/SxxH motif. It has been reported that the Thr and His residues in a TPLH tetrapeptide exhibit the potential to form hydrogen bonding within this tetrapeptide as well as between such tetrapeptides in neighboring ARs ( Figure 3.1B ), implying that the TPLH tetrapeptides presumably contribute to the formation of a hydrogen-bonding network within an ankyrin scaffold, thus affecting its conformational stability as well as target binding property (4). Yet such contributions remain to be further verified. Moreover, our statistical analyses of AR sequences in the aforementioned database show that there is a location preference for the presence of T/SxxH motifs in certain AR proteins. Figure 3.2 presents the probabilities of a T/SxxH motif appearing in different locations of AR proteins. For example, in proteins composed of 4 ARs, the probabilities of having a T/SxxH on AR2 and AR3 are significantly higher than those on AR1 and AR4, while AR2 of proteins containing 5 and

7 ARs are heavily favored for the presence of T/SxxH motifs with probabilities greater than 50%. This finding indicates that even though TPLH or T/SxxH (in general) tetrapeptides are involved in intra-repeat as well as inter-repeat hydrogen bonding networks within the whole molecule, they are not favored in certain locations of different

AR proteins, therefore, their contributions to the stability of the global structure could not be even. Evidently, it is of interest to explore the molecular bases underlying the contributions of the TPLH motifs to the stability, structure, and function of AR proteins and the location preference of such contributions.

1 10 20 30 D - -G –TPLHLA - - - G - - -VV – LLL - - GADVNA -

Figure 3.1. The consensus sequence of an AR motif and the TPLH-mediated hydrogen bonding network. A , a consensus AR motif and its secondary structure. Rectangles and arrows represent helices and β sheets, respectively. B, a close view of the hydrogen-bonding network in a typical TPLH as evidenced in the NMR structure of δ1 gankyrin (Yuan et al 2004). The potential hydrogen bonds (T108 H N-H111 N , T108 γ1 γ1 δ1 ε2 O -H111 H N, T108 H -H111 N , and H111 H -A140 O) are indicated by dashed magenta lines.

Figure 3.2. Probability of T/SxxH appearing on each AR of proteins containg three to twlve ARs. Probability at each AR of a certain protein group was calculated by dividing the actual number of total T/SxxHs found on a specific AR within the group by the number of proteins in this group. Some ARs have been shown to be favored by TPLH motifs, like AR2 and AR3 of 4-AR proteins, AR2 of 5-AR and 7-AR proteins, AR8 of 9- AR proteins, and AR5 and AR9 of 12-AR proteins.

In our current study, we investigated the roles of TPLH motifs through a “two- way” approach, in which existing TPLH motifs in an AR protein (gankyrin) were perturbed whereas novel TPLH motifs were introduced into another AR protein (P16).

Composed of seven and four ARs, respectively ( Figure 3.3), gankyrin and P16 are

structurally similar in the first four N-terminal ARs, and they compete with each other for

binding to cyclin-dependent kinase 4 (CDK4) (14). However, while gankyrin contains

six T/SxxH tetrapeptdies, no T/SxxH tetrapeptide is present in P16. We generated

fourteen gankyrin mutants with mutations in native T/SxxH motif(s) and five P16

mutants with novel T/SxxH motif (s) ( Table 3.1) and evaluated the mutagenic effect on

CDK4-binding/modulating, stability, and structure. Our results showed that in spite of

their minor or moderate contributions to CDK4 binding and modulating, T/SxxH

tetrapeptides play a major role in stabilizing the global structures of gankyrin and P16,

and their contributions to the stability of the whole molecule tend to vary with their locations. T/SxxH tetrapeptides in the middle of a long stretch of ARs are more likely to influence the stability positively, and T/SxxH motifs located at both N- and C- termini contribute little or even negatively to the stability. Our results provide novel insights into understanding the unique biochemical and biophysical properties of AR proteins, and are of significance in molecular evolution as well.

10 20 30 40 50 60

| | | | | |

P16 1 mepaagssmepsaDWLATAAARGRVEEVRAl~lEAGALPNAPNSYGR RPIQ VMm~mGSAR 58

Gankyrin 1 ~~~~~~megcvsnLMVCNLAYSGKLEELKEsilADKSLATRTDQDSR TALH WAcsaGHTE 54

70 80 90 100 110 120

| | | | | |

P16 59 VAELLLLHGAEPNCADPaTLT RPVH DAAREGFLDTLVVLHRAGARLDVRDAWGR LPVD LA 118

Gankyrin 55 IVEFLLQLGVPVNDKDD~AGW SPLH IAASAGRDEIVKALLGKGAQVNAVNQNGC TPLH YA 113

130 140 150 160 170 180

| | | | | |

P16 119 EELGHRDVARYLRAaaggtrgsnharidaaegpsdipd~~~~~~~~~~~~~~~~~~~~~~ 156

Gankyrin 114 ASKNRHEIAVMLLEgganpdakdhyea tamh raaakgnlkmihillyykastniqdtegn 173

190 200 210 220 230

| | | | |

P16 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Gankyrin 174 tplh lacdeerveeakllvsqgasiyienkeek tplq vakgglglilkrmveg 226

Figure 3.3. The TPLH network in Gankyrin and P16. A, Tertiary structures of human gankyrin (left) and P16 (right). In both proteins, the TPLH tetrapeptides as well as its close variants are highlighted in yellow color. Helices are in green, and β sheets are in gold. B, Structure-based sequence alignments of human gankyrin and P16. The TPLH tetrapeptides and its close variants in P16 and gankyrin are highlighted in blue and red, respectively. 53

water Name# Mutation ∆Gd D1/2 (M) m (kcal* Tm (kcal*mol -1) mol -1 *M -1) (°C)

gankyrin WT 3.99 1.61 2.47 46.8 GankTPLH1A T42A 4.30 1.60 2.70 47.6 GankTPLH1AB T42A/H45A 5.93 1.33 4.41 44.7 GankTPLH2A S75A 7.09 1.75 3.99 50.3 S75T 6.83 1.99 3.09 55.9 GankTPLH2AB S75A/H78A 5.53 1.53 3.54 47.2 GankTPLH3A T108A 1.31 1.30 1.04 44.7 H111Q 2.69 1.26 2.13 45.8 GankTPLH3AB T108A/H111A 1.12 1.20 0.86 41.9 GankTPLH4A T141A 2.69 1.40 1.87 43.8 GankTPLH4AB T141A/H144A N/A N/A N/A N/A GankTPLH5A T174A 3.23 1.39 2.27 47.6 GankTPLH5AB T174A/H177A 3.24 1.55 2.05 46.5 GankTPLH6A T207A 3.24 1.47 2.16 47.5 GankTPLH6AB T207A/Q210A 2.86 1.53 1.89 47.1 P16 WT 1.94 0.6 3.23 46.0 P16TPLH1 R47T/Q50H N/A N/A N/A N/A P16TPLH2 R80T N/A N/A N/A N/A P16TPLH3 L113T/D116H 4.229 0.96 4.10 50.9 P16TPLH1-2 R47T/Q50H/R80T 2.487 0.62 4.13 44.4 P16TPLH2-3 R80T/L113T/D116H 1.903 0.55 4.35 36.3

Table 3.1. Conformational stability of TPLH mutants of gankyrin and P16

3.2 Results.

With regards to the abundance and functional diversity of AR proteins, it is important to evaluate the contributions of the conserved and prevalent TPLH tetrapeptide to the unique biochemical and biophysical properties of AR proteins. Towards this aim, we first rationally designed fourteen gankyrin mutants (Table 3.1), in which each of six

TPLH motifs in gankyrin was perturbed by replacing the conserved Thr (or Ser in a 54

SPLH variation) or both Thr and His with Ala residues. Except GankTPLH4AB, which was insoluble during expression in bacteria, the other thirteen mutants were soluble, and were evaluated for potential mutagenic effect on the function, stability, and structure of gankyrin using CDK4 binding assay, Circular dichroism (CD), and NMR, respectively.

Subsequently, we introduced novel TPLH motifs into P16 on the basis of a structure- orientated sequence alignment between P16 and the first four N-terminal ARs of gankyrin ( Figure 3.2B ). We also generated two P16 mutants with engineered TPLH motifs in the second/third ARs and the third/fourth ARs of P16 to explore the potential cooperation between TPLH motifs in P16. Of these five P16 mutants, two precipitated during protein expression and purification due to poor solubility, and the rest three were characterized as previously described.

3.2.1 Mutagenic effect on gankyrin-CDK4 association.

To evaluate the mutagenic effect of these gankyrin mutants on its function, we first performed the binding assay on various gankyrin proteins with the (His) 6-tagged

CDK4-cyclin D2 holoenzyme. As shown in Figure 3.4A , when gankyrin wild type (WT) was present in a reaction mixture containing no (His) 6-tagged CDK4-cyclin D2, there

was no detectable gankyrin protein in the pull-down product (lane 1). However, gankyrin

was detected in the pull-down product from a reaction mixture including both gankyrin

WT and (His) 6-tagged CDK4-cyclin D2 [gankyrin:(His) 6-tagged CDK4-cyclin D2, 2:1]

(lane 2), indicating that gankyrin associates with (His) 6-tagged CDK4-cyclin D2 in vitro .

Similarly, all tested gankyrin mutant proteins were detected in the pull-down products

(lanes 3-13), implying that all these gankyrin mutants retain comparable CDK4-binding

ability. Therefore, mutations in these TPLH motifs do not significantly impair the CDK4- binding ability of gankyrin.

1 20

1 00

40 P 16 W T

CDK4 Inhibition(%) P16 TPLH2 P16 TPLH3 20 P16 TPLH2-3

0 0 200 400 600 800 1000 1200 1400 1600 Concentration of P16 [nM]

Figure 3.4. Functional analyses of gankyrin and P16 mutants. A , pull-down assays to assess the interaction between gankyrin proteins and the CDK4-cyclin D2 complex. Lanes: 1, the control reaction only containing gankyrin wild type (WT) and bivine serum albumin (BSA); 2-13, the reaction mixtures containing the CDK4-cyclin D2 complex (1.0 µM) and various gankyrin proteins (at the concentration of 2.0 µM) as indicated. B, in vitro CDK4 kinase assay to evaluate the CDK4-inhibitory activities of P16 mutants. Each reaction mixture contained about 0.3 g of recombinant CDK4/cyclin D2 holoenzyme, 50 ng of GST-Rb791-928, 5 Ci [ γ-32 P] ATP, and varying concentrations of P16 proteins. After incubation at 30 ºC for 15 minutes, proteins in the reaction mixture were separated by SDS-PAGE, and the incorporation of 32 P into GST-Rb791-928 was evaluated using a PhosphorImager (Molecular Dynamics). IC 50 was defined as the concentration to achieve 50% of the maximal inhibition of the kinase activity, and a >2- fold change in the IC 50 value was regarded as significant. 56

3.2.2 Mutagenic effect on the conformational stability of gankyrin.

Subsequently, guanidinium hydrochloride (GdnHCl)-induced unfolding of gankyrin WT and mutants was monitored by following the changes in ellipticity at 222 nm. The unfolded fraction, defined as the ratio of the difference in ellipticity at 222 nm between the tested sample and the native state and the difference between the completely unfolded state and the native state, was derived from the raw data and plotted against the concentration of denaturant ( Figure 3.5A ). The unfolding curves of these AR proteins are

well fitted to a model with a two-state transition between native and unfolded proteins,

indicating that GdnHCl-induced unfolding of these proteins is cooperative. The values of

water ∆Gd (the denaturation free energy in water), D 1/2 (the denaturant concentration at the

midpoint of transition), and the slope m were obtained on the basis of two-state approximation ( Table 3.1). These thirteen soluble gankyrin TPLH mutants can be approximately categorized into four groups based on their behavior in GdnHCl-induced unfolding. The first group includes mutants GankTPLH 1A, 2AB, 5A, 5AB, 6A, and

water 6AB. GankTPLH 1A, 5A, 5AB, and 6A have m values and Gd values close to those of gankyrin WT, and it is safe to claim that they are comparable with gankyrin WT in conformational stability. As for GankTPLH 2AB and 6AB, both their m values and

water Gd values are considerably different from the corresponding values of gankyrin WT,

water thus the difference in the ∆Gd values of these proteins cannot be directly interpreted

as the difference in conformation stability. However, their D 1/2 values are almost identical to that of gankyrin WT, indicating that their structural stabilities in GdnHCl-induced unfolding could be similar to that of gankyrin WT. The second group includes

GankTPLH 1AB, 3A, 3AB, 4A, 6AB, and H111Q. While GankTPLH 1AB, 3A, 3AB,

and 4A have m values largely different from that of gankyrin WT, the D 1/2 value of

gankyrin WT (1.61 M) is higher than those of GankTPLH 1AB, 3A, 3AB, and 4A (1.33

M, 1.30 M, 1.20 M, and 1.40 M, respectively), suggesting that GankTPLH 1AB, 3A,

3AB, and 4A are less structured at high concentrations of denaturant GdnHCl. In

water comparison with gankyrin WT, H111Q has a similar m value but a decreased ∆Gd value, indicative of lower conformational stability. The last group includes S75A

(GankTPLH2A), and S75T2A. These two mutants have similar m values as gankyrin

water -1 WT but their ∆Gd values, 7.09 and 6.83 kcal*mol , are remarkably higher than that

of gankyrin WT, implying that these two substitutions in this specific SPLH motif

substantially stabilize the global structure of gankyrin.

Figure 3.5. Chemical- and heat-induced unfolding of representative gankyrin and p16 INK4a mutants monitored by far-UV CD. A, GdnHCl-induced unfolding. Samples contained about 7.5-10.0 M proteins and were incubated with different amounts of guanidinium hydrochloride on ice overnight. The ellipticity at 222 nm was monitored by far-UV CD (190-260 nm) at 25 ºC. The concentration of GdnHCl was determined using its refraction index. The fraction unfolded, defined as (the ellipticity at 222 nm at a denaturant concentration-the ellipticity at 222 nm at the native state) / (the ellipticity at 222 nm at the fully unfolded state-the ellipticity at 222 nm at the native state), was plotted against the GdnHCl concentration. The unfolding curve was fitted to a model of two-state approximation. B, Heat-induced unfolding. Thermal melting spectra were recorded at 222 nm by heating from 5 °C to 65 °C with a rate of 1°C per minute and a 1 °C interval. The fraction unfolded was defined as (ellipticity at 222 nm at certain temperature - ellipticity at 222 nm at the native state) / (ellipticity at 222 nm at the fully unfolded state - ellipticity at 222 nm at the native state). Tm, the temperature at the midpoint of transition, was obtained through fitting the melting curve to a two-state transition model.

The above results are further supported by an NMR study, in which the stability of tertiary fold, not the unfolding of secondary elements in CD analyses, was monitored

by 2D 1H-15 N HSQC upon the addition of GdnHCl to gankyrin WT, T42A

(GankTPLH1A), S75A (GankTPLH2A), and T108A (GankTPLH3A). On one hand, as

self-evident in Figure 3.6, all four proteins show an all-or-none behavior with the addition of GdnHCl and there is no detectable partially folded intermediate, indicating that ARs in gankyrin WT and mutants are not sequentially unfolded. These results support a two-state denaturation process as assumed in the above CD analysis. On the other hand, the stability follows the order of S75A (GankTPLH2A) > T42A

(GankTPLH1A) ~ gankyrin WT > T108A (GankTPLH3A), and their melting points fall

water in the range of 1.2-1.4, 1.4-1.6, and 1.6-1.8 M, respectively. Accordingly, the ∆Gd values for S75A (GankTPLH2A), T42A (GankTPLH1A), gankyrin WT, and T108A

(GankTPLH3A) are 7.09, 4.30, 3.99, and 1.31 kcal*mol -1, respectively.

Figure 3.6. 2D 1H-15 N HSQC spectra of gankyrin WT showing the all-or-none unfolding: (A) the overlap of the spectra recorded at 0 (black) and 1.4 M (red) GdnHCl, respectively; (B) the spectrum at 1.6 M GdnHCl (blue). The sample still retains native fold at 1.4 M GdnHCl, but denatures at 1.6 M GdnHCl. The transition point is thus determined to be in the range of 1.4-1.6 M. Similar behavior was observed in gankyrin mutants T42A, S75A, and T108A, except with identical or different transition point. 60

Heat-induced unfolding of gankyrin WT and mutants also follow a two-state transition ( Figure 3.4B ). The Tm values, which are temperatures at the midpoint of transition, are listed in Table 1. The Tm values of GankTPLH 1A, 2AB, 5A, 5AB, 6A,

6AB, and H111Q mutants are comparable (within 1°C) to that of gankyrin WT (47.6°C,

47.2°C, 47.6°C, 46.5°C, 47.5°C, and 47.1°C, 45.8°C versus 46.8°C), suggesting that these mutants are as stable as gankyrin WT in heat-induced unfolding. In contrast,

GankTPLH 1AB, 3A, 3AB, and 4A all have Tm values lower than gankyrin WT, and

appear to be less stable than gankyrin WT in heat-induced unfolding. Interestingly, the

Tm values of S75A (GankTPLH2A) and S75T are 50.3°C and 55.9°C, respectively,

indicating that like in GdnHCl-induced unfolding, these two substitutions significantly

increase the stability of gankyrin in heat-induced unfolding.

3.2.3 Mutagenic effect on the structure of gankyrin.

Out of thirteen soluble gankyrin TPLH mutants, three gankyrin mutants, T42A

(GankTPLH1A), S75A (GankTPLH2A), and T108A (GankTPLH3A) were chosen to

represent gankyrin mutants with conformational stability comparable, higher, and lower

than that of gankyrin WT, respectively, and were subjected to in-depth NMR studies to

investigate the potential structural perturbations after mutations in the TPLH motifs.

These include evaluation of (1) overall conformational changes through analysis of 3D

15 N-edited NOESY, which also led to the backbone amide assignments in 2D 1H-15 N

ε2 HSQC; (2) local conformational changes around TPLH via analysis of His H resonances by 2D 1H-homonuclear NOESY; (3) changes in conformational flexibility by

H/D exchange monitored by 2D 1H-15 N HSQC.

The assignment of 3D 15 N-edited NOESY spectra is based on the previous NMR

α studies on gankyrin WT by using the sequential NOE assignment such as H (i)-HN(i+1) and H N(i)-HN(i+1), as well as by inspecting NOE pattern that entails three-dimensional conformational information. A total of 214 of 219 possible backbone NH protons have been assigned for each gankyrin mutant. The combined 1H and 15 N pair wise chemical

shift differences ( ∆ppm) with respect to the wild type were calculated as

2 2 1/2 1 [( ∆δ HN ) +( ∆δ N*αN) ] , in which αN is the scaling factor (0.17) used to normalize the H

and 15 N chemical shifts. The results are shown in Figure 3.7, which taken together with

NOE pattern were used to evaluate the conformational changes in three mutants. First, it

is validated that gankyrin is fairly tolerated to the Thr to Ala mutation in a TPLH, which

does not induce global conformational changes. Excluding the mutation residue (A42,

A75 and A108, respectively), the average chemical shift changes are 0.03, 0.02, and 0.03

ppm for T42A, S75A, and T108A mutants, respectively. Secondly, all these mutations

induce similar local chemical shift perturbation pattern centered on the mutation site. In

specific, the largest chemical shift perturbations are around the mutation site, followed by

ripple effect on the loop residues preceding the next TPHL motif. This behavior is in-

consistent with the modular nature of ankyrin repeat proteins. It is interesting to note that

S75 to Ala mutation introduced a relatively large perturbation on T108 ( ∆ppm = 0.48

ppm). Lastly, the NOE patterns were retained remarkably for the amide protons in these

mutants. For example, in T108A mutant, the NOE between A108 H N and H111 H N is of

comparable intensity as the corresponding one in WT, indicative of retaining helical

conformation for this stretch of residuals. These observations, while facilitating the above

sequential assignments, are in strong support of no major structural changes for these mutants.

3 (A) * 2 ppm ∆ ∆ ∆ ∆ 1

0 (B) * 3

2 ppm ∆ ∆ ∆ ∆ 1 T108

3 (C) * 2 ppm ∆ ∆ ∆ ∆ 1

0 0 20 40 60 80 100 120 140 160 180 200 220

Residue Number

Figure 3.7. Residue pairwise chemical shift difference between gankyrin mutant and WT: (A) T22A (GankTPLH1A), (B) S75A (GankTPLH2A), and (C) T108A (GankTPLH3A). The mutation site in each mutant is marked by asterisk, while T108 experiencing comparatively large perturbation in mutant S75A is labeled. The combined 1H and 15 N pair wise chemical shift differences ( ∆ppm) with respect to the wild type were 2 2 1/2 calculated as [( ∆δ HN ) +( ∆δ N*αN) ] , in which αN is the scaling factor (0.17) used to normalize the 1H and 15 N chemical shifts.

2D 1H-homonuclear NOESY experiments were also recorded on these mutants as

well as S75T and H111Q to investigate the histidine in the TPLH motif, as its imidazole

ε2 ring is important for the hydrogen bond network ( Figure 3.8). First, the histidine H resonances in the five TPLH motifs are clearly identified in all five mutants, albeit that

ε2 H111 H resonance is significantly weaker in T108A mutant and absent in H111Q mutant. Secondly, these resonances in each mutant retain similar NOE pattern as that in

ε2 gankyrin WT including the one in the mutated TPLH motif, e.g. H48 H in T45A

ε2 α mutant. Most notably, the identification of His H and (His+30) H suggests the

ε2 retention of inter-repeat hydrogen bonding between His N and (His+29) O in all these mutants, e.g. H45-W74. This is further supported by other inter-repeat NOEs such as

ε2 β ε2 those between H78 H and A101 H and between H78 H and N103 H N in S75A

mutant. Lastly, it is apparent that the Thr to Ala mutation in one TPLH motif induces

ε2 little perturbation on the adjacent TPLH motifs. For example, in T42A mutant H78 H resonance appears to be little perturbed in contrast to large chemical shift change of H45

ε2 ε2 H . As shown in previous NMR studies (27), the histidine H region is a good indicative of the loop conformations and conformational stability. From the above analysis, T108A

(Figure 3.8E ) may induce more destabilizing effect on gankyrin than T42A ( Figure

3.8B ) and S75A ( Figure 3.8C ). Nevertheless, the single Thr to Ala mutation disrupts the

intra-repeat hydrogen bonding that needs Thr hydroxyl group but likely retains the inter-

repeat interactions involving His side chain. From this perspective, mutations in His

residue of one TPLH motif could cause more profound effect than corresponding

mutations at Thr residue since a substitution at His residue disrupts the intra-repeat Thr-

His hydrogen bonding as well as the inter-repeat one involving the Thr hydroxyl group in

the neighboring AR. Indeed, H111Q ( Figure 3.8F ) brings about the largest perturbation

to the TPLH network while S75T ( Figure 3.8D ) induces least changes.

Figure 3.8. Selected spectral regions from 2D NOESY spectra on gankyrin: (A) WT, (B) T42A (GankTPLH1A), (C) S75A (GankTPLH2A), (D) S75T, (E) T108A (GankTPLH3A), and (F) H111Q mutants . The cross-peaks designated “a”, “b”, and “c” ε2 ε1 δ2 α are NOEs from His H in a TPLH motif to His H , His H , and (His+30) H , respectively. The cross-peaks designated “d” (in panel A and B) and “e” (in panel A and ε2 ε2 C) are NOEs of H45 H – D68 H N, and H78 H – N103 H N, respectively, further indicating the retention of a structured imidazole ring in T42A and S75A, respectively.

In addition, these three gankyrin mutants are further subjected to H/D exchange monitored by 2D 1H-15 N HSQC in comparison with gankyrin WT. All four samples

display similar behavior ( Figure 3.9). The slowly exchanging NH protons observed after

25 hour H/D exchange are identified in the helix 4, 5, 6, 7, 9, 10, and 11, except A134

and A140 located in the AR4/AR5 loop ( Figure 3.10 ). The results indicate that the single mutation is not enough to perturb the most stable hydrophobic core formed by the helical segments. For example, despite relatively large chemical shift perturbation of L59 in

T42A, I79 in S75A, and L126 in T108A, these residues are visible under the same H/D exchange condition as gankyrin WT.

Figure 3.9. 2D 1H-15 N HSQC of gankyrin proteins after 25 hour H/D exchange at room temperature: (A) WT; (B) T42A (GankTPLH1A); (C) S75A (GankTPLH2A); and (D) T108A (GankTPLH3A). The slow-exchanging backbone amide cross-peaks are labeled. It is clear from these results that the most stable hydrophobic core formed by the helical segments is hardly perturbed by T42A, S75A, or T108A mutation.

Figure 3.10 . Ribbon diagram showing the slow exchanging amide proton detected in gankyrin T42A (GankTPLH1A). No major differences were observed among gankyrin WT and three mutants, T42A (GankTPLH1A), T75A (GankTPLH2A), and T108A (GankTPLH3A), as evidenced in Figure 9.

Taken together, our studies with gankyrin TPLH mutants show that perturbations in the TPLH motifs of gankyrin mainly impact the conformational stability of gankyrin, and such influence varies with the location of the TPLH motifs. Perturbations in the

TPLH motifs located between AR1 and AR2, AR5 and AR6, AR6 and AR7 do not bring about notable changes in the stability of the global structure; similar perturbations in the

TPLH motifs between AR3 and AR4, AR4 and AR5 considerably destabilize the global structure; mutations in the TPLH motif between AR2 and AR3 leads to a more stable structure. In other words, TPLH motifs in the middle of gankyrin, i.e. between AR3 and

AR4, AR4 and AR, stabilize the global structure, while TPLHs at other locations does not contribute or even contribute negatively to the conformational stability of gankyrin.

To further confirm these findings, we used a "reverse" approach in which novel TPLH motifs were introduced into P16 and their impact on the function, stability, and structure was evaluated.

3.2.4 Mutagenic effect on P16.

The expression of P16 and its mutants has been hampered by the unstable nature of P16 (61). Mutations on the second AR of P16, P16TPLH1 and P16TPLH1-2, yielded unstable proteins which formed inclusion body in E. coli and were not subjected to further studies. The CDK4-inhibitory activities of three soluble P16 mutants were quantitatively evaluated using in vitro CDK4 kinase assays ( Figure 3.4B ). The IC 50 values of P16TPLH2, P16TPLH3, and P16TPLH2-3 are 35 ± 10 nM, 290 ± 32 nM, and

110 ± 21 nM, respectively. In comparison, the IC 50 value of P16 WT is 72 ± 15 nM.

Since a two-fold change in IC 50 is regarded as significant (Byeon et al 1998), the CDK4- inhibitory activities of P16TPLH2 and P16TPLH3 are moderately increased and decreased, respectively, while P16TPLH2-3 exhibits a CDK4-inhibitory activity comparable to that of P16 WT.

In GdnHCl-induced unfolding, three soluble P16 mutants, P16TPLH2,

P16TPLH3, and P16TPLH2-3, all exhibited two-state transitions ( Figure 3.5A ).

water -1 However, P16TPLH2 has a ∆Gd value of 4.229 kcal*mol and a D 1/2 value of 0.96M

(Table 3.1) that are significantly higher than the corresponding values of the other two

P16 mutants and P16WT, indicating that P16TPLH2 is more stable than P16WT, i.e. the 68

introduction of the TPLH motif in the third AR of P16 increased the conformational stability of P16. Contrarily, P16TPLH2-3 and P16TPLH3 behave similarly as P16 WT in

water GdnHCl-induced unfolding as evidenced with comparable ∆Gd and D 1/2 values.

These findings are further supported by results from heat-induced unfolding ( Figure 3.5B

and Table 3.1). The Tm value of P16TPLH2 is 50.9 °C, significantly higher than that of

P16TPLH2-3, P16TPLH3, and P16 WT (44.4°C, 36.3 °C, and 46.0°C, respectively).

Moreover, the Tm value of P16TPLH3 is 36.3 °C, much lower than that of P16WT,

suggesting that the introduction of the TPLH motif in the fourth AR of P16 destabilizes

P16 in heat-induced unfolding.

The mutagenic effect on the structure of P16 was evaluated by 1D proton and 2D

NOESY NMR as previously described. There is no significant perturbation to the global

structure of P16 upon the introduction of TPLH motifs (data not shown), whereas notable

changes occur in the local conformation of the corresponding region. For example, the

R80T substitution in P16TPLH2 introduces a novel TPVH motif in the third AR of P16.

Figure 3.11 shows the selected regions from 2D NOESY spectra on P16 WT and

P16TPLH2. Apparently, there are more and stronger NOEs involving H83 in the spectra

of P16TPLH2, indicating that the imadazole ring of H83 in P16TPLH2 is better

structured. Such local conformation change does not generate novel contacts with CDK4

but stabilizes the global structure of P16 thus leading to the aforementioned moderate

increase in the CDK4-inhibitory activity of P16TPLH2.

Figure 3.11 . Selected spectral regions from 2D NOESY spectra on (A) P16 WT, (B) mtant R80T (P16TPLH2). The cross-peaks designated a, b, c, d and e are NOEs from ε2 α δ1 ε1 H83 H to L113 H , H83 H , H83 H , D108 H N, L113 H N, V106 H N, and R107 H N, respectively. Data were recorded under identical experimental conditions, including sample concentration and NMR experimental parameters. Apparently the imidazole ring of H83 in R80T (P16TPLH2) mutant is better structured, evidenced by more and stronger NOEs.

3.3 Discussion.

In addition to their abundance and functional diversity, AR proteins have recently gained a lot of attention due to their unique biochemical and biophysical features

(6,7,68). One such unique feature is that the global structure of an AR protein is stabilized by the accumulation of local, short interactions originating from each individual residue; hence, the prevalently existing TPLH (or T/SxxH) tetrapeptides have been assumed to play certain roles in maintaining the structures and functions of AR proteins (7). Here, we report the first in-depth study on the contributions of TPLH motifs to the structure, function, and stability of AR proteins. While experiments were 70

performed on two specific AR proteins, gankyrin and P16, our findings could be applied to other AR proteins in general.

First, the major contribution of TPLH motifs is to stabilize the global structure of

AR proteins (4). As described earlier, all gankyrin and P16 TPLH mutants except

GankTPLH4AB, P16TPLH1, and P16TPLH1-2 retained virtually unperturbed global structures, while only little or moderate change in their CDK4-binding or inhibition activity was observed. In contrast, even single substitutions in specific TPLH motifs of gankrin and P16 brought about significant changes in the stability of the global structures.

These results indicate that instead of being directly involved in forming the structure and

CDK4 binding/modulating, TPLH motifs function to impact the conformational stabilities of gankyrin and P16 as well as other biophysical properties. From this perspective, GankTPLH4AB, P16TPLH1, and P16TPLH1-2 could be regarded as extreme cases, in which TPLH mutations caused radical changes in the stability thus leading to the loss of the global structure of gankyrin or P16. On one hand, as shown in

Figure 3.1A , there are two conserved stretches in an AR motif, the TPLH motif in

positions 4-7 and the V/I-V-X (hydrophilic)-L/V-L-L motif in positions 17-22, both of

which are important in forming the canonical helix-turn-helix conformation of an AR (4).

While the V/I-V-X (hydrophilic)-L/V-L-L stretch forms the second α-helix of an AR, the

TPLH tetrapeptide forms a tight turn and initiates the first α-helix. In an AR protein,

helices from different ARs are packed into helix bundles, and the loops connecting

neighboring ARs orientate in such that they are perpendicular to the helical axes (6,7,63).

Accordingly, the TPLH tetrapeptide is located within the relatively flexible loop region,

and its contribution to the global structure is secondary to those helical residues, such as

the V/I-V-X (hydrophilic)-L/V-L-L stretch, which is the central piece of the helix bundles. On the other hand, it has been well demonstrated that most of contacts between

AR proteins and their targets are located in the helical regions as well as in the “tips” of the loops, which are defined as the first residue of an AR (position 1) and the last residue of the preceding AR (position 30 or 34) (7). TPLH tetrapeptides could moderate the contacts in their proximity but not directly associate with targets. Of note, both gankyrin and P16 are able to interact with proteins other than CDK4, and our results do not rule out the possibility that mutations in TPLH motifs may affect the interactions with non-

CDK4 proteins.

Secondly, contributions of TPLH motifs to the conformational stability are

location-dependent. In gankyrin, six TPLH tetrapeptides form a hydrogen-bonding

network which connects neighboring ARs thus facilitating the formation of the core

structure of helice bundles. Hence, one may assume that all these TPLH tetrapeptides

contribute to the stability similarly. However, results from our current study revealed

that the TPLH network plays more complicated roles in the conformational stability of

gankyrin and such roles depend on the location of the TPLH motifs. Basically, the

gankyrin TPLH mutants can be put into three groups with distinct locations: mutants in

TPLH1, TPLH5, and TPLH6 have comparable conformational stability with gankyrin

WT; mutants in TPLH2 are more stable than gankyrin WT; mutants in TPLH3 and

TPLH4 are less stable than gankyrin WT (GankTPLH4AB is regarded as the extreme

case in this group). Mutants in TPLH1, TPLH5, and TPLH6 contain disruptive TxxH

mutations on AR2, AR6, and AR7 of gankyrin, it appears that the TPLH motifs located at

two ends of the molecule do not contribute much to the stability of the molecule. On the

contrary, TPLH motifs in the middle of the molecule, TPLH3 and TPLH4 are very important for the stability of gankyrin, as evidenced by significant decrease in the stability caused by GankTPLH3A, 3AB and 4A. Extremely, GankTPLH4AB even precipitated during the protein expression process (see above). Interestingly,

GankTPLH2A and GankTPLH2AB are more stable than gankyrin WT in both chemical- and heat- induced unfolding. All these results demonstrate that TPLH motifs positioned in the middle (AR2, AR3 and AR4) of gankyrin are very important to its stability. The strong hydrogen bonds between these middle ARs hold them together and form a core, which stabilizes the whole linear structure of gankyrin. This notion is further supported by thermodynamic studies of P16 TPLH mutants. As shown in Table 2, P16TPLH2 is much more stable than P16 WT in both chemical- and heat- induced unfolding, which indicates adding this TPVH motif does stabilize AR3 of P16 and in turn makes the whole protein more stable. Contrarily, P16 TPLH3 is much less stable than P16 WT in heat- induced unfolding (Tm: 36.3 °C vs 46.0 °C), implying that the introduction of a TxxH motif into AR4 of P16 is not favored at least in heat-induced unfolding. Apparently, introductions of TPLH motifs into AR3 and AR4 of P16 bring about opposite effect in

water the stability of P16. More interestingly, the values of ∆Gd and Tm of P16 TPLH2-3 are between the corresponding values of P16 TPLH2-3 and P16 TPLH2, indicating that

P16 TPLH2-3 is more stable than P16TPLH3, but less stable than P16 TPLH2. Hence, to certain extent, the contributions from TPLH motifs at different locations are additive.

Thirdly, the above location-dependent contribution of TPLH motifs to the

conformational stability of an AR protein is not against the fact that the structural,

functional, and biophysical roles of TPLH motifs in AR proteins are integrated. Indeed,

there is an intricate structural basis underlying the contribution of TPLH motifs to the conformational stability. On one hand, the threonine residue in a TPLH plays three major roles: an intra-AR hydrogen bonding interaction involving the hydroxyl group and the neighboring His residue, an inter-AR hydrogen bonding interaction involving its side chain and His residue in the neighboring AR, and a hydrophobic interaction attributed to its methyl group with other residues, e.g. the residue at (Thr+26) position. Mutations in a

TPLH motif may impair these three interactions differently, thus bringing about different effect on the conformational stability. For example, a single S75A mutation disrupts the intra-repeat hydrogen bonding interaction (between 75 Ser and 78 His) but re-introduces

75 β missing hydrophobic interactions between Ala CH 3 and its surrounding residues

including 101 Ala in the next AR. This may partially explain the unexpected high stability of gankyrin S75A. Accordingly, gankyrin S75T retains all aforementioned interactions,

water -1 and has an almost identical ∆Gd value to S75A (6.83 vs 7.09 kcal*mol ) but higher

Tm (55.9 vs 50.3 °C). In contrast, a H111Q substitution eliminates the intra-AR hydrogen-bonding between the hydroxyl group of 108 Thr and 111 His, which consequently

destabilizes the global structure. With regard to this issue, it is also important to note that

in addition to the location preference, a T/SxxH tetratpeptide prefers Thr over Ser at the

first position of this tetrapeptide. Out of 2359 T/SxxH-containing AR sequences, 1801

AR sequences have TxxH motifs, while the rest 558 AR sequences harbor SxxH, imply

that Thr is more favored than Ser in this conserved tetrapeptide. One plausible

explanation for such “Thr” preference is that Thr can use the extra methyl group to form

hydrogen-bonding with His residue in the TPLH motif of the preceding AR, thus

contributing to the stability more than Ser in most of cases. 74

On the other hand, it could be safer to say that the contribution a TPLH motif to the stability of the global structure depends on the "local" structure, instead of the location itself. Generally, ARs at both N- and C-termini depart from the consensus sequence ( Figure 3.1A ) more than ARs in the middle of the molecule. Moreover, the

"local" structure of a TPLH motif at a specific location varies with AR proteins. For

example, the contributions of the first TPLH motif, i.e. located at AR2, to the stability of

gankyrin and P16 are totally different. While GankTPLH1 (T42A) did not caused

detectable changes in the stability of gankyrin, P16TPLH1 and P16TPLH1-2 precipitated

during expression and purification. As revealed in the solution structure of P16, the

conformation of AR2 in P16 deviates from the typical helix-turn-helix confromation of

most of ARs. The first half of AR2 of P16 consists of only a short helical turn (residues

47-51) ( Figure 3.2B ), not a regular helix, indicating that mutations in P16TPLH1 and

P16TPLH1-2 may not lead to an structured TxxH motif engaging in hydrogen-bonding network but rather significantly impair the stability and solubility of P16.

In conclusion, as a part of the conserved AR, the T/SxxH motif does not only have the preference for location, but also contribute to the conformational stability of the global structure in a location-dependent manner. This location-dependent variation of the influence of T/SxxH motifs on the stability could bring about a novel strategy to improve the stability of AR proteins of pharmaceutical potential, that is, through introducing

“stability-favorable” T/SxxH motifs in certain locations and removing “stability- unfavorable” T/SxxH motifs in other locations. For example, P16 is an important tumor suppressor of potentials in cancer therapy, but it is notorious for its low stability (61). An engineered P16 with increased stability but intact function and structure, like P16TPLH2

could be of a better choice than P16 WT. Apparently, this approach is arguably complementary to the popular consensus design approach, which has been used to generate several AR proteins with extremely high stability (in comparison with natural

AR proteins with the same number of ARs) (69-71). In consensus-designed AR proteins, a number of identical “synthetic” ARs are packed together with or without two hydrophilic “capping” ARs at the N- and C-termini, and TPLH motif is present in each of these consensus ARs (71). On one hand, since the contributions of these TPLH motifs to the stability of consensus-designed AR proteins could be opposite as revealed in our current study, refinements in different TPLH motifs could further improve the stability of these AR proteins. On the other hand, a lot of natural AR proteins, such as gankyrin, interact with multiple target proteins with distinct functions in cells; however, consensus- designed proteins usually don’t exhibit such multi-functionalities. Additionally, since most of TPLH modifications don’t cause significant perturbations to the global structure and function of AR proteins, TPLH-modified AR proteins could be more like native AR proteins than consensus-designed AR proteins, which are totally “foreign” proteins to the immune system. Hence, natural AR proteins with TPLH modifications could be more appropriate than consensus-designed AR proteins in certain cases with regards to potential clinical application.

3.4 Materials and Methods.

3.4.1 Database analysis.

The source of the 5786 ankyrin repeats in 1008 proteins (as of May 2008) was downloaded from the Swiss-Prot database ( http://www.expasy.org/sprot/ ). All sequence

analysis including the sequence of each ankyrin repeat was performed using JAVA- or

PERL-based programs.

3.4.2 Cloning, expression, and purification of human gankyrin, p16 INK4a and their

mutants.

Human gankyrin cDNA was cloned into pGEX-6p-2 vector (Amersham) and the

glutathione-S-transferase (GST) -fusion protein was expressed in Escherichia coli BL21

(DE3) Codon plus cells (Novagen) upon IPTG induction. After sonication, GST-fusion

gankyrin was purified from the cell lysate using a reduced glutathione-agarose (G beads)

column (Sigma). After washing the column with TES buffer (10 mM Tris-HCl, 1 mM

EDTA, 1 mM β-mercaptoethanol, and 150 mM NaCl at pH 7.5), the protein-bound G

beads were suspended in 30 ml of TES buffer in a 50 ml tube, and 100 units of

PreScission protease (2 units/ l; Amersham) were added to remove GST. After incubation at 4 °C for 24 h, the G beads were re-packed on a column and the flow through was collected and further purified by an S100 column (Pharmacia) equilibrated with 5 mM HEPES, 1 M EDTA, and 1 mM DTT (pH 7.5). After SDS–PAGE analysis, fractions containing free gankyrin protein were pooled, concentrated, and lyophilized for further analyses. All gankyrin mutants were generated using PCR-based site-directed mutagenesis (Stratagene), and were expressed and purified essentially the same as gankyrin WT.

Human p16 cDNA was cloned into a pTG vector and expressed in bacteria as

GST-fusion protein as previously described (Byeon et al. 1998). Free P16 protein was purified similarly as gankyrin except that 100 units of Thrombin (Sigma) were used to

remove the GST tag and the incubation was performed at room temperature for 2 hours.

All P16 mutants were created using site-direct mutagenesis, and were expressed and purified as P16 WT.

3.4.3 Pull-down assay.

To investigate the interaction between gankyrin proteins (including WT and mutant proteins) and the CDK4-cyclin D2 complex, 50 µg of the CDK4-cyclin D2 complex and 100 µg of free gankyrin proteins were incubated at 4 °C in 1.0 mL of PBS

(pH 7.4) for 2-4 hours. Of note, there is an (His) 6 tag at the C-terminus of CDK4 as

previously described (5). The concentrations of CDK4-cyclin D2 and gankyrin are 1.0

µM and 2.0 µM, respectively. Subsequently, 250 µL of fresh TALON resin (Qiagen)

(pre-equilibrated with PBS at 4 °C) was added into each reaction mixture, and the incubation was continued for another four hours. The reaction mixture was loaded onto a spin column and after centrifugation at 4 °C, 1500 rpm for 2 minutes, washed with PBS

(three times), PBS with 50 mM imidazole (one time), and PBS with 100 mM imidazole

(one time). Proteins bound to the TALON resin were eluted out of the column with 80

µL of PBS containing 1 M imidazole, and were further analyzed with Western blot using

a primary antibody against human gankyrin (PW8325, BIOMOL Int’l) and a AP-

conjugated secondary antibody (sc-2034, Santa Cruz Biotechnology).

3.4.4 In vitro CDK4 kinase assay.

The in vitro CDK4 activity assay was performed as previously described (27).

Briefly, each reaction mixture contained about 0.3 g of recombinant CDK4/cyclin D2 78

holoenzyme and varying concentrations of P16 proteins in 15 L of the kinase buffer (50 mM HEPES, 10 mM MgCl 2, 2.5 mM EGTA, 0.1 mM Na 3VO 4, 1 mM NaF, 10 mM β– glycerolphosphate, 1 mM DTT, 0.2 mM AEBSF, 2.5 mg/mL leupeptin, and 2.5 mg/mL aprotinin). After incubation at 30 ºC for 30 minutes, 50 ng of GST-Rb791-928 and 5 Ci

[γ-32 P] ATP were added in the reaction mixture and the whole reaction mixture was

incubated at 30 ºC for another 15 minutes. Subsequently, proteins in the reaction mixture

were separated by SDS-PAGE, and the incorporation of 32 P into GST-Rb791-928 was

quantitatively evaluated using a PhosphorImager (Molecular Dynamics). The IC 50 value

was defined as the concentration of a kinase inhibitor required to achieve 50% of the

maximal inhibition of CDK4, and measurements were performed in triplicate.

3.4.5 Circular dichroism (CD) analyses.

Recombinant proteins were dissolved in 20 mM sodium borate buffer (pH 7.4)

containing 40 M DTT and dialyzed against this borate buffer at 4 °C overnight.

Samples containing 7.5–10.0 M proteins were incubated with different amounts of

GdnHCl (in a stock solution of 8.5 M) on ice overnight and then equilibrated at 25 °C just prior to CD analysis. The rotation at 222 nm was measured on an AVIV far-UV spectropolarimeter using a quartz microcell (Helma) of 0.1 cm light pass length, and the exact concentrations of GdnHCl were determined using the refractive index. For each sample, three scans were averaged. The ellipticity at 222 nm, an indicator of the existence of α–helical secondary structure was taken as the measure of the degree of structure

present in the protein at each GdnHCl concentration, and the free energy of protein

denaturation in aqueous condition was obtained on the basis of two-state approximation.

Heat-induced unfolding experiments were performed using 10 M proteins in the

borate buffer (pH 7.4) with 1 nm bandwidth and a 10 second response time. Thermal

melting spectra were recorded at 222 nm by heating from 5 °C to 65 °C at the rate of 1 °C

per minute and a 1 °C interval followed by cooling down to 5 °C at the same rate. Tm

was defined as the temperature at the midpoint of transition.

3.4.6 NMR analyses.

All uniform 15 N-labeled gankyrin and P16 proteins, including WT and mutants were prepared as previously described. Each NMR sample contained about 0.4 mM protein, 5 mM HEPES, 1 mM DTT, 5 µM EDTA in 90% H 2O/10% D 2O at pH 7.5.

NMR experiments, including 2D 1H-homonulcear NOESY, 2D 1H-15 N HSQC, and 3D

15 N-edited NOESY, were performed at 27 °C, on a Bruker DRX-600 or a Bruker DRX-

800 spectrometer equipped with a cryoprobe and z-axis gradient. The NOE mixing time was 150 ms. Data were processed with NMRPipe and analyzed with NMRView.

To perform the 2D 1H-15 N HSQC of WT gankyrin, T42A, S75A, and T108A mutants in the presence of GdnHCl, each 15 N-labeled protein was lyophilized and then dissolved in 5 mM HEPES, 1mM DTT, 5 µM EDTA (pH 7.5) with various amounts of

GdnHCl (from a 8.5 M stock solution) in 90% H 2O/10% D 2O. For a specific protein,

each denaturation mixture has a fixed amount of 15 N-labeled protein of about 0.2 mM.

The mixtures were incubated on ice overnight, followed by 2D 1H - 15 N HSQC at 20 °C.

The real GdnHCl concentration in each reaction mixture was determined by measuring

the refractive index as previously described.

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