Altering dimerization specificity by changes in surface electrostatics

Abstract

Arc repressor forms a homodimer in which the subunits intertwine to create a single globular domain. To obtain Arc sequences that fold preferentially as heterodimers, variants with surface patches of excess positive or negative charge were designed. Several but not all oppositely charged sequence pairs showed preferential heterodimer formation. In the most successful design pair, ␣ helix B of one subunit contained glutamic acids at positions 43, 46, 47, 48, and 50, whereas the other subunit contained lysines or arginines at these positions. A continuum electrostatic model captures many features of the experimental results and suggests that the most successful designs include elements of both positive and negative design.Arc repressor ͉ continuum electrostatics ͉ heterodimer ͉ negative design ͉ protein stability A central challenge of protein science is to understand the relationship between amino acid sequence and the specification of tertiary structure (1-3). Attempting to design proteins that exhibit novel properties provides one means of testing and refining this understanding. Progress has been made in the design of coiled-coil and helical-bundle structures, and computational design algorithms have been applied successfully in several instances to the redesign of globular proteins (4-9). It is clear, nevertheless, that our understanding of the determinants of protein folding is far from complete (10-11).In this paper, we use P22 Arc repressor, a member of the ribbon-helix-helix family of DNA-binding proteins, to probe the sequence determinants of tertiary and quaternary specificity. Arc is a small 53-residue protein that folds as a homodimer. The structure of the Arc dimer has been determined by solution NMR and x-ray crystallography (12)(13)(14). Unlike many dimers, subunits in Arc do not form independent domains but rather intertwine to form one domain with a single hydrophobic core (Fig. 1A). As a result, folding and dimerization of Arc are coupled, and tertiary and quaternary structure are formed concomitantly (15).Arc subunits interact with a dissociation constant of approximately 10 nM but do not form detectable heterodimers with related ribbon-helix-helix proteins that adopt the same tertiary fold. Clearly, the specificity of Arc dimerization depends on a set of detailed sequence-encoded interactions. We sought to design pairs of Arc variants that folded preferentially as heterodimers. Toward this end, we produced and characterized mutants with surface patches of positive or negative charge at high local densities. Some pairs of variants with complementary patterns of positive and negative charge exhibited preferential heterodimer formation that could be rationalized by using a simple method based on continuum electrostatics (16). The dimeric nature of this system allows certain positive-and negative-design issues to be quantified, because homodimers formed by individual sequences can be compared with heterodimers formed by sequence pairs. Materials and MethodsMutagenesis and Purification. Mutations i...

Breaking and Restoring the Hydrophobic Core of a Centromere-binding Protein

“…It appears sufficient for predicting their importance to dimerization that the key residues make extensive non-polar interactions in the dimer, and will contribute substantially to the experimental K d (ϳ1.0 M) previously determined for the protein (48). With respect to charge-charge interactions at the dimer interface, while they do not figure in binding hot spots, it is possible that modulation of charge patterns could contribute to specificity in homo-and hetero-interactions in the RHH family, as seen for the Arc repressor (57).…”

Intra- versus Intermolecular Interactions in Monellin: Contribution of Surface Charges to Protein Assembly

“…Coulombic interactions are found to play a significant role in ligand binding, for instance inducing chelation of metal ions [7][8][9] and binding of substrate in enzyme catalysis. [10][11][12][13] Coulombic interactions are believed to control specificity, 14,15 cooperativity 9,16 and kinetics 17,18 of protein interactions. Another important aspect of Coulombic interactions is the avoidance of unspecific association, aggregation and amyloid formation, [19][20][21] which is of utmost importance in the high-density environment of the cell.…”

Specificity versus stability in computational protein design

“…Protein-protein interactions have been successfully reengineered to alter binding specificity both by using and ignoring negative design (7)(8)(9)(10)(11)(12)(13)(14)(15). How is success possible in the absence of negative design?…”