Electrostatic interactions in the reconstitution of an SH2 domain from constituent peptide fragments

Abstract

Fragment complementation has been used to delineate the essential recognition elements for stable folding in Src homology 2 (SH2) domains by using NMR spectroscopy, alanine scanning, and surface plasmon resonance. The unfolded 9-kD and 5-kD peptide fragments formed by limited proteolytic digestion of the N-terminal SH2 domain from the p85␣ subunit of phosphatidylinositol 3Ј-kinase fold into an active nativelike structure on interaction with one another. The corresponding 5-kD fragment of the homologous Src protein, however, was not capable of structurally complementing the p85 9-kD fragment, indicating that fragment complementation among these SH2 domains is sensitive to the sequence differences between the Src and p85 domains. Partial complementation and folding activity could be recovered with hybrid sequences of these SH2 domains. Complete alanine scanning of the 5-kD p85 fragment was used to identify the sequence recognition elements required for complex formation. The alanine substitutions in the p85 5-kD fragment that abolished binding affinity with the cognate 9-kD fragment correlate well with highly conserved residues among SH2 domains that are either integrally involved in core packing or found at the interface between fragments. Surprisingly, however, mutation of a nonconserved surface-exposed aspartic acid to alanine was found to have a significant effect on complementation. A single additional mutation of arginine to aspartic acid allowed for recovery of native structure and increased the thermal stability of the designed Src-p85 chimera by 18°C. This modification appears to relieve an unfavorable surface electrostatic interaction, demonstrating the importance of surface charge interactions in protein stability.Keywords: Fragment complementation; SH2 domain; electrostatic interactions; protein stability Supplemental material: See www.proteinscience.org.Extensive site-directed mutagenesis studies of intact proteins have been used to reveal the fundamental interactions that stabilize protein structures. Buried hydrophobic interactions clearly play an essential role in stabilizing specific folds (Richards 1977;Kellis et al. 1989;Sandberg and Terwilliger 1989;Bowie et al. 1990;Shortle et al. 1990;Lim and Sauer 1991;Eriksson et al. 1992;Jackson et al. 1993;Cordes et al. 1996;Dahiyat and Mayo 1997;Xu et al. 1998;Chen and Stites 2001), whereas the role of surface interactions is less clear Tidor 1994, 1999;Meeker et al. 1996;Suckow et al. 1996;Schwehm et al. 1998;Sindelar et al. 1998;Cordes and Sauer 1999;Xiao and Honig 1999;Spector et al. 2000;Strop and Mayo 2000;Perl and Schmid 2001). Fragment complementation is another useful and sensitive phenomenon that can be used to analyze the interactions that stabilize protein structures. Certain single domain proteins retain the ability to form a folded non-covalent complex when cleaved into one or more fragments (for review, see de Prat-Gay 1996). These complexes Reprint requests to: Deborah S. Wuttke, Department of Chemistry and Biochemistry, UCB 215, ...

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