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

Chapter 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 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 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). Ephephrin 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 and (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 rasdependent 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 10 the transverse relaxation rate on the strength(ωeff or 1/τcp) of the refocusing RF field defines a relaxation dispersion profile, from which exchange rates, populations and differences in chemical shift can be extracted by a curve fitting(A.G. Palmer, 2001). CPMG relaxation dispersion experiments in which exchange effects are quenched through the application of pulses are often the method of choice for the study of millisecond dynamics in protein (A.G. Palmer, 2001). A similar quenching effect can be achieved by applying a continuous radio-frequency field that is allowed to vary both in magnitude and in frequency, and this approach is used to study faster processes (kex up to 100000s-1). Many recent NMR studies have provided evidence for dynamics on the microsecond timescale in structural regions of biomolecues that are known to have an in vivo function, such as macromolecular recognition and ligand binding (Mulder FAA, 2001; Volkman BF, 2001; van Tilborg PJ, 1999; Mittermaier A, 1999; Malmendal A, 1999; Ishima R, 1999; Feher VA, 1999; Kristensen SM, 2000; Lu J, 2000; McIntosh PB, 2000; Rozovsky S, 2001; Zhu L, 2001; Katahira M, 2001; Botuyan MV, 2001) and cytalysis (Hoogstraten CG, 2000; Osborne MJ, 2001; Wang L, 2001; Cole R, 2002; Eisenmesser EZ, 2002). 1.3.2.2 Measuring dynamics on the pico- to nanosecond time scale Consider a protein that is tumbling in solution such that every orientation is equally probable. When internal motions and molecular tumbling cause the reorientation of the 1H-15N bond vector with respect to the external magnetic field, the local magnetic field at the site of the 15 N spin that derives from the directly attached 1H magnetic dipole will fluctuate. It can be shown that, although the local dipolar interaction 29 between 1H and 15 N spins averages to zero because of the molecular tumbling, the time-dependent variations in the field lead a spin system that has been perturbed by radio frequency pulses to return or relax to a thermal equilibrium. Because the fluctuations of the local magnetic fields are sensitive to internal motions, a measurement of the NMR relaxation rates provides a direct way of extracting dynamics parameters. The 15 15 N relaxation experiments monitor either the recovery of N Z-magnetisation to its equilibrium postion (T1), or the decay of magnetisation orthogonal to the Z-axis to its zero equilibrium value (T2). The 1H spin has a magnetogyric ratio (γ) that is ten times larger than that of 15 N, and the inherent sensitivity of the NMR experiment scales are as γ5/2; therefore, experimental sensitivity is optimised by shuffling magnetisation from an amide 1H to its directly coupled 15N and then back again to 1H for detection as a 2D data set. Here, one peak is obtained for each (1H-15N) pair in the protein, with an intensity proportional to exp(-τ/Ti), where Ti is that T1 or T2 value of the particular 15 N mu cleu s an d τ is a variable relaxation delay; relaxation times are measured by recording a series of spectra and fitting the peak intensities as a funcation of τ. The values of Ti so obtained are usually interpreted in terms of generalised order parameters that describe the amplitude of bond vector motions, but specific models can be used as well (T. Bremi, 1997). In addition to these experiments, many experiments that provide complementary information to the 15N studies described above have been developed, such as those that measure the correleated motions of successive residues in proteins (P. Pelupessy, 2003; P. Lundstrom, 2005) or the dynamics of backbone carbonyl-Cα bonds (T.Wang, 2005). 30 Side chain motions in proteins are also amenable to study with the use of spin relaxation techniques (D. M. LeMaster, 1996; D. R. Muhandiram, 1995). In particular, the use of 2H spectra as a probe of side chain methyl group dynamics has seen substantial advances in recent years. Due to the poor resolution of 2H spectra, uniformly 13C labelled, fractionally deuterated protein need to overcome the obstacle. One exciting application of the methodology described above is in the use of order parameters as restraints in molecular dynamics calculations to produce an ensemble of structures that satisfies both structural and dynamical NMR data. 1.3.2.3 Relaxation data analysis Nuclear magnetic relaxation data on macromolecules in solution contain information concerning the nature of internal motions that are of fundamental importance for their biological functions, stability and folding. 15N and 13C NMR relaxation measurements provide unique experimental data for the characterisation of intramolecular motions in a wide range of time scales from pico- to milliseconds. The experimental transverse and longitudinal relaxation rate and NOEs can be expressed in terms of the spectral density function J(ω), which is the Fourier transformation of the reorientation correlation function C(t). In the model-free approach, the dynamic information on fast internal motions contained in an NMR experiment is essentially specified by two parameters: a generalised order parameter, S, which is a measure of the degree of the spatial restriction of motion, and an effective correlation time τe, which is measure of the rate (time scale) of the motion. These two parameters were defined in a modelindependent way. For both isotropic and anisotropic overall motion, expressions for appropriate spectral density in terms of S and τe were derived without invoking a specific model for internal motion. 31 The observable quantities in the NMR relaxation experiment are determined by certain linear combinations of spectral density, J(ω), evaluated at different frequencies. J(ω) is given by ∞ J (ω ) = ∫ C (t ) cos ωtdt (1) where C(t) is the appropriate correlation function. Given that the three experimentally determined parameters R1, R2 and NOE depend on the spectral density function at five different frequencies, it is not possible to calculate the spectral density values at thee frequencies without an assumption about the form of the spectral density function. This problem has been approached in two ways, the reduced spectal densiy mapping, in which the relaxation rates are directly translated in the spectral density at three different frequencies, and second by use of a physical model to describe the spectral density function. In the model-free approach, C(t) = C0(t)C1(t) (2) where C0(t) describes the overall motion and C1(t) is the correlation function for internal motion, given by C1(t) = S2 + (1-S2)e-t/τe (3) where S2 (0[...]... that the second and third SH3 domain interact with the C-terminal of DOCK180 In terms of binding 12 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 18 19 -18 36 In addition, two of the other weak binding sites were pinpointed at the region of 17 93 -18 10 and 18 35 -18 52 respectively Finally, these... between the activated PDGF receptor and the actin polymerisation machinery (Aspenstrom P, 2002) 15 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... 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, 6 41 653) 18 1. 2.2.2 SH3 binding specificity and affinity... 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. .. motion, the spectral density is given as follows: J (ω ) = (1 − A) S 2τ 2 A (1 − S 2 )τ ' (1 − A) (1 − S 2 )τ ''  2  AS 2τ 1 + + +   5 1 + (ωτ 1 )2 1 + (ωτ 2 ) 2 1 + (ωτ ') 2 1 + (ωτ '') 2  (10 ) where τ = τ e 1 + τ 1 1 and τ = τ e 1 + τ 2 1 ' 1 '' 1 The first step in the analysis of experimental data is to establish the nature of overall motion For isotropic macromolecular motions, τM can be determined... to the SH3 ligand binding, selective specificity and protein stability 1. 3.3 Folding study of SH3 domain by NMR NMR spectroscopy is the ideal method for determining the structural details of unfolded and partially folded states of proteins that are difficult to study through other methods NMR spectroscopy can be applied not only directly for characterising disordered states of proteins populated at the. .. motions and molecular tumbling cause the reorientation of the 1H -15 N bond vector with respect to the external magnetic field, the local magnetic field at the site of the 15 N spin that derives from the directly attached 1H magnetic dipole will fluctuate It can be shown that, although the local dipolar interaction 29 between 1H and 15 N spins averages to zero because of the molecular tumbling, the time-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... 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., 19 93; Weng et al., 19 95) 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. .. pathways and growth factor signalling (Tu Y, 19 98) 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, 19 98) 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 . length Grb4. The main binding region of DOCK180 was located at region 18 19 -18 36. In addition, two of the other weak binding sites were pinpointed at the region of 17 93 -18 10 and 18 35 -18 52 respectively 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. 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