Protein stabilization by specific binding of guanidinium to a functional arginine-binding surface on an SH3 domain

Abstract

Guanidinium hydrochloride (GuHCl) at low concentrations significantly stabilizes the Fyn SH3 domain. In this work, we have demonstrated that this stabilizing effect is manifested through a dramatic (five- to sixfold) decrease in the unfolding rate of the domain with the folding rate being affected minimally. This behavior contrasts to the effect of NaCl, which stabilizes this domain by accelerating the folding rate. These data imply that the stabilizing effect of GuHCl is not predominantly ionic in nature. Through NMR studies, we have identified a specific binding site for guanidinium, and we have determined a dissociation constant of 90 mM for this interaction. The guanidinium-binding site overlaps with a functionally important arginine-binding pocket on the domain surface, and we have shown that GuHCl is a specific inhibitor of the peptide-binding activity of the domain. A different SH3 domain possessing a similar arginine-binding pocket is also thermodynamically stabilized by GuHCl. These data suggest that many proteins that normally interact with arginine-containing ligands may also be able to specifically interact with guanidinium. Thus, some caution should be used when using GuHCl as a denaturant in protein folding studies. Since arginine-mediated interactions are often important in the energetics of protein-protein interactions, our observations could be relevant for the design of small molecule inhibitors of protein-protein interactions.

Figure 1.

Chevron plots of (A) wild-type Fyn SH3 domain and (B) its R40N mutant in 0.4 M GuHCl. (•) Chevron plots in the absence of GuHCl.

Figure 2.

The correlation between ΔG_u and kinetic m_ku values for the wild-type Fyn SH3 domain (•) and the R40N mutant (▴) at different concentrations of GuHCl. The ΔG_u values plotted were determined at the GuHCl concentrations shown in Table 1.

Figure 3.

Overlays of ¹⁵N-¹H correlation maps collected for wild-type Fyn SH3 domain samples containing (A) 0–0.85M GuHCl and (B) 2 M urea or 1MNaCl. The peak marked with an asterisk in A corresponds to a residue in the C-terminal 6-his tag region of the construct that shifted significantly upon GuHCl addition. Fitting the data from this peak yielded a K_d value of 560 ± 70 mM. This K_d value is only threefold different from that reported for the nonspecific protein :GuHCl interactions and is unlikely to report on a specific interaction between the 6-his tag and GuHCl. (C) Magnitude of total baseline-corrected peak shifts (ΔΩ = [ΔΩ_H² + ΔΩ_N²]^1/2), color coded on a ribbon-representation of the Fyn SH3 domain (generated using the coordinates of the X-ray crystal structure [Noble et al. 1993] and the program MOLMOL [Koradi et al. 1996]).

Figure 4.

The guanidinium binding curves determined from NMR peak shift analysis. The fraction of protein bound is plotted as a function of GuHCl concentration with best-fit curves calculated according to Equation 2. Values were obtained from analyses of shifts in peak position in the ¹H dimension. Results shown are for four different residues as indicated in the figure.

Figure 5.

The interactions of the conserved arginine in SH3 domain ligands. (A) The guanidino moiety of the arginine side chain is involved in forming hydrogen bonds with the side chains of T14, T16, and D17 in the Src SH3 domain. (B) The aliphatic moiety of the arginine side chain donated by the target is also closely stacked against the side chain of W36. Figures were generated using the PDB file 1QWF (Feng et al. 1995) and the program PyMOL (DeLano Scientific). (C) The consensus sequence of phage display targets that have affinity for the Fyn SH3 domain is given along with the sequence of the Vsl12 ligand and the position nomenclature. The symbol X in the consensus sequence is any residue, and Z denotes hydrophobic residues. The conserved arginine residue at the P₋₃ position is underlined.

Figure 6.

The effect of GuHCl on the affinity of the wild-type Fyn SH3 domain (hatched bars) and the T14R mutant (open bars) for the Vsl12 target peptide. Black bars indicate the effect of arginine on the affinity of the wild-type Fyn SH3 domain for this ligand. The reported “fold change” in K_d was calculated by normalizing the K_d value obtained at any concentration of GuHCl or arginine to the value obtained in the absence of GuHCl or arginine. Depicted error bars in each case indicate fitting errors. The K_d of T14R in 0.4 M GuHCl was not determined. The interaction between wild-type Fyn SH3 domain and target peptide in 0 M GuHCl has a K_d value of 0.22 ± 0.01 μM.

Figure 7.

The influence of arginine and guanidinium binding on the affinity of the SH3 domain for its target peptide. Affinity of the wildtype Fyn SH3 domain for target peptide is plotted at corresponding concentrations of arginine and GuHCl.

Figure 8.

Influence of GuHCl on the T_m of the wild-type Fyn SH3 domain (black bars), the Sho1 SH3 domain (hatched bars), and the Abp1 SH3 domain (open bars). All of the ΔT_m values are with respect to the T_m in 0 M GuHCl. A negative ΔT_m value is indicative of destabilization. Error bars indicate uncertainties associated with fitting denaturation profiles to the appropriate equations as described in Materials and Methods. The Sho1 SH3 domain did not exhibit cooperative unfolding at GuHCl concentration of 0.4 M or higher. The wild-type Fyn, Abp1, and Sho1 SH3 domains in 0 M GuHCl have T_m values of 76.9 ± 0.11, 53.43 ± 0.20, and 47.75 ± 1.37 (°C), respectively.