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

Chapter 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 54 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 Stage I Stage II 95℃, 95℃, 95℃, 95℃, 95℃, 95℃, 55℃, 45 s ~55℃, 45 s (*Tm-5℃)*, 30 s 72℃, 72℃, 72℃, Go to step2, 25 cycles Go to step2, 30 cycles Go to step 2, cycles 72℃, 10 72℃, 10min 72℃, 5min *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. 55 To characterise the binding interactions between the hNck2 SH3 domains and prolinerich peptides, two-dimensional 1H-15N HSQC spectra of the 15 N-labelled SH3 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: (1) Kd = k − [ E ][ L] (2) = k + [ EL] [ EL] ( ET + LT + Kd ) − ( ET + LT + Kd ) − ETLT (3) PEL = = LT LT For protein resonance, PEL represents the fraction population of EL. ET: total protein concentration LT: total ligand concentration Kd: dissocation constant 56 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. δobs =PLδL + PELδEL (4) R2,obs=PER2,E+PELR2,EL (5) PEL = δobs − δL (6) δEL − δL δL: chemical shift at free state δEL: chemical shift at bound state δabs: chemical shift observed Combined with equation (3), δobs − δL ( ET + LT + Kd ) − ( ET + LT + Kd ) − ETLT (7) = δEL − δL LT With known ET, δL, the variant LT changes as the function of δobs, and the two parameters fitting can then be carried out for Kd and δEL. In case of moderately fast exchange, the exchange contribution (the last part of equation (8)) to the dissociation constant should also be considered. The following equation is used, R 2, obs =PLR 2, L + PELR 2, EL + PELPL2 4π(δEL − δL ) (8) k −1 When k-1 values are very large (fast exchange), the equation (8) reduced to equation (5). 57 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 novosynthesized 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 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 15Nlabelled 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. 58 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. 59 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 HSQCTOCSY, and HCCH-TOCSY spectra. With the input of the dihedral angles predicted by TALOS and NOE distances derived from three-dimensional and 13 15 N HSQC-NOESY C 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 [...]... K52 70.8947 D34 63. 751 SH32 _Wrch2 88. 137 7 V27 96.5146 W46 1 23. 238 D 33 124.097 V56 109.97 Q 43 148.82 N41S SH 33 _Wrch2 406.67 L49 1 73. 982 V27 31 8 .39 7 E14 155.492 T24 32 3.478 N 53 198.85 V47 555.6 93 S11 62.49 43 G34 33 8.47 Y 73 57. 939 D34 Average 115.12955 259.146 53 SH32 _mWrch1 80.0857 W35s 186.499 W46 99.7 539 G38 75.5967 L17 114.014 K30 79.196 R37 89.2772 N50 55.44 03 Y51 97.48285 The final average Kd values... Spectra of free (blue) and bound states (red) are superimposed 66 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... for both SH3- 2 domain (Figure 3. 10) and SH3- 3 domain (Figure 3. 11) 15 N NMR backbone 75 Figure 3. 9 CD characterization of the hNck2 SH3- 2 and SH3- 3 domains (a) Far-UV CD spectra of hNck2 SH3- 2 (black) and SH3- 3 (gray) which were collected at a protein concentration of 20 μM in 50 mM phosphate buffer (pH 6.8) at 20 °C on a Jasco J-810 spectropolarimeter (b) Near-UV CD spectra of hNck2 SH3- 2 in the absence... those of SH3- 2, indicating a more restricted motion of the HN bond vector of SH3- 3, as compared with SH3- 2 However, the J(H) values of SH3- 3 are quite close to those of SH3- 2 Due to the fact that high-frequency motion contributes more to J(H) in the situation of high mobility, all residues of SH32 and SH3- 3 are dominated by restricted motions, except for a few residues in the Cterminal of SH3 domains. .. 20 03) In this study, the structures and dynamic properties of the second and third SH3 domains were determined, and the binding profiles were characterized by heteronuclear NMR Spectroscopy and ITC Although both hNck2 SH3 domains adopt the conserved tertiary fold common to all SH3 domains (Figure 3. 3), they have distinctive dynamic properties The SH3- 2 domain is characterised by well-structured 83 state... 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 31 0-helix However, for SH3- 3, the most- perturbed residues were Thr 13 and Glu14 on the RT loop, Glu32, Asn 33, and Asp34 on the n-Src loop, 65 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... 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... acquired for SH3- 2 (Figure 3. 9b) and SH3- 3 (Figure 3. 9c) in the absence and presence of 8 M urea The significant difference between the spectra under the native condition and 8 M urea indicated that like SH3- 2, SH3- 3 also has a very tight tertiary packing, consistent with the presence of very upfield NMR signals at ca -0.5 ppm in the one-dimensional 1 H NMR spectrum However, the SH3- 3 domain was thermodynamically... we conducted further ITC titration studies on the binding of SH3- 2 to mWrch1 and Wrch2, as well as the binding of SH3- 3 to Nogo-A and Wrch2 Figure 3. 8 presents the ITC titration data of mWrch1 -SH32 after subtraction of the blank Except for the result with the SH3- 3Wrch2 interaction which gave rise to a very unusual profile, the remaining data could be fitted to generate thermodynamic binding parameters... 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 . atoms 0.48 0 .37 65 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. superimposition of hNck2 SH3- 2 and Nck1 SH3- 2 domains recently 61 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. 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