Structural studies on DdCAD 1 a ca2+ dependent cell cell adhesion protein

Summary DdCAD-1 is a novel Ca2+-dependent cell adhesion molecule from the social amoeba Dictyostelium discoideum because it lacks a hydrophobic signal peptide and a transmembrane domain

STRUCTURAL STUDIES ON DDCAD-1:

LIN ZHI (M.Sc.)

A THESIS SUBMITTED FOR DOCTOR OF PHILOSOPHY

DEPARTMENT OF BIOLOGICAL SCIENCES

NATIONAL UNIVERSITY OF SINGAPORE

2006

Acknowledgements

I would like to express my sincere appreciation to my enthusiastic supervisor, Associate Professor Yang Daiwen, for his guidance, inspiration, patience and trust throughout this project

Special thanks to Prof Siu Chi-Huang as well as my collaborators, Sriskanthadevan Shrivani and Huang Haibo, from Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto Without their efficient collaboration it would not have been possible for me to complete this project in time

I would also like to express my appreciation to Dr Song Jianxing, Dr Mok Keung and other QE committee members, for their helpful advice and critical suggestions Thank Dr Mok for his kindly providing an over-expression vector used

Yu-in this work Thanks were also due to Dr Fang JYu-ingsong for his assistance Yu-in NMR experiments

I wish to take this opportunity to express my gratitude to my fellow graduates, postdoctoral fellows, friends, brothers and sisters from department of biological sciences and other departments/institutes Their friendship made my research life at the NUS a pleasant learning experience In particular, I’d like to thank B C Karthik,

Chan Siew Leong, Dr Du Ning, Dr, Huang Weidong, Jiang Naxin, Dr K P Manoharan, Liu Yang, Li Kai, Dr Pung Yuh fen, Qiu Wenjie, Ran Xiaoyuan, Dr Ru mingbo, Siu Xiaogang, Wang Wun Long, Xu Xingfu, Xu Ying, Dr Xu Yingqi, Yang Shuai, Dr Zhang Jingfeng, Dr Zhang Xu, Dr Zhang Yonghong, Zhang Yuning, and Zheng Yu for their kind help in experiments and/or data analysis

Although any words are not even enough to express my heartfelt gratitude to

my family in China, I would still like to thank my parents for their sustaining family love and support Praise the Lord for His amazing grace and unfailing love Without this everlasting love, I would not have been able to accomplish or even start this thesis

Lastly, the financial assistance in the form of a research scholarship provided by NUS is gratefully acknowledged

2.1 S TUDIES ON D D CAD-1 OF D ICTYOSTELIUM DISCOIDEUM: A C A 2+

-DEPENDENT CELL ADHESION PROTEIN FROM EUKARYOTE 7

2.1.1 Biological background of D discoideum 7

2.1.1.2 The striking feature of D discoideum development 8

2.1.2.1 Molecular Characterization of DdCAD-1 10

2.1.2.2 Functional characterization of DdCAD-1 13

2.1.2.2.1 Adhesion properties of DdCAD-1 13

2.1.2.2.3 A Novel Transport Pathway 15

2.1.2.2.4 Involvement of DdCAD-1 in the regulation of developmental

2.2 S TRUCTURAL STUDIES ON P ROTEIN S OF M YXOCOCCUS X ANTHUS, A C A 2+

-DEPENDENT SELF - ASSEMBLY MOLECULE FROM PROKARYOTE 19

2.2.2.1 NMR structural studies on protein S 21

2.2.2.2 X-ray structure of Ca 2+ -bound NPS 24

2.2.3 Models for protein S multimerization 25

2.3 S TRUCTURAL STUDIES ON CADHERINS : THE C A 2+ - DEPENDEND CAM S FROM

2.3.1 The cadherin superfamily 25

2.4.2 Advantages of structural studies by NMR 47

2.4.3 The general strategy to NMR structure determination 48

2.4.3.5 Structure calculation and refinement 51

2.4.3.6 Evaluation of structural quality 52

3.2.2 Preparation of plasmid DNA from E coli 56

3.2.3 DNA digestion, DNA fragment purification and ligation 56

3.2.4 DNA sequencing 57

3.3.1 Preparation of E coli competent cells 60

3.3.2 Transformation of E coli competent cells 60

3.4.1 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 61

3.4.4 Protein secondary structure evaluation by Circular Dichroism

3.5 E XPRESSION AND P URIFICATION OF D D CAD-1 64

3.5.2.1 Expression of unlabeled DdCAD-1 64

3.5.2.2 Stable-isotopic labeling of DdCAD-1 65

3.9 NOE ASSIGNMENT , STRUCTURAL CALCULATION AND REFINEMENT 78

3.10 S TRUCTURE - BASED ALIGNMENTS AND S TRUCTURAL COMPARISON 79

CHAPTER 4: RESULTS 81

4.1 E XPRESSION AND PURIFICATION OF D D CAD-1 82

4.1.1 Expression of His-DdCAD-1 in E coli 82

4.1.2 Determination of solubility of over-expressed His-DdCAD-1 82

4.2 I N - VITRO C A 2+ - BINDING PROPERTY OF D D CAD-1 89

4.3 T HERMAL M ELTING P ROPERTIES OF D D CAD-1 93

4.4.1.1 Backbone assignment of Ca 2+ -free DdCAD-1 98

4.4.1.2 Backbone assignment of Ca 2+ -bound DdCAD-1 98

4.4.4 Secondary structure characterization from backbone assignment

109

4.5.1 NMR Structure of Ca 2+ -free DdCAD-1 117

4.5.2 NMR Structure of Ca 2+ -bound DdCAD-1 118

4.6 S TRUCTURAL COMPARISON TO OTHER CELL ADHESION PROTEINS 126

4.7 C A 2+ - DEPENDENT HOMO - ASSOCIATION OF D D CAD-1 IN VITRO 136

4.10 S TRUCTURAL MODEL OF THE C A 2+ - BOUND D D CAD-1 DIMER 147

5.1 H EAT - INDUCED AGGREGATION OF D D CAD-1 153

5.3 M ECHANISM OF C A 2+ - DEPENDENT ADHESION MEDIATED BY D D CAD-1 155 5.4 D ISTINCT ROLES FOR THE TWO DOMAINS OF D D CAD-1 156

APPENDIX I: STATISTICS ON PROTON-PROTON DISTANCES 191 APPENDIX II: REPRESENTATIVE SLICES FROM 13 C & 15 N-EDITED

APPENDIX III: CONSTANT-TIME 13 C- 1 H HSQC OF 13 C, 15 N-LABELED MBP

194 APPENDIX IV: SUMMARY OF ASSIGNMENTS OF AROMATIC

RESONANCES OF SAM, DDCAD-1 AND MBP 195 APPENDIX V: DIFFERENT FUNCTIONS OF THE TWO DOMAINS OF

List of Figures

Figure 2.1 The development of D discoideum and the expression of

CAMs 9

Figure 2.4 Schematic overview of the cadherin superfamily depicting

representative molecules for the respective subfamilies 27

Figure 2.6 The general flowchart for determining solution structures of

and in the presence of Ca2+ (blue) with high absorbance at 280

nm 87Figure 4.5 SDS PAGE of final purified DdCAD-1 88Figure 4.6 The final construct of DdCAD-1 sample 90

Figure 4.8 In-vitro Ca2+-binding properties of DdCAD-1 92Figure 4.9 Thermal denaturation curves of Ca2+-free/Ca2+-bound DdCAD-

Figure 4.10 The effect of temperature on the conformation of Ca2+

-free/Ca2+-bound DdCAD-1 as measured with CD 95

Figure 4.11 CD spectra of Ca2+-free DdCAD-1 after temperature

denaturation 96

Figure 4.12 Apparent hydrodynamic molecular weight (MW) of Ca2+-free

DdCAD-1 aggregate from DLS measurement at 20 ºC 97

Figure 4.13 C , C connectivity for a stretch of residues from P93 to F96 99

Figure 4.14 1H-15N HSQC spectrum of DdCAD-1 acquired at 500 MHz and

30 C on a sample of 0.8 mM protein and pH 6.2 100

Figure 4.15 1H-15N HSQC spectra of DdCAD-1 at 30 ºC and pH 6.2 in the

absence (blue) and presence (red) of Ca2+ 101

Figure 4.16 Representative slices of aliphatic sidechain assignments of

Figure 4.17 Representative slices from the 13C and 15N-edited NOESY 105

Figure 4.18 Constant-time 13C-1H HSQC of 13C, 15N-labeled Ca2+-free

Figure 4.19 Combined 13C /13C chemical shift index plot of Ca2+-free

Figure 4.26 Ramachandran Plot of Ca2+-free DdCAD-1 121

Figure 4.27 Ramachandran Plot of Ca2+-bound DdCAD-1 122

Figure 4.28 Proline conformation 124Figure 4.29 Chemical shift changes induced by Ca2+ binding to Ca2+-free

DdCAD-1 125Figure 4.30 Mn2+-binding properties of DdCAD-1 127Figure 4.31 Mn2+-induced relaxation effects in 1H-15H HSQC spectra of 0.2

mM Ca2+-free DdCAD-1 in the presence 0-100 µM Mn2+ (I) 128Figure 4.32 Mn2+-induced relaxation effects in 1H-15H HSQC spectra of 0.2

mM Ca2+-free DdCAD-1 in the presence 0-100 µM Mn2+ (II) 129Figure 4.33 Mn2+-induced relaxation effects in 1H-15H HSQC spectra of 0.2

mM Ca2+-free DdCAD-1 in the presence 0-100 µM Mn2+ (III) 130Figure 4.34 NMR 1H or 15N chemical shift titration curves for the binding

Figure 4.35 Structural comparisons with protein S and cadherins 133

Figure 4.37 Structure-based sequence alignments 137Figure 4.38 Apparent hydrodynamic radius Rh from DLS measurements at

Figure 4.39 15N R1, 15N R2, and heteronuclear NOEs for DdCAD-1 in the

absence of Ca2+ (red cycle) and in the presence of Ca2+ (black

asterisk) 140Figure 4.40 Gel filtration profiles of subdomains 141

Figure 4.42 Mutations mapped onto the surface of DdCAD-1 145Figure 4.43 CD spectra of representative mutants 146Figure 4.44 Structural model of Ca2+-bound DdCAD-1 dimer 150Figure 4.45 Proposed mechanism for cell-cell adhesion mediated by

List of Tables

Table 4.1 Experimental NMR data and structure statistics for Ca2+-free/bound

DdCAD-1 120Table 4.2 Statistics of structure-based alignments of DdCAD-1, protein S and

Table 4.3 Structural statistics for the 10 lowest-energy Ca2+-bound DdCAD-1

dimers 149

List of Abbreviations

1D/2D/3D/4D One-/Two-/Three-/Four-dimensional

cAMP Cyclic adenosine monophosphate

CD Circular dichroism

D I The N-terminal domain of DdCAD-1

D II The C-terminal domain of DdCAD-1

Da (kDa) Dalton (kilodalton)

DdCAD-1 Dictyostelium discoideum Ca2+ dependent cell adhesion molecule-1

DTT Dithiothreitol

EDTA Ethylenediaminetetraacetic acid

FID Free induction decay

g/mg/μg Gram/Milligram/Microgram

HSQC Heteronuclear single quantum coherence

IPTG Isopropyl- -D-1-thiogalactopyranoside

l/ml/μl Liter/ Milliter/Microliter

min Minute

NOESY Nuclear Overhauser enhancement spectroscopy

NPS The N-terminal domain of protein S

PAGE Polyacrylamide gel electrophoresis

PCR Polymerase chain reaction

ppm Parts per million

RMSD Root mean square deviation

SDS Sodium dodecyl sulphate

TOCSY Total correlation spectroscopy

T1 Longitudinal relaxation time

T1 Spin-lattice relaxation time in the rotating frame

T2 Transverse relaxation time

Tris 2-amino-2-(hydroxymethyl-1,3-propanediol

uv Ultraviolet

Summary

DdCAD-1 is a novel Ca2+-dependent cell adhesion molecule from the social

amoeba Dictyostelium discoideum because it lacks a hydrophobic signal peptide and a

transmembrane domain, which are present in the most of cell adhesion proteins in eukaryotes It is synthesized as a soluble protein and then transported to the plasma membrane by contractile vacuoles In order to understand more how this adhesion molecule functions, we sought to investigate its structural and mechanistic characteristics

In this study, we described the novel features of the NMR solution structures of

Ca2+-free and Ca2+-bound DdCAD-1 in monomeric forms based on multi-dimensional NMR spectroscopy Three Ca2+-binding sites with similar Ca2+-binding affinities were also identified DdCAD-1 contains two β-sandwich domains, resembling –crystallins and cadherins, respectively Distinct binding interactions are ascribed for the two domains of DdCAD-1 The N-terminal domain plays a major role in homophilic binding, while the C-terminal domain tethers the protein to the cell membrane The evolutionary relationship to the proteins with similar structures was also explored Based on structural and mutagenesis analyses, we proposed a model for the Ca2+-bound DdCAD-1 dimer as a basis for understanding DdCAD-1-mediated cell-cell adhesion at the molecular level Our results provide new insights into Ca2+-dependent mechanism for cell-cell adhesion, which may constitute common structural principles underlying Ca2+-dependent cell-cell adhesion in eukaryotic cell

In addition to structural and functional studies, we developed a new strategy for the assignment of aromatic side-chain resonances of 13C, 15N-labeled proteins The aromatic assignment can be achieved using a single 3D 13C- and 15N-edited NOESY experiment on the basis of prior assignment of backbone and aliphatic side-chain resonances About 75% and 57% aromatic CH and NH groups in DdCAD-1 (24 kDa) and MBP (a maltose-binding protein, 42 kDa) were assigned based on the proposed strategy, respectively This strategy can improve the precision of protein structures, especially for large proteins

Chapter 1: INTRODUCTION

Chapter 1: Introduction

Development of eukaryotes is characterized by the coordinated expression of a variety of cell adhesion systems, which provide the mechanical forces that regulate cell shape, cell motility and the formation of three-dimensional tissue structures Cell adhesion molecules (CAM) not only contribute to the formation of specialized junctional complexes that maintain tissue integrity, but also can serve as signaling centers that regulate cell proliferation, differentiation, apoptosis and many important cellular processes Dysfunction of cell adhesion molecules often leads to diseases and abnormalities in fetal development Recent studies of metazoan and non-metazoan adhesion systems have revealed the diversity of roles played by adhesion molecules

as well as common principles that underlie the mechanism and function of this

important class of molecules (Bowers-Morrow et al., 2004)

The social amoeba Dictyostelium discoideum is a non-metazoan related to both

animals and fungi, situated favorably at an evolutionary position close to

Acanthamoebae and the acellular slime mold Physarum polycephalum (Baldauf et al.,

2000; Baldauf et al., 1997) For several decades, Dictyostelium has been a favorable

model organism for multi-faceted study of cell-cell adhesion since it is amenable to

both genetic and biochemical manipulations (Coates et al., 2001)

Early studies distinguished two major classes of cell adhesion sites expressed

during Dictysotelium development (Beug et al., 1973) One class is sensitive to low

concentrations of EDTA, while the other is stable in the presence of 10-15mM EDTA

The EDTA-resistant sites are mediated by csA/gp80 and LagC/gp150 at the

aggregation stage and the mound stage of development (Noegel et al., 1986; Dynes et

al., 1994) Expression of the Ca2+/Mg2+-independent cell adhesion molecule gp80 is induced at the onset of cell aggregation, and becomes maximal at the mid-aggregation stage gp80 molecules preferentially associate with raft-like domains in the plasma membrane and they mediate cell-cell adhesion via a homophilic binding mechanism Upon the formation of loose aggregates, cells express another Ca2+/Mg2+-independent cell adhesion molecule, gp150, which mediates cell-cell adhesion by heterophilic binding and is likely to be involved in the sorting out of prespore cells and prestalk cells

The EDTA-sensitive cell adhesion sites can be divided into two subtypes, the EDTA/EGTA-sensitive adhesion sites and the EDTA-sensitive/EGTA-resistant adhesion sites The EDTA-sensitive/EGTA-resistant sites appear at 2 hours of development and they are probably dependent on Mg2+ (Fontana, 1993) The EDTA/EGTA-sensitive sites are mediated by the cell adhesion molecule DdCAD-1/gp24, which is encoded by the cadA gene and appears soon after the initiation of

development (Brar et al., 1993)

DdCAD-1 is a unique Ca2+-dependent cell adhesion molecule because it lacks a

hydrophobic signal peptide and a transmembrane domain (Wong et al., 1996) It is

synthesized as a soluble protein of 213 amino acids and then transported to the

plasma membrane by contractile vacuoles (Sesaki et al., 1997) DdCAD-1 molecules

on the cell surface can be induced to form ‘caps’ by antibody crosslinking, suggesting

that they are linked to the cytoskeleton by a transmembrane component (Wong et al.,

1996) 125I-Labeled DdCAD-1 binds to cells in a dose-dependent and saturable manner Furthermore, precoating cells with anti-DdCAD-1 Fab fragments blocks the binding of labeled DdCAD-1 to cells, suggesting a homophilic mode of interaction DdCAD-1 shows limited sequence similarities with the spore coat protein, protein

S, of Myxococcus xanthus and classical or type I cadherins Similar to cadherins and

protein S, DdCAD-1 is a Ca2+-binding protein and its adhesive activity is dependent

on Ca2+ Genetic knockout of the cadA gene, which encodes DdCAD-1, not only

results in the loss of the Ca2+-dependent cell-cell adhesion but also gives rise to

aberrant cell sorting and a reduction in spore yield (Wong et al., 2002) These results

indicate that, in addition to cell-cell adhesion, DdCAD-1 plays an important role in

cell type proportioning and pattern formation during Dictyostelium development

However, the structural details of DdCAD-1 and mechanism of its homophilic interaction are not clear Moreover, how Ca2+-binding pocket is constituted and how many Ca2+-binding sites still remain unknown Identifying the number and nature of

Ca2+-binding sites in DdCAD-1 is of general interest because the functions of this molecule are regulated by Ca2+

This study covered the fields from NMR methodology to structural and functional characterization of DdCAD-1 The major objectives of this study were:

1 to develop a new strategy for sequence-specific assignment of aromatic side chain resonances of uniformly 1H, 15N and 13C-labeled DdCAD-1

2 to determine the three dimensional solution structures of Ca2+-free and Ca2+

-bound DdCAD-1

3 to identify Ca2+-binding sites

4 to explore evolutionary relationships of adhesion molecules and the characteristics of their structure-function relationships

5 to investigate homophilic interaction of DdCAD-1 in vitro

6 to model the dimer structure based on mutagenesis study

7 to discover the Ca2+-dependent mechanism of cell-cell adhesion mediated by DdCAD-1

In this thesis, we report the novel features of the NMR solution structures of Ca2+free and Ca2+-bound DdCAD-1 in monomeric forms Distinct binding interactions have been ascribed for the two domains of DdCAD-1 In addition, we describe a model structure for the Ca2+-bound DdCAD-1 dimer as a basis for understanding DdCAD-1-mediated cell-cell adhesion at the molecular level Our results suggest an alternative Ca2+-dependent mechanism for cell-cell adhesion existing in eukaryotic cell, which may constitute common structural principles underlying Ca2+-dependent cell-cell adhesion and provide a new framework for understanding the homophilic interaction

-Chapter 2: LITERATURE REVIEW

-dependent cell adhesion protein from eukaryote

2.2 Structural studies on Protein S of Myxococcus Xanthus, a

from eukaryotes

2.4 Protein structural determination by NMR

Chapter 2: Literature Review

2.1 Studies on DdCAD-1 of Dictyostelium discoideum: a Ca2+ -dependent cell

adhesion protein from eukaryote

2.1.1 Biological background of D discoideum

2.1.1.1 The genome of D discoideum

The amoebozoa are notable as representing one of the earliest branches from the last common ancestor of all eukaryotes Each of the existing branches of the crown group of eukaryotes provides an example of the ways in which the ancestral genome has been adapted by gene duplication, divergence and deletion Very recently, the

genome of D Dictyostelium, as the best-studied member of the group of amoeba and the first free-living protozoan, has been fully sequenced (Eichinger et al., 2005) The

gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts A proteome-based phylogeny shows that the amoebozoa diverged from the animal–

fungal lineage after the plant–animal split, but Dictyostelium seems to have retained

more of the diversity of the ancestral genome than have plants, animals or fungi The

complete protein catalog of Dictyostelium provides a new perspective for studying its cellular and developmental biology Systematically, Dictyostelium provides a level of

complexity that is larger than the yeasts, but much simpler than plants or animals Hence, detailed molecular analyses in this system may disclose control networks that are hard to investigate in more complex systems, and may further portend regulatory

strategies used in higher organisms

2.1.1.2 The striking feature of D discoideum development

The soil-dwelling social amoeba, Dictyostelium discoideum, has been studied for

the past 5 decades, which has contributed significantly to the understanding of

cellular motility, signalling and interaction (Kessin, 2001) Dictyostelium has a

simple and well-defined life cycle (Figure 2.1a) that permits biochemical, genetic, and cell biological analysis of the dynamic processes during multicellular

development (Otto et al., 2001; Weijer, 1999; Siu et al., 2004) Dictyostelium cells

have adopted a strategy for multicellular development that differs from that of

metazoa In their vegetative stage, Dictyostelium cells are single-celled amoebae that feed on bacteria and multiply by binary fission The striking feature of Dictyostelium

amoebae is that, when their food source is depleted, they undergo a switch in behavior to form a fruiting body This is a highly differentiated multicellular structure composed of spore cells supported by a skeleton of stalk cells that are arranged as a stalk and a basal disc, which anchors the stalk to the substratum Although the fruiting body possesses only a small number of cell types, its development shows much of the complexity as seen in metazoa There is one fundamental difference: animals develop

from a single cell by a combination of growth and differentiation Dictyostelium

development, however, requires no growth, and multicellularity is achieved by aggregation of many unicellular amoebae This greatly simplifies the study of development and provides an easy route to examine cell-cell adhesion through studies

Figure 2.1 The development of D discoideum and the expression of CAMs (a)

Amoebae proliferate as single cells during the growth phase Upon starvation, cells undergo chemotactic migration in response to cAMP During aggregation, cells coalesce into adherent cell ‘streams’ that eventually come together to form the mound, the first stage of multicellular development The mound compacts to form

a tight aggregate and then develops a ‘tip’, which coordinates further development After extension to form the first finger, the developing structure either immediately forms a fruiting body, the process of culmination or forms a motile slug that migrates to seek conditions favourable for culmination Scale shows relative timing of development Expression of CAMs during development is

shown below (from Coates et al., 2001; Siu et al., 2004) (b) Schematic drawing

showing contractile vacuole–mediated transport of DdCAD-1 from the cytosol to

the plasma membrane (from Sesaki et al., 1997).

b

a

of aggregation and cell-surface binding

During Dictyostelium development, cell multicellularity is maintained by the

expression of several cell adhesion systems The pioneering work of Gerisch (1961) led to the discovery of two classes of cell–cell adhesion sites based on their sensitivity

to EDTA Subsequent studies have particularly divided the adhesion systems into four types as follows

i) A Ca2+-dependent (EGTA/EDTA-sensitive) adhesion system mediated by DdCAD-1/gp24 is expressed soon after the initiation of development (Figure 2.1a)

ii) A Mg2+-dependent (EDTA-sensitive/EGTA-resistant) adhesion system is expressed a couple of hours after the initiation of DdCAD-1

iii) A Ca2+-independent/Mg2+-independent (EDTA/EGTA-resistant) adhesion system mediated by gp80 appears at the onset of cell aggregation, cells acquire iv) A second Ca2+-independent/Mg2+-independent (EDTA/EGTA-resistant) adhesion system mediated by LagC/gp150 is expressed on cells in the early post-aggregation stage

2.1.2 Characterization of DdCAD-1

2.1.2.1 Molecular Characterization of DdCAD-1

DdCAD-1, also named contact sites B (csB) or gp24 (Beug et al., 1973; Yang et

al., 1997), is encoded by the cadA gene that contains two introns Its full-length

cDNA was cloned by immunological screening of gt11 expression libraries (Wong,

ATGTCTGTTGATGCAAATAAAGTAAAATTCTTCTTTGGTAAAAACTGCACTGGT 54

M S V D A N K V K F F F G K N C T G 18 GAATCATTTGAATACAACAAAGGTGAAACTGTAAGATTCAACAATGGTGATAAA 108

E S F E Y N K G E T V R F N N G D K 36 TGGAATGATAAATTCATGTCATGTTTGGTTGGTTCAAATGTTAGATGTAACATT 162

W N D K F M S C L V G S N V R C N I 54 TGGGAGCATAATGAAATTGATACTCCAACTCCAGGAAAATTCCAAGAATTGGCT 216

W E H N E I D T P T P G K F Q E L A 72 CAAGGCAGTACAAACAATGATTTAACCTCAATAAATGGTCTTTCAAAGTTCCAA 270

Q G S T N N D L T S I N G L S K F Q 90 GTCTTACCAGGAGCTTTTCAATGGGCAGTTGATGTTAAAATTGTCAACAAAGTT 324

V L P G A F Q W A V D V K I V N K V 108 AATTCAACTGCTGGTTCATATGAAATGACCATTACTCCATACCAAGTTGATAAG 378

N S T A G S Y E M T I T P Y Q V D K 126 GTTGCTTGCAAAGATGGTGATGACTTTGTTCAATTGCCAATTCCAAAACTTACT 432

V A C K D G D D F V Q L P I P K L T 144

CCACCAGATTCTGAAATTGTTAGCCATTTAACAGTTCGTCAAACACATACACCA 486

P P D S E I V S H L T V R Q T H T P 162 TATGACTATGTTGTAAATGGAAGTGTTTACTTTAAATACTCCCCAACAACTGGC 540

Y D Y V V N G S V Y F K Y S P T T G 180 CAAGTTACAGTTATTAAAAAAGATGAGACATTCCCAAAGAATATGACTGTTACA 594

Q V T V I K K D E T F P K N M T V T 198 CAAGATGATAATACATCTTTCATCTTTAACTTAAACTCTGAAAAATAA 639

Q D D N T S F I F N L N S E K * 213

Figure 2.2 Sequence of DdCAD-1 (a) The nucleotide sequence of the DdCAD-1

cDNA and its deduced amino acid sequences (b) Hydropathy profile of DdCAD-1

(from Wong et al., 1996)

a

b

residues than basic ones, giving rise to a negatively charged protein with a predicted isoelectric point (pI) of 5.30 (Figure 2.2a) Although DdCAD-1 has been shown to be

a cell adhesion molecule associated with the plasma membrane, the Kyte & Doolittle (1982) hydropathy profile of DdCAD-1 indicates a typical soluble protein(Figure 2.2b) The amino-terminal region is relatively hydrophilic and does not appear to contain a signal peptide sequence The entire protein has only two short hydrophobic segments at amino acid positions 40–50 and 85–105 The first segment is too short to span a lipid bilayer, and the second segment contains several charged residues and has

a relatively low hydrophobicity index to qualify for a transmembrane domain Therefore, the hydrophobicity plot of DdCAD-1 predicts a soluble cytosolic protein, rather than an integral membrane protein

DdCAD-1 shows sequence similarities with the spore coat protein, protein S, of

Myxococcus xanthus and classical or type I cadherins (Wong et al., 1996) Sequence

alignment shows that the first 97 residues of DdCAD-1 can be aligned with the terminal domain of protein S with 70% sequence similarity The full length DdCAD-1 shows 19% sequence identity and 43% sequence similarity with protein S DdCAD-1 shows a slightly lower degree of sequence identity (14~15%) with members of the cadherin family The carboxy-terminal half of the protein shows limited but significant sequence similarities with the extracellular (EC) repeats of classical cadherins Similar to E-cadherin, DdCAD-1 contains multiple Ca2+-binding sites and mediates Ca2+-dependent cell–cell adhesion by homophilic binding These findings suggest that both protein S and DdCAD-1 could be primitive members of the

N-cadherin superfamily

2.1.2.2 Functional characterization of DdCAD-1

2.1.2.2.1 Adhesion properties of DdCAD-1

Knecht (1987) first reported that DdCAD-1 (gp24) is involved in early adhesion

of Dictyostelium discoideum The localization of DdCAD-1 at the cell surface was

shown by quantitative binding of the anti-DdCAD-1 antibodies to intact cells To demonstrate the cell binding activity of DdCAD-1, the binding of solubilized DdCAD-1 to intact cells was examined 125I-Labeled DdCAD-1 bound to cells in a dose dependent and saturable manner, and the binding was displaced specifically by unlabeled DdCAD-1 Purified DdCAD-1 was capable of inhibiting the re-association

of dispersed cells previously undergoing EDTA-sensitive aggregation Moreover, precoating cells with anti- DdCAD-1 IgG and Fab fragments blocked the binding of

125

I-labeled DdCAD-1 to cells Collectively, these in vitro assays provide direct

evidence that DdCAD-1 is a cell adhesion molecule that functions through a homophilic mode of interaction The binding of DdCAD-1 to cells is sensitive to EGTA/EDTA, suggesting that the activity of DdCAD-1 may involve calcium ions Direct binding studies showed that 45Ca2+ could bind to DdCAD-1 blotted onto nitrocellulose membrane In addition, pre-incubation of the native protein with calcium ions resulted in a shift in its gel mobility Therefore DdCAD-1 is characterized to mediates cell-cell interactions via a Ca2+-dependent mechanism,

rendering DdCAD-1 the first cell adhesion molecule in D.discoideum to utilize a

Ca2+-based adhesion system

2.1.2.2.2 Expression of DdCAD-1

DdCAD-1 is the first adhesion molecule to be expressed during development and

is not seen in cells growing on bacteria It appears soon after the beginning of starvation and it accumulates rapidly during the first four hours of development

(Knecht et al., 1987; Yang et al., 1997) DdCAD-1 expression is stimulated by the

prestarvation factor (PSF, such as autocrine) that signals nutrient depletion during vegetative growth in axenic cultures and by nanomolar pulses of cAMP during

development (Rathi et al., 1991) The promoter region of the cadA gene contains

several cAMP-response elements, similar to the ones found in the csaA gene that

encodes gp80 (Desbarats et al., 1992; Wong et al., 2002) Developing cells were also

found to secrete a large protein complex called counting factor (CF), which is part of

a negative feedback loop that regulates the expression of DdCAD-1 and gp80

(Roisin-Bouffay et al., 2000) It is possible that CF may utilize signaling pathways

that involve cAMP Although CF indeed affects the cAMP-induced cAMP and cGMP pulses, changing the size of exogenous cAMP pulses administered to cells does not phenocopy the effects of CF on cell–cell adhesion, suggesting that CF regulates

DdCAD-1 and other CAMs via a pathway independent of cAMP pulses (Tang et al.,

2001)

At the onset of development, DdCAD-1 is present uniformly throughout the cytoplasm As the cells enter the aggregation stage, high levels of DdCAD-1 partition

to the cell periphery and become associated with F-actin-containing membrane protrusions, such as filopodia and lamellipodia that emanate from the leading edge of

migrating cells (Sesaki et al., 1996) DdCAD-1 is enriched at initial

filopodia-mediated cell–cell contacts, and more stable contacts are formed with the subsequent entry of gp80 Interestingly, DdCAD-1 does not remain in cell–cell contacts but re-distributes away as gp80 moves into the contact regions The re-distribution of

DdCAD-1 may reflect the dynamic nature of Dictyostelium morphogenesis, which

requires weaker interactions that permit the constant “breaking” and “re-making” of contacts as the cells migrate to form large aggregates The contrasting distribution patterns of DdCAD-1 and gp80 are especially apparent in multicellular streams and early aggregates, where DdCAD-1 is absent in the interior region but present at high levels at the periphery These observations suggest that DdCAD-1 is involved primarily in the recruitment of cells into cell streams, while gp80 maintains contact stability among cells Nevertheless, how DdCAD-1 is excluded from cell–cell contacts by gp80 is not known At the post-aggregation stage, DdCAD-1 is absent from cell–cell contact regions of cells However, the cellular level of DdCAD-1 remains more or less constant throughout the second half of the developmental cycle and it is present predominantly in the cytoplasm It remains to be elucidated how DdCAD-1 is internalized The unique distribution of DdCAD-1 suggests that, in addition to cell–cell adhesion, it may participate in other developmental processes

2.1.2.2.3 A Novel Transport Pathway

A substantial amount of DdCAD-1 is present on the cell surface and an enrichment of DdCAD-1 in intercellular contacts is also observed, consistent with the role of a cell-cell adhesion molecule These observations thus raise the question of how DdCAD-1 is transported and anchored to the cell surface since DdCAD-1 lacks

of a signal peptide or transmembrane domain Sesaki (1997) provided the first evidence for a nonclassical protein transport mechanism that uses contractile vacuoles, which are intracellular organelles and known to function exclusively in osmoregulation in cells, to target a soluble cytosolic DdCAD-1 to the cell surface Immunofluorescence microscopy and subcellular fractionation revealed a preferential association of DdCAD-1 with contractile vacuoles Proteolytic treatment of isolated contractile vacuoles degraded vacuole-associated calmodulin but not DdCAD-1, demonstrating that DdCAD-1 was present in the lumen The use of hyperosmotic conditions that suppressed contractile vacuole activity led to a dramatic decrease in DdCAD-1 accumulation on the cell surface and the absence of cell cohesiveness Shifting cells back to a hypotonic condition after hypertonic treatments induced a rapid increase in DdCAD-1–positive contractile vacuoles, followed by the accumulation of DdCAD-1 on the cell membrane 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a specific inhibitor of vacuolar-type H+-ATPase and thus of the activity of contractile vacuoles, also inhibited the accumulation of DdCAD-1 on the cell surface

Furthermore, an in vitro reconstitution system was established, and isolated

contractile vacuoles were shown to import soluble DdCAD-1 into their lumen in an ATP-stimulated manner Based on these morphological and biochemical evidences, a

novel path for DdCAD-1 transportation was proposed (Figure 2.1b) DdCAD-1 is transported to the cell surface via contractile vacuoles, which are normally involved

in osmoregulation As the vacuoles fuse with the plasma membrane to release its content, DdCAD-1 moves out of the vacuole membrane and becomes part of the plasma membrane Therefore, the accumulation of DdCAD-1 on the cell surface is influenced by the osmolarity of the environment

In Dictyostelium, DdCAD-1 is secreted into the medium throught contractile vacuoles during development (Siu et al., 1997) Normally, only a portion of DdCAD-

1 molecules is associated with the lumenal surface of contractile vacuoles Upon fusion with the plasma membrane, the unbound DdCAD-1 is released into the

medium as a soluble protein (Sesaki et al., 1997) Since DdCAD-1 mediates cell–cell

adhesion by binding homophilically, excessive DdCAD-1 in the medium has an adhesive effect, which can be eliminated by incubation of the conditioned medium

anti-with an anti-DdCAD-1 antibody (Siu et al., 1997) Therefore, membrane-bound

DdCAD-1 has an adhesive role, while secreted DdCAD-1 may serve to modulate the adhesive interactions

2.1.2.2.4 Involvement of DdCAD-1 in the regulation of developmental events

Genetic manipulations, such as over-expression or knockout mutation of the protein during vegetative growth, have confirmed the function of DdCAD-1, as well

as gp80 and gp150, in cell–cell adhesion Detailed analyses of the phenotype of

knockout mutants have revealed additional developmental roles for DdCAD-1 (Wong

et al., 2002)

First, cadA − cells display defects in morphogenesis and culmination is delayed by

6 h Although cadA − strains are capable of completing the developmental cycle and

can form mature fruiting bodies, the spore yield is reduced by 50% Cell-type analysis shows a 2.5-fold increase in prestalk cells in the slug stage The wild-type

phenotype is restored when null cells are transfected with a cadA-expression vector

These results implicate a role for DdCAD-1 in cell-type proportioning and cell differentiation Since DdCAD-1 is completely internalized in the post-aggregation stage, it is likely that DdCAD-1 functions intracellularly The Ca2+-binding properties

of DdCAD-1 raise the possibility that the cytosolic DdCAD-1 is involved in the regulation of Ca2+ homeostasis by sequestering Ca2+ Thus, the loss of DdCAD-1 in

cadA − cells can lead to higher levels of free Ca2+

in the cytosol As high levels of free

Ca2+ have been associated with a propensity to prestalk cell differentiation (Saran et

al., 1994; Cubitt et al., 1995; Schlatterer et al., 2001), the aberrant cell-type

proportioning seen in cadA − cells may be related to increased levels of free Ca2+

2.1.3 Summary

Ca2+-dependent cell-cell adhesion mediated by DdCAD-1 has been well-studied

for the past two decades Similar to other CAM (Yap et al., 2003), DdCAD-1 also

play roles in signaling networks that regulate cell-type proportioning, cell differentiation, and cell sorting However, the knowledge of the underlying mechanisms is still very limited Future studies in this area will be important for a

better understanding of the dynamic adhesive and cellular processes during

discoideum development

DdCAD-1 also resembles other Ca2+-dependent adhesion proteins, especially protein S and cadherins, in many aspects, which strongly suggests that DdCAD-1 could be structurally and functionally related to these proteins The following two sections will review structural studies on protein S from prokaryote and cadherins from eukaryotes

2.2 Structural studies on Protein S of Myxococcus Xanthus, a Ca2+ -dependent

self-assembly molecule from prokaryote

2.2.1 Biological context

Myxococcus Xanthus is a Gram-negative soil bacterium that is an excellent

prokaryotic system for the study of the regulation of development (Inoye et al., 1979; Inouye et al 1981; Teintze et al., 1991) It has a complex life cycle including a

temporal sequence of cellular aggregation, mound formation and myxo-sporulation

(Kaiser, 1979; Dworkin et al., 1985; Shimkets, 1987) It displays social behavior similar to that of Dictyostelium discoideum, a eukaryotic mound When nutrients are

depleted in a solid culture medium, cells begin to undergo aggregation to form mounds The rod-shaped vegetative cells then convert to round or ovoid spores During development, protein S is induced and accumulated in very large amounts Protein S is an acidic (pI 4.5) heat-stable Ca2+-binding protein It is synthesized as a soluble protein that is transported in some way to the outer surface of cells and

assembled into a spore surface coat During fruiting body formation, protein S synthesis constitutes a major component of total soluble protein synthesis Purified protein S will be able to self-assemble around protein S-deficient spores in the presence of Ca2+ Each spore can contain about 2.5×106 molecules Thus protein S is

probably the most abundant protein produce during development in M.xanthus Since

protein S is not synthesized in vegetative cells or in cells induced with glycerol, its production is likely to be strongly developmentally regulated

Protein S appears to be quite different from the spore coat proteins found in

Gram-positive bacteria, which are highly crosslinked by SS bonds (Aronson et al., 1976) The full functions of protein S inside the developing M.xanthus cell still remain

largely unknown Electron micrographs of spores containing reconstituted surface coats support the notion that protein S may be necessary for cell-cell interaction during fruiting body formation It may function together with other spore coat

proteins such as myxobacterial hemagglutinin (Nelson et al., 1981), proteins C (McCleary et al., 1991) and protein U (Gollop et al., 1991) by forming protective,

multilayer spore surface assemblies which additionally act as a cell-cell adhesive in a

calcium-dependent manner (Inouye et al., 1979)

The gene encoding protein S of 173 amino acids is separated by a short spacer

from a gene which encodes a protein isoform called protein S1 (Teintze et al., 1985; Downard et al., 1984) The two proteins have 88% sequence identity In contrast to protein S, synthesis of protein S1 is induced late in the M.xanthus developmental

cycle at the onset of sporulation Protein S1 must therefore be retained in the cell,

which is already surrounded by the impenetrable spore coat These two proteins may

be able to partially substitute for one another in their intracellular functions (Teintze

et al., 1985)

2.2.2 Structures of protein S

2.2.2.1 NMR structural studies on protein S

The three-dimensional solution structure of Ca2+-bound full-length protein S was

first determined using multi-dimensional heteronuclear NMR spectroscopy (Bagby et

al., 1994a,b,c) Protein S consists of two -sandwich domains (residues 1-86 and

92-173) connected by a short linker (Figure 2.3a) The overall folds of the two domains are essentially identical and the N-terminal domain is better defined than the C-terminal one Each domain comprised of two similar Greek-key motifs and a short -helix The Greek key motifs form two -sheets, each sheet consisting of strands a, b and d from one motif and strand c from the other motif The two sheets face one another to form a wedge arrangement, with an angle of ~70° between the pairs of sheets in the N-terminal and the C-terminal domains The protein S fold is similar to that of the -crystallins and can be clustered into the same protein superfamily

(Blundell et al., 1981; Wistow et al., 1983; Wistow et al., 1985; White et al., 1989; Bax et al., 1990) The presence of -helices is the major difference between the

protein S and B -crystallin (Figure 2.3b) This superfamily, including spherulin 3a, was also thought to be an evidence for evolutionary pragmatism where an ancient fold was able to adapt different functions in different organisms

Figure 2.3 Structures of Protein S (a) A ribbon representation of the

solution structure of full-length Protein S from Myxococcus Xanthus (PDB

ID: 1PRS); (b) Linear sequence alignment, based on the most highly

conserved residues (marked with vertical dashed lines), of protein S (S) and

B-crystallin (G) (from Bagby, 1994c); (c) A ribbon representation of the

crystal structure of the N-terminal domain of protein S complexed with

Ca2+(PDB ID: 1NPS) Calcium ions are shown as spheres

a

b

c

Figure 2.3a also shows two putative Ca2+- binding sites in the N-terminal and the C-terminal domains These two binding sites occupy similar positions relative to the backbone skeleton of the respective domains One molecule of protein S is supposed

to bind two Ca2+ ions, with apparent dissociation constants in the range from 27 -76

µM (Teintze et al., 1988) The side chain oxygen atoms of each of Glu10 and Glu71,

and their C-terminal domain homologues, Glu99 and Thr159, are proposed to ligate

Ca2+ based on a search of the protein S structure for clusters of backbone and chain oxygen atoms suitable for Ca2+ binding These two sites are located at the molecular surface and have no structural similarity to those of calmodulin However,

side-no experimental evidences were obtained to support these two Ca2+-binding sites in the full-length protein S

In comparison with the Ca2+-bound protein S, approximately 20% of backbone amide cross peaks in the 1H-15N HSQC spectrum of Ca2+-free protein S disappeared and many of the remaining peaks significantly shifted to random-coil frequencies The loss of structural integrity in the absence of Ca2+ suggests that the role of Ca2+ is

at least to partly stabilize the structure Later studies using NMR and optical spectroscopy demonstrate that the Ca2+-free N-terminal domain of protein S (NPS)

forms two equilibrium folding intermediates that are in slow exchange (Bagby et al.,

1998) The intermediates arise from differential calcium-dependent folding of subdomains which are not contiguous along the polypeptide chain The structures of these intermediates are incompatible with several previously proposed folding

mechanisms for Greek key -barrel domains (Richardson et al., 1977; Haynie et al.,

1993) A hypothesis of the folding mechanism for the N-terminal domain of protein S was further proposed, which involves three nucleation sites (α-helix, EF -hairpin, and tyrosine corner) for folding and sequential acquisition of native long-range interactions Interaction between the sites stabilizes the native-like structure of the calcium-insensitive subdomain Folding of the calcium-dependent subdomain requires calcium binding, which stabilizes the subdomain and reduces charge repulsion between carboxylates of calcium-ligating residues

2.2.2.2 X-ray structure of Ca 2+ -bound NPS

The crystal structure of the N-terminal domain of protein S (NPS) has also been also determined by X-ray diffraction at 1.8 Å resolution, using molecular replacement

with the NMR structure as a search model (Wenk et al., 1999) The overall topology

of NPS was nearly the same as in intact protein S (Figure 2.3c) Two Ca2+-binding sites that bind two Ca2+ ions were determined in NPS The first site is made up by the oxygen atoms of the side-chains of Ser80 and Asn37, the backbone carbonyl-oxygen atoms of Gln54 and Asn78, and one water molecule; the second one consists of sidechains of residues Ser40 and Asn77, the carbonyl-oxygen atoms of Thr38 and Tyr8, and another water molecule These two binding sites are totally different from the putative ones proposed by Bagby (1994c) The high-resolution X-ray structure of NPS provides a significant new insight into the structural details of Ca2+-binding sites

and these findings agree with the mutagenesis studies (Teintze et al., 1988)