Ligand-induced changes in dynamics in the RT loop of the C-terminal SH3 domain of Sem-5 indicate cooperative conformational coupling

Abstract

We report the effects of peptide binding on the (15)N relaxation rates and chemical shifts of the C-SH3 of Sem-5. (15)N spin-lattice relaxation time (T(1)), spin-spin relaxation time (T(2)), and ((1)H)-(15)N NOE were obtained from heteronuclear 2D NMR experiments. These parameters were then analyzed using the Lipari-Szabo model free formalism to obtain parameters that describe the internal motions of the protein. High-order parameters (S(2) > 0.8) are found in elements of regular secondary structure, whereas some residues in the loop regions show relatively low-order parameters, notably the RT loop. Peptide binding is characterized by a significant decrease in the (15)N relaxation in the RT loop. Concomitant with the change in dynamics is a cooperative change in chemical shifts. The agreement between the binding constants calculated from chemical shift differences and that obtained from ITC indicates that the binding of Sem-5 C-SH3 to its putative peptide ligand is coupled to a cooperative conformational change in which a portion of the binding site undergoes a significant reduction in conformational heterogeneity.

Figure 1.

Assigned ¹H-¹⁵N HSQC spectra of Sem-5 C-SH3 without peptide (green) and with peptide (red), taken using a 750-MHz Varian spectrometer at 25°C.

Figure 2.

(A) The magnitude of differences in ¹H (top graph) and ¹⁵N (bottom) chemical shifts for each residue between the free and complexed state. (B) Color mapping of the ¹H chemical shift difference in the NMR minimized average structure with the peptide overlayed in the minus orientation and the same position as seen in the X-ray structure. (C and D) Binding curves of chemical shift changes for sample of residues (Ser170, 362 ± 46 μM and Asn206H₂N^e, 427 ± 53 μM in ¹⁵N dimension; Ser205, 397 ± 36 μM and Leu173, 355 ± 34 μM in ¹H dimension) as a function of peptide concentration. Lines are drawn as the best-fitted lines as calculated (see text). T156 and Q160 are residues not involved in binding and do not show chemical shift changes during the titration of the ligand.

Figure 2.

(A) The magnitude of differences in ¹H (top graph) and ¹⁵N (bottom) chemical shifts for each residue between the free and complexed state. (B) Color mapping of the ¹H chemical shift difference in the NMR minimized average structure with the peptide overlayed in the minus orientation and the same position as seen in the X-ray structure. (C and D) Binding curves of chemical shift changes for sample of residues (Ser170, 362 ± 46 μM and Asn206H₂N^e, 427 ± 53 μM in ¹⁵N dimension; Ser205, 397 ± 36 μM and Leu173, 355 ± 34 μM in ¹H dimension) as a function of peptide concentration. Lines are drawn as the best-fitted lines as calculated (see text). T156 and Q160 are residues not involved in binding and do not show chemical shift changes during the titration of the ligand.

Figure 3.

Calorimetric titration of Sem-5 C-SH3 domain with the Sos peptide (Ac-VPPPVPPRRR-NH₂) in 50 mM NaOAc and 100 mM NaCl, and 10 mM CaCl₂ (pH 4.8) at 25°C.

Figure 4.

Representatives of the worst (L162, E169, E155, G201) and the best (N206, E172, W191, D187) fits of ¹⁵N−T₁ (A) and T₂ (B). Fitting errors are <2% and <1% for the worst and best fits, respectively. Duplicate points are taken for each time delay and shown in the same graph.

Figure 5.

¹⁵N-relaxation data for Sem-5 C-SH3 domain both in the unliganded (open circles) and liganded state (filled circles). Elements of secondary structure are the same for both states. β-Sheets are represented as arrows, 3₁₀ helix by a cylinder, loops by straight black lines, and turns by bent lines.

Figure 6.

(A) Order parameters (S₂) profile both for the free (open circles) and complexed (filled circles) C-SH3 domain with Sos peptide. (B) Difference between the order parameters of the free and complexed C-SH3 domain.

Figure 7.

(A) Internal correlation times (τ_e) profile both for the free and complexed C-SH3 domain with Sos peptide. (B) Residues, which exhibit conformational exchange (R_ex), both for the free and complexed C-SH3 domain.

Figure 8.

Space-filling model representation of the crystal structure determined (Lim et al. 1994b) for Sem-5 C-SH3 (gray) with and without the Sos peptide (yellow). Residues belonging to the binding interface but showing no dynamic changes in the picosecond timescale on binding are shown in green, and residues in red are those showing dynamic changes. Residues shown in blue (S170 and G171) are those that do not interact with the peptide in the complex, but which nonetheless experience significant changes in chemical shift and dynamics on binding.