Effect of pH and salt bridges on structural assembly: Molecular structures of the monomer and intertwined dimer of the Eps8 SH3 domain

Abstract

The SH3 domain of Eps8 was previously found to form an intertwined, domain-swapped dimer. We report here a monomeric structure of the EPS8 SH3 domain obtained from crystals grown at low pH, as well as an improved domain-swapped dimer structure at 1.8 Å resolution. In the domain-swapped dimer the asymmetric unit contains two "hybrid-monomers." In the low pH form there are two independently folded SH3 molecules per asymmetric unit. The formation of intermolecular salt bridges is thought to be the reason for the formation of the dimer. On the basis of the monomer SH3 structure, it is argued that Eps8 SH3 should, in principle, bind to peptides containing a PxxP motif. Recently it was reported that Eps8 SH3 binds to a peptide with a PxxDY motif. Because the "SH3 fold" is conserved, alternate binding sites may be possible for the PxxDY motif to bind. The strand exchange or domain swap occurs at the n-src loops because the n-src loops are flexible. The thermal b-factors also indicate the flexible nature of n-src loops and a possible handle for domain swap initiation. Despite the loop swapping, the typical SH3 fold in both forms is conserved structurally. The interface of the acidic form of SH3 is stabilized by a tetragonal network of water molecules above hydrophobic residues. The intertwined dimer interface is stabilized by hydrophobic and aromatic stacking interactions in the core and by hydrophilic interactions on the surface.

Simple but predictive protein models

“…In crystallographic studies of the Eps8 SH3 domain, Kishan et al [38] uncovered a domain swapped Eps8 SH3 that is similar visually to our domain swapped Src SH3 dimer (Figure 2) [17], and a root-mean-square deviation between the predicted and observed structures of !6 Å determined by Pymol (http://www.pymol.org). Similarity between predicted and experimental values for domainswapped SH3 homologs indicates that the principal assumptions in our model reflect actual interactions between similar proteins.…”

Domain swapping is a consequence of minimal frustration

“…For illustration, we chose an SH3 domain of the epidermal growth factor receptor pathway substrate 8 (Eps8), which has been observed in both the monomeric and dimeric domain-swapped conformations (28). The monomeric SH3 (MSH3) of Eps8 is a relatively small protein, formed by two orthogonal ␤-sheets connected by a hinge named the n-src loop (Fig.…”

Protein Oligomerization Through Domain Swapping: Role of Inter-molecular Interactions and Protein Concentration

“…1 The exchangeable domain is usually appended to a flexible hinge region and this hinge allows the placement of the swapped region in both intramolecular and intermolecular configurations. More than 40 proteins have been found to form domain-swapped configurations, such as the human prion, 12,13 the SH3 domain, 14,15 p13suc1, [16][17][18][19] cystatins, [20][21][22][23][24] and interleukin-10 and interferon g. 25,26 These domain-swapped proteins display diverse sequences and secondary structures of the swapped domains, and flexibility and diversity in the hinge regions. This diversity suggests that almost any protein may oligomerize via domain swapping.…”