Electrostatic Effects in the Folding of the SH3 Domain of the c-Src Tyrosine Kinase: pH-Dependence in 3D-Domain Swapping and Amyloid Formation

The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form intertwined dimers and amyloid fibrils at mild acid pHs. In this work, we show that a single mutation of residue Gln128 of this SH3 domain has a significant effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils. The Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acid pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates under any of the conditions assayed. We have also solved the crystallographic structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to the hexagonal space group P6522 and the asymmetric unit is formed by one chain of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0, crystals belong to the orthorhombic space group P212121, with two molecules at the asymmetric unit showing the characteristic fold of the SH3 domain. Analysis of these crystallographic structures shows that the residue at position 128 is connected to Glu106 at the diverging β-turn through a cluster of water molecules. Changes in this hydrogen-bond network lead to the displacement of the c-Src-SH3 distal loop, resulting also in conformational changes of Leu100 that might be related to the binding of proline rich motifs. Our findings show that electrostatic interactions and solvation of residues close to the folding nucleation site of the c-Src-SH3 domain might play an important role during the folding reaction and the amyloid fibril formation.This research was funded by the Spanish Ministry of Science and Innovation and Ministry of Economy and Competitiveness and FEDER (EU): BIO2009-13261-C02-01/02 (ACA); BIO2012-39922-C02-01/02 (ACA); CTQ2013-4493 (JLN) and CSD2008-00005 (JLN); Andalusian Regional Government (Spain) and FEDER (EU): P09-CVI-5063 (ACA); and Valentian Regional Government (Spain) and FEDER (EU): Prometeo 2013/018 (JLN). Data collection was supported by European Synchrotron Radiation Facility (ESRF), Grenoble, France: BAG proposals MX-1406 (ACA) and MX-1541 (ACA); and ALBA (Barcelona, Spain) proposals 2012010072 (ACA) and 2012100378 (ACA)

Hydrogen-bond distances in the water network at the distal loop and diverging β-turn.

<p>Hydrogen-bond distances in the water network at the distal loop and diverging β-turn.</p

Overall fold of the monomeric structure of the c-Src-SH3 domain.

<p>Overall fold of the monomeric species of the WT c-Src-SH3 domain (WT_M, PDB code 4JZ4). The AU is composed by two chains of the SH3 domain; both chains are represented as a cartoon (white). The n-Src loop residues in chains A and B are shown in red. In chain B, the poor electronic density in the difference maps does not allow to model residues 114-115. Both chains show a nickel-binding site at the N-terminal formed by the residues His83-Ser82-Gly81, with slight differences in the conformation and in the axial ligand (nickel ion is represented with a green sphere). All the figures were performed using the program Pymol 1.7 (distributed by Schrödinger).</p

Thermal stability of WT and mutants of the c-Src-SH3 domain.

a<p>Errors are fitting errors to the two-state denaturation model.</p><p>Thermal stability of WT and mutants of the c-Src-SH3 domain.</p

Nucleation site of the WT c-Src SH3 domain.

<p>Hydrogen-bond interactions among the residues belonging to the diverging β-turn and those of the distal loop are shown in green dotted lines. WT_M (PDB code 4JZ4) chains A (panel A) and B (panel B) are shown in blue and cyan, respectively. (C) Intertwined dimer structure of the WT c-Src SH3 domain (PDB code 4JZ3), residues at chain A are shown in white sticks and those belonging to the symmetry related molecule (chain B) are in magenta sticks.</p

X-ray data collection and refinement statistics.

<p>Statistics for the highest-resolution shell are shown in parentheses.</p><p>X-ray data collection and refinement statistics.</p

DLS experiments.

<p>(A) Average <i>R</i>_h as a function of c-Src-SH3 domain concentration, in 0.1 M sodium acetate (pH 5.0) at 25°C. Symbols represent measured data in the presence of 5% PEG300 (open circles) and absence of PEG300 (filled circles). (B) Aggregation kinetics of c-Src-SH3 followed by DLS. The protein at a concentration of 25 mg·ml⁻¹ (3.6 mM) in 0.1 M sodium acetate pH 5.0 was incubated at 25°C (square) containing 5% PEG300 and without PEG300 at 20°C (triangles) and 25°C (circles) as a function of time.</p

Comparisons in terms of RMSD (Å).

a<p>Alignment performed taking into account only non-hydrogen atoms.</p>b<p>Alignment performed taking as reference residues 84-110 of the monomer.</p>c<p>Alignment performed taking as reference residues 117-140 of the monomer.</p><p>Comparisons in terms of RMSD (Å).</p

Superposition of the monomeric structures of the c-Src-SH3 domain.

<p>Superposition of the crystallographic structures of the monomeric c-Src-SH3 domain (WT_M, chain A blue and B clear blue, PDB code 4JZ4; Q128E_M, chain A red and B clear red, PDB code 4OMO) with that solved by NMR (PDB code 1SRL) (grey).</p

Intertwined dimer structures of the c-Src-SH3 domain.

<p>(A) Structure of the intertwined dimer of the c-Src-SH3 domain. Open chain of the WT c-Src-SH3 domain is shown in cartoon (blue). Chain B, which generates the dimer, was obtained by symmetry (light-blue). (B) Superposition of the intertwined structures of the WT_D (blue) and Q128E_D (red), Q128K_D (yellow), Q128R_D (green) mutants. (C) Superposition of the open chain of the intertwined dimer structure of the WT c-Src-SH3 domain (blue) (PDB code 4JZ3) to chain A of the monomeric structure of the WT (PDB code 4JZ4): the overlay of the N- and C-terminal regions is shown in cyan and white, respectively.</p