NMR study of the human NCK2 SH3 domains structure determination, binding diversity, folding and amyloidogenesis 1

1.1 Ephrin signalling pathway and the roles of ephrins/Ephs 1.1.1 Ephrin signalling pathway The Eph receptor and ephrins are membrane-bound proteins that function as a receptor-ligand

Chapter 1 Introduction

In multicellular organisms, a multitude of different signal transduction processes are required for coordinating the behaviour of individual cells to support the function of the organism as a whole The organism’s sensing of both the external and internal environments at the cellular level relies on signal transduction Eph receptors and ephrins have captured the interest of the biology community in recent years The ephrin signalling pathway plays many important roles in cells: segmentation, axon guidance, fascination, cell migration, angiogenesis, limb development and tumorigenesis Some proteins coordinate with ephrins and Eph receptors in these pathways One of them is Nck2 The Nck2 adaptor protein, which directly binds with the phosphorylation site on the cytoplasmic side of ephrinB/EphB through its SH2 domain, interacts with numerous downstream functional effectors Realising the importance of Nck2 in coordinating indirect ephrin/Eph-effector interactions and eventually a huge assembly to function properly, we chose this particular protein as our target to investigate bindings between SH3 domains that are independent tandem segments and effectors from a structural point of view In this thesis, the SH3 binding mechanism and its abnormal folding process of mutants were investigated through a series of biophysical methods

1.1 Ephrin signalling pathway and the roles of ephrins/Ephs

1.1.1 Ephrin signalling pathway

The Eph receptor and ephrins are membrane-bound proteins that function as a receptor-ligand pair There are 13 Eph receptors and 8 ephrins that have been

identified in mammals (Tuzi and Gullick, 1994; Orioli and Klein, 1997; Pasquale, 1997) Only one Eph receptor and one ephrin are found in Drosophila melanogaster and one Eph receptor and 4 ephrins are found in C elegants (Scully et al., 1999; Wang et al., 1999; Bossing and Brand, 2002) Eph receptors and ephrins can be divided into two groups based on a sequence and homology and binding preference Recent research has indicated that EphA receptor binds preferentially to ephrin-As (with the exception of EphA4), and EphB receptors have a preference to bind with ephrin-Bs However, cross class interactions have been found in specific contexts (Himanen et al., 2004) Ephrin-As and ephrin-Bs exhibit different structural features, because ephrin-As are tethered to the membrane by means of a glycosylphosphatidylinosito anchor, whereas ephrin-Bs span across a membrane with

a cytoplasmic domain In spite of the limited interactions between ephrin-As and ephrin-Bs, they are promiscuous within a class with different Eph receptors binding to

a given ephrin, and vice versa The promiscuity may guarantee functional

redundancies that have been observed in vivo for both ephrin and Eph receptors

(Reldheim et at., 2000)

Eph receptors can be regarded as one member of a superfamily of receptor tyrosine kinases They are autophosphorylated upon binding to their cognate ephrin ligands, and subsequently activate downstream signalling cascades The reverse signalling transduction can also be activated after the ephrin binds to Eph recepors, activating the downstream effector of ephrins Transmembrane ephrins can either be activated through the tyrosine phosphorylation of their cytoplasmic tail or through interaction with various signalling molecules Although the GPI-linked ephrin stimulation is still unclear, they have been shown to activate a member of the Src-family kinases In

addition, oligomerisation and clusting of Eph receptors and ephrins are essential for their signalling function and might be regulated by localisation in membrane microdomains (Cowan and Henkemeyer, 2002; Kullanderand Klein, 2002) Eph- ephrin complexes can progressively aggregate into larger clusters, the size of which might depend on the densities of Eph receptors and ephrins on the cell surface Several weak ephrin-ephrin and receptor-receptor interactions could promote the association of the complexes into an interconnected network (Elena B Pasquale, 2005)

Figure 1.1 Ephrin signalling pathway Eph receptor tyrosine kinases and their ephrin

receptors are recognised to regulate several important processes during development including axon guidance, cell migration, angiogenesis, synaptic plasticity, etc (Image from protein lounge signalling pathway database)

1.1.2 Roles of ephrins and Eph receptors

1.1.2.1 Axon guidance

The function of Eph receptors and ephrin ligands was firstly studied in axon guidance

It was shown that cells expressing Eph receptors avoided territories expressing ephrins, thus providing necessary cues to guide axons to their appropriate target (O’Leary and Wilkinson, 1999) Recently, it was also found that Eph receptors and ephrins can also regulate axon pathfinding through attractive interactions (Knoll et al., 2001; Kullander et al., 2001; Hindges et al., 2002; Mann et al., 2002; Eberhart et al., 2004) and ephrins can act as receptors on navigating axons Henkemeyer proposed that EphB2 acts as a ligand to activate an ephrin-induced reverse signalling and direct the migration of ephrin-expressing (Henkemeyer et al., 1996) Another Eph receptor, EphA4 (which can bind ephrin-B2 and ephrin-B3 in addition to ephrin-As), has also been shown to act as a ligand to control the formation of the anterior commissure tract (Kullander et al., 2001) Ephrin-B3 is a known ligand for EphA4, however no defects

of the anterior commissure were reported in the ephrin-B3 null mice (Kullander et al., 2001), raising the possibility that EphA4 is acting as a ligand for one of the ephrin-As ephrin-induced reverse signalling has also been implicated in retinal axon pathfinding (Birgbauer et al., 2000; Birgbauer,et al., 2000; Birgbauer et al., 2000; Hornberger et al., 1999)

1.1.2.2 Segmentation

Ephs and ephrins have been recognised early for their role in segmentation (Wilkinson, 2000) Initial expression studies of these proteins have shown that several members of both the Eph receptor family and the ephrin family are expressed in a

segmented pattern in the hindbrain and in somites, suggesting that Ephs and ephrins could have a role in segmentation during embryogenesis (Gale et al., 1996) Cells expressing ephrin-B2 are excluded from the Eph receptor-expressing rhombomeres, presumably after the activation of the receptors (Xu et al., 1999) Zebrafish mutant studies, in which the disruption of somite formation is associated with the loss of EphA4 and uniform ephrin-B2 expression in paraxial mesoderm, have shown that reverse signalling is required for the formation of boundaries during somite morphogenesis (Barrios et al., 2003) Forward signalling was shown to be responsible for the epithelialisation of the somite, in an autonomous and non-autonomous manner

It is still unclear whether the repulsion mechanism is utilised in the formation of the boundary in the paraxial mesoderm

1.1.2.3 Cell migration

Eph receptors and ephrins also regulate both cranial and trunk neural crest cell (NCC) migration (Holder and Klein, 1999; Wilkinson, 2000) Similarly, a cell’s autonomous forward signalling has been shown to regulate branchial NCCs’ migration in mice (Adams et al., 2001) It has been suggested that forward signalling in Eph-expressing NCCs was necessary and sufficient for proper branchial arch development, and ephrin-B2 expressed in the neural tube ( in r4 and r6 ) might be involved in the delamination of Eph-expressing NCCs (Adams et al., 2001) Ephrin-B1 is also required for the proper migration of branchial NCCs because mutant mice display a cleft palate, consistent with a defect in NCC (Davy et al., 2004) Ephrin-B1 also acts autonomously in the cell in NCC to regulate their targeted migration, and this reverse signalling involving the binding of PDZ containing protein is required for this function Very recently, forward and reverse signalling have been implicated in the

migration and adhesion of cells involved in the separation of the urorectal region (Dravis et al., 2004) Dravis proposed that the simultaneous activation of forward and reverse signalling in the same cell leads to adhesion, while the unidirectional activation of either forward or reverse signalling leads to repulsion

1.1.2.4 Angiogenesis

Several studies have implicated Eph receptors and ephrins in angiogenesis (Adams, 2002) Studies show that the deletion of ephrin-B2 and ephrin-B4 result in identical phenotypes, characterised by defective angiogenic remodeling (Wang et al., 1998; Adams et al., 1999; Gerety et al., 1999) Based on the fact that EphB4 is expressed on veins, while ephrin-B2 is restricted to arteries, Adams proposed that this receptor/ligand pair might be involved in setting up the arterial and venous identity of blood vessels, possibly by means of repulsion between ephrin-B2 and EphB4 expressing endothelial cells The same authors subsequently provided evidence that ephrin- induced reverse signalling was required for blood vessel remodelling , because the expression of a deleted form of ephrin-B2 lacking the cytoplasmic domain, was unable to rescue the angiogenesis defects associated with the loss of ephrin-B2 (Adams et al., 2001) The role of ephrin-induced reverse signalling in angiogenesis was considered to be quite controversial until recently However, the Cowan study demonstrated that angiogenesis proceeds normally in the absence of the ephrin-B2 cytoplasmic domain, inferring that forward signalling is sufficient for this process (Cowan et al., 2004)

1.1.2.5 GPI-anchored ephrins

GPI-linked ephrins could act as receptors and activate a reverse signalling pathway to regulate epidermal morphogenesis One study showed that GPI-linked ephrins act as receptors in a guidance decision affecting vomeronasal axons (Knoll et al., 2001; Knoll and Drescher, 2002) In stripe assays, vomeronasal axons prefer to grow on Eph receptors rather than a control protein, suggesting that ephrin-A5-expressing axons are attracted towards EphA6 expressing territories (Knoll et al., 2001)

1.2 Nck2 adaptor protein and its SH3 domain

1.2.1 Nck2 adaptor protein

Signal transduction involves the coordinated relay of information from extracellular cues to intracellular effectors The formation of multimeric protein complexes is a critical step in the activation of most intracellular signal transduction cascades In many cases, the proteins consisting of src homology 2 and 3 (domains) are very important in these processes The “adaptor” term is used to describe the features of these proteins that lack intrinsic enzymatic functions and consist almost entirely of SH2 and SH3 domains

Various biochemical and genetic analyses have identified the SH2/SH3 adaptor proteins as critical mediators in the activation of diverse signal responses The abnormal activation of these proteins resulted in developmental defects and the onset

of various abnormalities (Mayer BJ, 1988; Clark SG, 1992; Simon MA, 1993; Garrity

PA, 1996) There are many excellent papers reporting the detailed signal functions of these adaptor proteins, including p58, Grb2, Crk and nck2 The human Nck cDNA was originally cloned by (Lehmann et al., 1990) Using monoclonal antibodies that

Figure 1.2 Grb4 (Nck2) in the ephrin signalling pathway (Nature, 413, 13, 2001)

recognise the melanoma-specific MUC18 antigen, NCK was identified as a false positive during the screening of melanoma cDNA expression In order to identify the SH2-containing proteins, Margolis and colleagues later cloned the murine homolog of Nck, termed Grb4 The human and mouse Nck cDNA encode a 47 kDa protein consisting entirely of SH3 domains and single C-terminal SH2 domains Studies shows that the Nck proteins are expressed in many tissues and cell types (Park D, 1992; Li W, 1992), implying its function in diverse cellular events Nck SH2 Domain associates with the tyrosine phosphorylated proteins including EGP, PDGF, HGF and VEGF Here we should mention that Nck-associated proteins are involved in GTPase activation Many cell surface receptors utilise members of the Ras superfamily of low molecular weight GTP-binding proteins to regulate the activities of multicellular signalling cascades (Vojtek AB, 1995) Williams and colleagues first reported that Nck directly interacts with the SOS GTP exchange factor, resulting in enhanced transcription from a ras-dependant reporter gene This investigation shows that Nck and SOS constitutively associate in quiescent or growth factor stimulated cells via a second Nck SH3 domain and C-terminal proline-rich region of SOS Following PDGF stimulation, Nck and hyperphosphorylated SOS translocate to the activated RTK, leading to SOS-mediate ras activation However, Tanaka et al report that exogenous over-expressions of various dominant negative Nck proteins have little effect on the EGF activation One possible explanation of this discrepancy is that both ras-dependent and ras-independent signalling pathways control ERK activation In addition to the reports of association with ras, several groups have reported a functional link between Nck, the Paks and various Rho family members Two members of the Pak family Pak1 and Pak3 have been shown to be constitutively associated in quiescent or growth factor-stimulated cells with the second Nck domain

(Galisteo ML, 1996; Bagrodia S, 1995; Bokoch G, 1996; Lu W, 1997) Associated Rho-specific GTP exchange factors are most likely to be involved in this process, although the functional relationship between these factors and Nck or the PAKs remains to be determined Together, all data show that Nck proteins have a functional role in the activation of signal events involving members of the Ras superfamily in low molecular weight GTP-binding proteins

Recent evidence from different labs strongly supports the ability of different domains

of the Nck to regulate diverse signal transduction events Not only does it involve the cellular proliferation, but also the regulation of cellular migration and actin cytoskeletal dynamics Mutation studies in WASP show the reduced mobility of lymphoid immune cells, reflecting the fact that WASP and its relatives play a critical role in the organisation of the actin cytoskeleton (Mayer BJ, 2001; Machesky LM, 1999; Mullins RD, 2000)

1.2.1.1 Nck2 interaction with Bcr-Abl

Sunita Coutinho, et al identified Grb4 as an interacting protein using yeast 2-hybrid

in which the Bcr-Abl was used as the bait The interaction was tested both in vitro and

in vivo, and grb4 was an excellent substrate of the Bcr-Abl tyrosine kinase The

association of Grb4 and Bcl-Abl in intact cells was mediated by an SH2-mediated phosphotyrosine-depentent interaction as well as an SH3-mediated phosphotyrosine-indepentent interaction Subcellular localisation studies have shown that Grb4 localises to both the nucleus and cytoplasm A coexpression of kinase-active Bcr-Abl with Grb4 results in the translocation of Grb4 from the cytoplasm and nucleus to the cytoplasm, to colocalise with Bcr-Abl This coexpression also induced a redistribution

of actin-associated Bcr-Abl Sunita Coutinho et al finally concluded that Grb4 in conjunction with Bcr-Abl may be capable of modulating the cytoskeletal structure and negatively interfering with the signalling of oncogenic Abl kinases She also speculated that Grb4 may therefore play a role in the molecular pathogenesis of chronic myelogenous leukemia (Sunita Coutinho et al., 2000)

1.2.1.2 Nck2 acting as a nuclear repressor of transcription

Co-immunoprecipitation experiments have revealed that the co-expression of Grb4 and v-Abl results in a complex formation between v-Abl and Grb4 independent of the kinase activity of v-Abl The interaction was identified as SH3-dependent but independent of the SH2 domain of Grb4 The results showed that an overexpression

of Grb4 results in the significant inhibition of the v-Abl-induced transcriptional activation from the promitogenic enhancer elements such as activator protein 1(AP-1) and the serum-responsive element (SRE) A further mutational analysis revealed that the first two SH3 domains primarily mediate the inhibitory function Co-expression experiments indicated that the inhibitory activity of Grb4 was specific for c-jun/c-fos-regulated promoter elements Finally, based on all these results, Thomas Jahn, et al suggested a novel role for Grb4 in the inhibition of promitogenic enhancer elements,

such as the 12-O-tetradecanoylphorbol-13-acetate-responsive element and the SRE

(Jahn T, 2001)

1.2.1.3 Nck2 interaction with Dock180 – a signalling protein implicated in

regulation of membrane ruffling and migration

Using a yeast 2-hybrid screen, Yizeng, Tu et al identified one new signalling protein, DOCK180, which interacts with Grb4 An SPR analysis showed that the second and third SH3 domain interact with the C-terminal of DOCK180 In terms of binding

affinity, interactions mediated by the individual SH3 were much weaker that that of the full length Grb4 The main binding region of DOCK180 was located at region 1819-1836 In addition, two of the other weak binding sites were pinpointed at the region of 1793-1810 and 1835-1852 respectively Finally, these results showed that a new interaction between DOCK180 and Grb4 had been identified More importantly, the protein complex was mediated by multiple SH3 domains, enhancing the weak interactions mediated by each individual SH3 domain (Tu Y, 2001)

1.2.1.4 Nck2 interaction with focal adhesion kinase (FAK)

Recently, Silvia M Goicoechea et al reported that the Nck-2-FAK complex was mediated by interactions involving multiple SH2 and SH3 domains of Nck-2 The SH2 mediated interaction with FAK was dependent on phosphorylation of Tyr397, which was involved in the regulation of cell mobility An overexpression of Nck2 modestly decreased cell mobility, whereas an overexpression of a mutant form of Nck2 containing only the SH2 domain significantly promoted cell mobility (Goicoechea SM, 2002)

1.2.1.5 Nck2 interaction with PINCH- LIM only protein known to mediate

integrin signal

Recently, Tu et al have identified the Nck2 adaptor proteins as a potential convergence point between integrin signalling pathways and growth factor signalling (Tu Y, 1998) PINCH, which is widely expressed and evolutionally conserved, interacted with the Nck2 The interaction was mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2 (Tu Y, 1998) Also, Algirdas Velyvis et al showed that the PINCH LIM4 domain, while maintaining the conserved LIM scaffold, recognised the third SH3 domain of the Nck2 by using NMR spectroscopy A quite

weak binding affinity was obtained by using the NMR titration method, and got a 3.1

mM Kd value (Algirdas Velyvis, 2003)

1.2.1.6 Nck2 interaction with PDGFR beta regulates actin polymerisation

Min Chen, et al recently reported a specific role for the Nck2 in platelet-derived growth factor-induced actin polymerisation in NIH 3T3 cells Data showed that an overexpression of Nck2 blocked PDGF-stimulated membrane ruffling and formation

of lamellipoda The mutation of the SH2 domain or the middle SH3 domain of Nck2 abolished its interfering effects It was shown that Nck2 bound at Tyr-1009, which was different from the Nck1 binding site and did not compete with phosphatidylinositol-3 kinase for binding to PDGFR Constitutively membrane-bound Nck2 blocked Rac1-L62-induced membrane ruffling and the formation of lamellipodia, suggesting that Nck2 acted in parallel to or down-stream of Rac1 It was the first report of cross talk between receptor tyrosine kinase signalling and the actin cytoskeleton (Chen M, 2000)

1.2.1.7 Nck2 interaction with Tyrosine-phosphorylated Disabled 1 and

redistribution in Reelin-stimulated neurons

Recently, Nck2 was identified as a phosphorylated-dependent Dab1-interacting protein The tyrosine phosphorylation sites of the Disabled1 (Dab1) docking protein were essential for the transmission of the Reelin signal, which regulated neuronal placement The binding sites of Dab1 with Nck2 were identified as Y220 or Y232 Nck2 was coexpressed with Dab1 in the developing brain and in cultured neurons, where Reelin stimulation led to the redistribution of Nck2 from the cell soma into neuronal processes Albena Pramatarova et al found that tyrosine-phosphorylated Dab1 in synergy with Nck2 disrupted the actin cytoskeleton A morphological

experiment indicated that conserved interaction existed between the Disabled and Nck family members The author also proposed a model in which Dab1 phosphorylation led to the recruitment of Nck2 to the membrane, where it acted in remodelling the actin cytoskeleton (Pramatarova A, 2003)

1.2.1.8 Nck2 interaction with TrkB tyrosine kinase receptor

Shingo Suzuki, et al recently identified Nck2 as a binding partner with the TrkB tyrosine kinase receptor by using a yeast 2-hybrid screen The brain-derived neurotrophic factor (BDNF) bound to and activated the TrkB tyrosine receptor kinase

to regulate cell differentiation, survival and neural plasticity in the nervous system Three tyrosines Y694, Y695 and Y771 were believed to be crucial residues for this interaction The data indicated that BDNF stimulation promoted the interaction of Nck2 with TrkB in cortical neurons (Suzuki S, 2002)

1.2.1.9 Nck2 interacts with WASP-Binding Protein WIRE

Recently, Pontus Aspenstrom found that Nck2 interacted with WASP-Binding Protein WIRE which had a role in the regulation of the action filament system WIRE was localised to action filaments in transiently transfected PAE/PDGFRβcells, and in cells simultaneously expressing WIRE and WASP, WIRE relocalised WASP to actin filaments, a relocalisation that required direct interaction between the two proteins A PDGF treatment of cells ectopically expressing WIRE resulted in the formation of peripheral protrusions composed of filopodia and a lamelliodia-like structure All data showed that WIRE had a role in the WASP-mediated organisation of the actin cytoskeleton, and that WIRE was a potential link between the activated PDGF receptor and the actin polymerisation machinery (Aspenstrom P, 2002)

1.2.1.10 Nck2 interaction with wrch1, belonging to Cdc42 subfamily

Jan Saras recently identified that Wrch1 is one of the binding partners of Nck2 The interaction was shown to be mediated via PxxP motifs in the N-terminal of Wrch1 to the second and third SH3 domains of Nck2 Wrch1, which belongs to the Cdc42 subfamily, was one of the least characterised family members The Wrch1 had no

detectable GTPase activity in vitro and its intrinsic nucleotide exchange rate was very

high in comparison with Cdc42 Being tranfected with Wrch1, NIH3T3 cells showed

an up-rounded, retracted phenotype The serum stimulation of cells expressing Wrch1 induced vigorous membrane blebbing, a phenomenon dependent on the activity of ROCK (Jan Saras, 2004)

1.2.2 SH3 domain

1.2.2.1 The history of SH3 domain

SH3 domains were first noted by researchers as regions of sequence similarity between diverging signalling proteins such as the Src family of tyrosine kinase, the Crk adaptor proteins and phospholipase protein (Mayer, 1988) It was soon apparent that this ~60-residue region of similarity is present in many proteins Because the small size of the module seemed to preclude enzymatic activity, the search for function naturally focused on potential protein interactions, and a screening of expression libraries using isolated SH3 domains soon identified seemingly specific binding partners (Cicchetti et al., 1992) Earlier studies indicated that the region bound by SH3 domains is in all cases proline-rich and identified PxxP as a core conserved binding motif (Ren et al., 1993) A host of subsequent studies using

alanine-scanning mutagenesis of known binding sites, phage display, combinatorial chemistry and high-resolution structure determination have revealed the specifics of binding in great detail (Kay et al., 2000) What is clear is that the surface of the SH3 domain bears a relatively flat, hydrophobic ligand-binding surface, which consists of three shallow pockets or grooves defined by conserved aromatic residues The ligand adopts an extended, left-handed helical conformation, termed the polyproline-2 (or PPII) helix The PPII helix has three residues per turn; this means it is roughly triangular in cross-section, and the base of this triangle sits on the surface of the SH3 domain Two of the three ligand-binding pockets of the SH3 domain are occupied by two hydrophobic-proline (φP) dipeptides in register on two adjacent turns of the helix, whereas the third ‘specificity’ pocket in most cases interacts with a basic residue in the ligand distal to the φPxφP core Remarkably, different binding modules such as

WW domains and profilin have converged on very similar modes of interaction with proline-rich, PPII-helical ligands (Kay et al., 2000; Zarrinpar and Lim, 2000)

Figure 1.3 SH3 domain, binding pockets/grooves and binding ligand (A) The structure

shown is based on PDB accession code 1CKB The SH3 domain is depicted in ribbons, with secondary-structural elements shown in different colours and labelled The bound peptide, PPPALPPKKR, is shown in blue with side chains Interface residues on the SH3 domain are shown in pink, for aromatic residues, and in light blue, for non-aromatic residues The locations of the xP grooves and specificity pocket on the SH3 domain are identified by broken

arrows (B) A schematic representation of the same structure to highlight the characteristics of

the ligand-binding surface on the SH3 domain such as the enrichment of aromatic residues

The same colouring scheme is used in (A) and (B) for the purposes of comparison (Biochemical Journal, 2005, Volume 390, 641–653)

1.2.2.2 SH3 binding specificity and affinity

1.2.2.2.1 SH3 ligand consensus sequences

The target specificity of particular SH3 domain is so important issue that we can predict ligands for particular protein of interest and develop ways to inhibit the

interactions in vivo The PPII helix of SH3 ligand has the similar overall structure and

ligand can bind in either of two orientations: class I has general consensus +xφPxφP and class II have the general consensus φPxφPx+ (x stands for any amino acid; + stands for basic residue) (Feng et al 1994; Lim et al., 1994; Mayer and Eck, 1995) The dipeptides occupy different positions on the surface of SH3 domain relative to the helix axis The determinants for binding to φP pockets are a continuous surface presented by the alkylated amide nitrogen of proline residue, and the carbonyl, α carbon and a side chain of preceding residue (Nguyen et al., 1998) The xP dipeptide

is the only naturally occurring amino acid in which two alkylated backbone atoms are separated by one single carbon (Mayer, 2001) The recently published Hck SH3/Ligand complex structure suggests that the class I ligand consensus sequence +xφPxφP should be expanded by a ligand consensus sequence +xxφPxφP (Holger Schmidt, 2007)

1.2.2.2.2 Artificial ligands for SH3 domain

Several groups have used phage display or combinatorial chemistry approaches to identify optimal ligands for particular SH3 domains, and in general have been able to find consensus sequences showing some specificity for a particular domain (Cestra et al., 1999; Cheadle et al., 1994; Feng et al., 1994; Rickles et al., 1994; Sparks et al.,

1994; Sparks et al., 1996) For synthetic peptide ligands, KD values for ‘specific’

ligands tend to be around 10 mM, although in a few cases submicromolar affinities have been reported (Posern et al., 1998) Such studies have served as the foundation for an algorithm based on sequence comparison, contacts identified in crystal structures, and phage-display binding data, which is designed to predict specific binding sites for any SH3 domain from its primary sequence (Brannetti et al., 2000)

1.2.2.2.3 Contribution of non-core residues of SH3 ligand to interaction

Because the core φPxφP scaffold restricts the variability that can be used to generate specificity, efforts have addressed the contribution of a third specificity pocket to the binding specificity of the SH3 domain Although most SH3 domains prefer an arginine residue at this site, the Crk SH3 has high selectivity for class II sites that instead contain a lysine residue (Wu et al., 1995) The Abl SH3, which ironically was the first for which specific targets were identified, is atypical in that it prefers class I ligands in which hydrophobic residues contact the third specificity pocket; this is because it lacks conserved acidic residues found in other SH3 domains, which make specific contact with the arginine residues of typical ligands (Ren et al., 1993; Weng

et al., 1995)

The residues outside binding core region of the PPII helix sometimes contribute to the binding with the RT-loop and n-Src loop of the SH3 domain (Feng et al., 1995) For example, the binding residues for the second SH3 domain of the adaptor Nck have been carefully mapped, indicating a strong reliance on a serine residue downstream of the core class II binding motif (PxxPxRxxS) On the other hand, phosphoserine,

acidic and proline residues are not tolerated in positions immediately next to the terminal in the core motif (Zhao et al., 2000)

C-1.2.2.2.4 Evolutionary balance between affinity and specificity

The binding of the SH3 domain of the Src family kinase Hck to the HIV Nef protein illustrates how regions of a ligand protein distant from the PPII helix can also modulate specificity In this case, a single residue in the RT loop of the Hck SH3 confers high-affinity binding to the native Nef, but not to the isolated PxxP-containing peptide (Lee et al., 1995) After screening a library of mutant Hck SH3 domains (in which six RT loop residues were randomised) by phage display for binding to the native Nef, Saksela and colleagues were able to isolate mutant domains that have up

to a 40-fold higher affinity than the wild-type Hck SH3 (Hiipakka et al., 1999) They also generated mutant SH3 domains that have a very high affinity and selectivity for

an Nef variant bearing a point mutation in the region predicted to contact the RT loop This is an excellent example that it is possible to generate SH3 domains that bind very selectively to a specific target protein This possibly allows binding sites to be blocked while leaving others untouched There must be some evolutionary advantage

to maintaining a relatively low affinity and selectivity for SH3-mediated interaction (Mayer, 2001)

salt bridges involving the LIM4 R197/SH3-3 D257 pair and the LIM4 R198/SH3-3 E233 pair, and one hydrogen bond involving LIM 4 R198/SH3-3 and N250 This interaction was mainly electrostatic; however, aliphatic side chains made some hydrophobic contacts with SH3-3 (Vaynberg J, 2005)

1.2.2.2.6 N-substituted residues

It was proposed that the requirement for proline in the two φP dipeptdies of the φPxφP core might be due to the unique structure of proline: it is the only N-substituted amino acid Nguyen et al constructed synthetic peptoid ligands, in which one or both of the proline residues were replaced with various non-natural N-substituted residues Higher binding affinities and selectivities were observed for these non-natural residues compared with natural peptides (Nguyen et al., 1998; Nguyen et al., 2000) Numerous examples imply that SH3 domains do not generally recognise the entire ring of proline, but only a small portion of the ring near the backbone nitrogen Each groove accommodates two peptide residues The minimal and sufficient requirement at each binding groove is a pair of sequential Ca- and N-substituted residues The Cα/N-substituted pair may be required because, in this arrangement, substituent groups are separated by only a single backbone carbon atom, forming a relatively continuous ridge that can pack efficiently into the domain grooves (Fig 1.4b)

1.2.2.3 SH3, WASP and actin polymerisation

Mayer proposed that SH3-mediated interactions can help recruit the WASP to sites of tyrosine phosphorylation, directly activate the VASP-Arp2/3 complex in the absence

of the GTP-Cdc42, recruit myosin motor and additional Arp2/3-binding sites to sites