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Surface residues and non-additive interactions stabilize a consensus homeodomain protein

Despite the widely reported success of consensus design in producing highly stabilized proteins, little is known about the physical mechanisms underlying this stabilization. Here we explore the potential sources of stabilization by performing a systematic analysis of the 29 substitutions that we previously found to collectively stabilize a consensus homeodomain compared to an extant homeodomain. By separately introducing groups of consensus substitutions that alter or preserve charge state, occur at varying degrees of residue burial, and occur at positions of varying degrees of conservation, we determine the extent to which these three features contribute to the consensus stability enhancement. Surprisingly, we find that the largest total contribution to stability comes from consensus substitutions on the protein surface and that the largest per-substitution contributions come from substitutions that maintain charge state, suggesting that although consensus proteins are often enriched in charged residues, consensus stabilization does not result primarily from charge-charge interactions. Although consensus substitutions at strongly conserved positions also contribute disproportionately to stabilization, significant stabilization is also contributed from substitutions at weakly conserved positions. Furthermore, we find that identical consensus substitutions show larger stabilizing effects when introduced into the consensus background than when introduced into an extant homeodomain, indicating that synergistic, stabilizing interactions among the consensus residues contribute to consensus stability enhancement of the homeodomain. Significance Statement Proteins composed of consensus sequences from multiple sequence alignments are often more stable than extant proteins used to create them. Often about half the residues in a consensus protein differ from those of extant proteins. The contributions of these differences to stability are unknown. Here we substitute groups of residues with different properties (conservation, charge variation, solvent accessibility) to determine which substitutions lead to consensus stabilization. We find that surface and charge-conserving substitutions contribute to stability, that weakly-conserved substitutions make a significant collective contribution to stability, and that there is a significant non-additive contribution to stability in the consensus background. These results provide insights to the sequence origins of consensus stabilization and the evolutionary constraints that determine protein sequences.