How a highly acidic SH3 domain folds in the absence of its charged peptide target

Abstract

Charged residues on the surface of proteins are critical for both protein stability and interactions. However, many proteins contain binding regions with a high net-charge that may destabilize the protein but are useful for binding to oppositely charged targets. We hypothesized that these domains would be marginally stable, as electrostatic repulsion would compete with favorable hydrophobic collapse during folding. Furthermore, by increasing the salt concentration we predict that these protein folds would be stabilized by mimicking some of the favorable electrostatic interactions that take place during target binding. We varied the salt and urea concentrations to probe the contributions of electrostatic and hydrophobic interactions for the folding of the 60-residue yeast SH3 domain found in Abp1p. The SH3 domain was significantly stabilized with increased salt concentrations according to the Debye-Huckel limiting law. Molecular dynamics and NMR show that sodium ions interact with all 15 acidic residues but do little to change backbone dynamics or overall structure. Folding kinetics experiments show that the addition of urea or salt primarily affects the folding rate, indicating that almost all the hydrophobic collapse and electrostatic repulsion occurs in the transition state. After the transition state formation, modest yet favorable short-range salt-bridges are formed along with hydrogen bonds, as the native state fully folds. Thus, hydrophobic collapse offsets electrostatic repulsion to ensure this highly charged binding domain can still fold and be ready to bind to its charged peptide targets, a property that is likely evolutionarily conserved over one billion years. Statement for broader audienceSome protein domains are highly charged because they are adapted to bind oppositely charged proteins and nucleic acids. However, it is unknown how these highly charged domains fold as during folding there will be significant repulsion between like-charges. We investigate how one of these highly charged domains folds in the presence of salt, which can screen the charge repulsion and make folding easier, allowing us to understand how folding occurs despite the proteins high charge. Supplementary materialO_LISupplementary material document containing additional details on protein expression methods, thermodynamics and kinetics equations, and the effect of urea on electrostatic interactions, as well as 4 supplemental figures and 4 supplemental data tables. (Supplementary_Material.docx), 15 pages C_LIO_LISupplemental excel file containing covariation data across AbpSH3 orthologs (FileS1.xlsx) C_LI O_FIG O_LINKSMALLFIG WIDTH=104 HEIGHT=200 SRC="FIGDIR/small/532811v1_fig4.gif" ALT="Figure 4"> View larger version (21K): org.highwire.dtl.DTLVardef@185b8eeorg.highwire.dtl.DTLVardef@303c18org.highwire.dtl.DTLVardef@b98532org.highwire.dtl.DTLVardef@1d7ce68_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 4.C_FLOATNO Analysis of domain backbone dynamics. A. 1H-15N heteronuclear NOE of AbpSH3 domain at 0 and 800 mM NaCl. B. Backbone 15N order parameters calculated from Modelfree. Error bars come from the fit. C. Atomic fluctuations of the AbpSH3 domain in no salt, 300 mM NaCl, 800 mM NaCl, and 800 mM KCl simulations. Average standard deviations in the atomic fluctuations (0.2 [A], 0.2 [A], 0.3 [A], and 0.1 [A] respectively) are larger than the difference between the lines. C_FIG