NMR study of the human NCK2 SH3 domains structure determination, binding diversity, folding and amyloidogenesis 3

To characterise the binding interactions between the hNck2 SH3 domains and rich peptides, two-dimensional 1H-15N HSQC spectra of the 15N-labelled SH3 proline-domains were acquired with a

Chapter 3

Structural insight into binding diversity of hNck2 SH3

domains

Part of this chapter has been published in:

Liu J, Li M, Ran X, Fan JS, Song J

Structural insight into the binding diversity between the human Nck2 SH3 domains

and proline-rich proteins Biochemistry 2006 Jun 13;45(23):7171-84

PMID: 16752908

3.1 Materials and Methods

The hNck2 SH3-2, SH3-3 and peptides DNA fragments were obtained by PCR-based

de novo design under procedures in Table 3.1 The primers used are listed in the

supplementary section of TableS.1

Table 3.1 PCR parameters for SH3 de novo design

SH3

Peptides

5 Go to step2, 25 cycles Go to step2, 30 cycles Go to step 2, 2 cycles

*Tm of overlapping region of forward and reverse primer pair

The expression and purification of proteins/peptides were described in chapter two, except that SH3-2 was further purified on the Mono-S ion exchange

To characterise the binding interactions between the hNck2 SH3 domains and rich peptides, two-dimensional 1H-15N HSQC spectra of the 15N-labelled SH3

proline-domains were acquired with a protein concentration of ~100 μM in the absence or

presence of peptides at a molar ratio of ~1:2 (SH3:peptide) as previously described If peptides bind with SH3 domains after titrations, the shifted residues of SH3 were assigned by superimposing the HSQC spectra in the absence and presence of the peptides The degree of perturbation was evaluated by an averaged chemical shift according to the formula [(Δ 1H)2 + (Δ15N/4)2]1/2 (ppm) The obtained averaged chemical shifts were then plotted against molar ratios and fitted to equation (3) by the script developed by K Gardner (freedom7.swmed.edu/NMRview/titration.html) to

estimate the dissociation constant (Kd)

The following descriptions will be couched primarily in terms of chemical shift changes, which are most widely used for the measurement of dissociation constants:

For protein resonance, PEL represents the fraction population of EL

ET: total protein concentration

LT: total ligand concentration

Kd: dissocation constant

When the exchange rate is very fast, the chemical shifts and relaxation rates of the nuclei of the ligand and the protein are completely averaged between the bound and free states and the weighted average spectrum is observed

δL: chemical shift at free state

δEL: chemical shift at bound state

δabs: chemical shift observed

Combined with equation (3),

3.2 Result

3.2.1 Gene Synthesis and Protein and Peptide Production

A high protein expression level was achieved in E coli BL21 cells for the de

novo-synthesized DNA constructs encoding hNck2 SH3 domains and the proline-rich peptides The entire 380-residue hNck2 protein was constructed and expressed as a soluble protein, although it precipitated above a concentration of 1 mg/mL This has indicated that the presence of the C-terminal SH2 domain or/and the loop linking the third SH3 domain to the SH2 domain might enhance the solubility of the first SH3 domain Moreover, to assess its structural properties of two domains as a whole, hNck2 (115-256), which included both SH3-2 and SH3-3, was also expressed In the HSQC of hNck2 (115-256), cross peaks corresponding to residues of SH3-2 domain can be well superimposed to those of SH3-2, whereas cross peaks corresponding to residues of SH3-3 underwent slight shifts We have also labelled hNck2 (115-256) with 15N and 13C and recorded a series of triple-resonance spectra Unfortunately, the spectra could not be assigned due to the extensive disappearance of resonance peaks, because of the conformational exchange on the microsecond to millisecond time scale Comparison of the binding profiles between the isolated and connected domains with the Nogo peptides showed no significant difference Consequently, in this study, we put our focus on the SH3-2 and SH3-3 domains and have successfully generated 15N-labelled and 15N- and 13C-labelled samples for the determination of NMR structures as well as study of their interactions with nine proline-rich peptides as listed in Figure3.1

Figure 3.1 Sequence alignments of SH3 and PPII peptides The upper panel shows the

alignment between Nck1 and Nck2 and the SH3s in Nck2 Secondary structure elements are also schematically presented The lower panel shows the alignment of PPII peptides Interactions between PPII peptides and the SH3 domain are shown as red arrows

3.2.1 NMR Structure Determination of the Second and Third hNck2 SH3 Domains

As shown in Figure 3.1, the hNck2 SH3-2 domain consists of 57 residues while the SH3-3 domain contains 59 residues Backbone assignments of both SH3 domains were successfully achieved for all non-proline residues with the analysis of a pair of triple-resonance experiments, CBCA(CO)NH and HNCACB, and the assigned HSQC spectra are shown in Figure 3.2 Side chain carbon and proton assignments were also completed for most residues on the basis of analysis of CCCONH, 15N-edited HSQC-TOCSY, and HCCH-TOCSY spectra With the input of the dihedral angles predicted

by TALOS and NOE distances derived from three-dimensional 15N HSQC-NOESY and 13C NOESY experiments as well as two-dimensional NOESY experiments for aromatic side chain connectivity, the NMR structures of the hNck2 SH3-2 and SH3-3 domains were calculated by CYANA and further refined by CNS Table 3.2 summarizes the constraints used and structural statistics for the 10 lowest-energy NMR structures of both domains accepted by the CNS protocol, with distance violations of <0.2 Å and dihedral angle violations of <5° Figure 3.3 shows the superimposition of the 10 selected structures of the SH3-2 and SH3-3 domains Both hNck2 SH3 domains have a conserved tertiary architecture adopted by all SH3 domains consisting of five β-strands organized into two β-sheets packed at right angles Between β-strands 1 and 2 are the long RT loops arranged in an anti-parallel manner, and between β-strands 2 and 3 are the shorter n-Src loops (Figure 3.3A and D) Both RT and n-Src loops were less defined than the secondary structure regions,

as evident from the large backbone rms deviation over these regions (Figure 3.3B and E) The four-residue fragment starting from S52 to V55 between strands 4 and 5 assumes a 310-helix characteristic of all SH3 domains Figure 3.3C shows a superimposition of hNck2 SH3-2 and Nck1 SH3-2 domains recently

Figure 3.2 HSQC and sequential assignments of SH3-2 and SH3-3 A shows the HSQC

and sequential assignment of SH3-2 B shows the HSQC and sequential assignment of SH3-3 Both spectra were acquired on an 800 MHz NMR spectrometer in a 50 mM phosphate buffer (pH6.8) at 20℃ The assigned residues are labelled in the spectra

Figure 3.3 Three-dimensional structures determined by NMR spectroscopy (A)

Superimposition of the ten lowest-energy NMR structures of the hNck2 SH3-2 domain in ribbon (A) and sausage (B) modes (C) Comparison of the structures of the hNck2 SH3-2 domain (blue) and Nck1 SH3-2 domain (2CUB, green) (D and E) Superimposition of the ten lowest-energy NMR structures of the hNck2 SH3-3 domain in ribbon (D) and sausage (E) modes (F) Comparison of the structures of the hNck2 SH3-3 domain (blue) and 1WX6 (yellow) with additional extensions, as well as the 1U5S (green) complexed with the LIM4 domain Three characteristic regions previously established to be critical for binding affinity and specificity are indicated; namely, the RT-Src loop, the n-Src loop and the 3 10 -helix

deposited (PDB entry 2CUB) Overall, the two structures are very similar, with a backbone rms deviation of ~1.35 Å over the secondary structure regions while the characteristic 310-helix is missing in the Nck1 SH3-2 structure, instead with a helix-like loop in this region Furthermore, the structure of the hNck2 SH3-3 domain determined here was also compared with the structure solved recently (PDB entry IU5S and 1WX6, in Figure 3.3F) Although both 1WX6 and 1U5S included extended sequences at both termini, the three structures are very similar, with a pairwise backbone rms deviation of ~1.00 Å over the secondary structure regions The main difference between the hNck2 SH3-3 structure determined here and 1U5S in complex with the LIM4 domain is over the N- and C-terminal regions, probably because 1U5S

is a complex structure which uses a non-classic region that consists of N- and

C-termini to bind LIM4 in an ultra-weak manner (with a Kd of 3 mM)

Table 3.2: NMR Constraints and Structural Statistics of the Ten

Lowest-Energy Structures of the Second and Third hNck2 SH3 domains

Second SH3 domain Third SH3 domain Restraints for calculation

Final energies (kcal/mol)

3.2.2 HSQC identification of the residues involved in binding of the hNck2 SH3

Domains

The interactions between hNck2 SH3 domains (SH3-2 and SH3-3) and their preferred proline-rich peptides were evaluated by HSQC perturbation experiments with a molar ratio of 1:2 (SH3: peptide) In fact, four peptides, cWrch1, WIRE, CAP-1, and CAP-2, showed no detectable binding to the second or third SH3 domain, indicating that these peptides may bind the first SH3 domain because they were all previously demonstrated to interact with the Nck2 protein On the other hand, the mWrch1 peptide was found to specifically bind the SH3-2 domain, while the Nogo-A and Prk2 peptides bind the SH3-3 domain exclusively On the basis of HSQC titration, the Wrch2 and SOS1 peptides were able to bind both SH3 domains The chemical shift changes induced by peptides at a molar ratio of 1:2 were measured by superimposing the HSQC spectra of the SH3 domains in the free state and in the presence of the peptides and the averaged chemical shifts are shown in Figure 3.5A Generally for both SH3 domains, the significantly affected residues were located over three regions, namely, the RT loop, the n-Src loop, and the 310-helix, consistent with the previously accepted notion that both the affinity and the specificity of the SH3-ligand interaction are mainly mediated by an interfacial region constituted by residues scattered over these three regions These residues in the spatial close proximity constitute three pockets designated as p1, p2 and p3 which are shown in Figure 3.5A However, a closer examination revealed that the two SH3 domains have distinctive sets of binding perturbed residues As shown in Figure3.4 and Figure 3.5a, for the SH3-2 domain, the significantly affected residues included Val5, Phe7, Arg13, Asp15, and Glu16 on the

RT loop, Lys30, Cys31, and Gly34 on the n-Src loop, and Phe47, Ser49, and Tyr51 close to or on the 310-helix However, for SH3-3, the most- perturbed residues were Thr13 and Glu14 on the RT loop, Glu32, Asn33, and Asp34 on the n-Src loop,

Figure 3.4 HSQC perturbation experiments of SH3-3 SH3-3 were titrated by

DOCK180(A), WIRE(B), Prk2(C), SOS1(D), nWrch1(E) and Wrch2(F) peptides with a

molar ratio 1:2 Spectra of free (blue) and bound states (red) are superimposed

Figure 3.5 Δδ as function of residue in perturbation experiments and electrostatic potential surface presentations of SH3-2 and SH3-3 A and B show the Δδ (see Chapter two)

as a function of the residue of SH3-2 and SH3-3, respectively E and F show the electrostatic potential surface of SH3-2 and SH3-3, respectively P1 and P2 stand for Pocket 1 and Pocket

2, which are labelled accordingly for SH3-2 and SH3-3 P3 stands for the specific pocket that normally binds to positively charged residues in the C-terminal of PPII peptides

and Lys52 on the 310-helix It appears that for SH3-2, more hydrophobic residues were involved in ligand binding, while for SH3-3, more hydrophilic, particularly negatively charged residues such as Asp and Glu were engaged in binding Interestingly, upon introduction of the Wrch2 peptide which is capable of binding both SH3 domains, the most drastically perturbed residues were located on the 310-helix, namely, a hydrophobic/aromatic residue (Tyr51) for SH3-2 and a positively charged residue (Lys52) for SH3-3

To gain further insight into the binding of Nogo-A peptide with SH3-3 identified in this study, we labelled these peptides with 15N and subsequently monitored its HSQC peak shifts induced by adding an excess of the unlabelled SH3-3 domain The results indicated that upon binding the majority of the HSQC peaks shifted (Figure 3.6) In particular, HSQC cross-peaks of Nogo-A Ala5 and Arg8 were significantly shifted

As judged from its sequence, Nogo-A peptide belongs to the class II SH3 ligands which have a consensus sequence of xPx#PxR While x can be any amino acid, # is a hydrophobic residue (usually Leu, Pro, or Val) The significant shift of the Arg8 peak was anticipated because this residue was thought to play a critical role in binding with SH3 domains through charge-charge interactions By contrast, Ala5 with a dramatic peak shift was not commonly found at that position of the class II ligands Interestingly, Prk2, another high affinity ligand for the hNck2 SH3-3 domain, also has

an Ala at that position (Figure 3.1) This observation implied that the Ala residue at this position might play a pivotal role in specifying the ligand preference for the hNck2 SH3-3 domain

Figure 3.6 HSQC perturbation experiment of Nogo-A peptide The Nogo-A peptide was

15 N labelled and titrated with unlabelled SH3-3 proteins The spectrum before adding the SH3-3 protein (blue) and the spectrum after adding the SH3-3 (red) are superimposed The residues are labelled accordingly

3.2.3 K d determination through HSQC titration

A series of titration experiments were carried out to fit the Kd curves for SH3-2 and SH3-3 with different ligands Four series titration experiments were performed, which included SH3-2-mWrch, SH3-2-Wrch2, SH3-3-Sos1 and SH3-nogo A series of titrations with corresponding molar ratios ( protein:ligand): 1:0, 1:0.5, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:12 and 1:14, were carried out to obtain chemical shift changes at different ligand concentrations for SH3-3-Nogo; and 1:0, 1:0.5, 1:1, 1:1.5, 1:2.0, 1:2.5, 1:3, 1:4 and 1:6 for the SH3-2-mWrch1 Figures 3.7A and B show the superimposition of three selected HSQC spectra at a molar ratio of 0, 1:1 and 1:14 of SH3-3-nogo, and a molar ratio of 0, 1:1, 1:6 of SH3-2-mWrch1, respectively The SH3-2-Wrch2 and SH3-3-Wrch2 titration HSQC spectra are not shown here The fitting equation used in the Kd value fitting is listed below (a full description is included in the Materials and Methods section):

Figure 3.7 Series of titration of SH3 and fitting curves A and B show the superimposition

of spectra with only three different titration ratios for SH3-3 and SH3-2, respectively The small boxes show the traces of E14 and W35S in the series of titration experiments C and D show the final fitting curves of most perturbed residues in SH3-3 and SH3-2

δav = δf + (δb-δf)*{(L+PT+Kd) - ((L+PT+Kd)2-(4*L*Kd))1/2}/(2*L)}

L = Ligand concentration

PT = Total protein concentration

Kd = Equilibrium dissociation constant

δav = Observed chemical shift

δf = Chemical shift change at free state

δb= Chemical shift change at bound state

The fittings that were generated by GRACE are shown in Figures 3.7 C and D Different colours are correlated to different residues selected by the fitting script Four series of Kd values were fitted and are listed in the following Table 3.3

Table 3.3 K d Values determined by NMR fitting method

49.7307 L49 88.1377 V27 406.67 L49 80.0857 W35s 67.704 E32 96.5146 W46 173.982 V27 186.499 W46 68.8609 E14 123.238 D33 318.397 E14 99.7539 G38 66.3663 N33 124.097 V56 155.492 T24 75.5967 L17 57.0062 L17 109.97 Q43 323.478 N53 114.014 K30 63.1266 T13 148.82 N41S 198.85 V47 79.196 R37

338.47 Y73 57.939 D34 Average

The final average Kd values after 50 iterations fitting were 63.751, 115.130, 259.147 and 97.483 μM for the SH33-Nogo, SH32_Wrch2, SH33_Wrch2 and SH32-mWrch1, respectively In order to fully saturate the binding between SH3 and PRP during titration, a molar ratio as high as 1:14 was reached in each peptide titration experiment Interestingly, most cross-peaks of the SH3 domain were saturated except for the Trp side-chain cross peaks