Deep Analysis of Residue Constraints (DARC): identifying determinants of protein functional specificity

Protein functional constraints are manifest as superfamily and functional-subgroup conserved residues, and as pairwise correlations. Deep Analysis of Residue Constraints (DARC) aids the visualization of these constraints, characterizes how they correlate with each other and with structure, and estimates statistical significance. This can identify determinants of protein functional specificity, as we illustrate for bacterial DNA clamp loader ATPases. These load ring-shaped sliding clamps onto DNA to keep polymerase attached during replication and contain one delta, three gamma, and one delta' AAA+ subunits semicircularly arranged in the order delta-gamma(1)-gamma(2)-gamma(3)-delta'. Only gamma is active, though both gamma and delta' functionally influence an adjacent gamma subunit. DARC identifies, as functionally-congruent features linking allosterically the ATP, DNA, and clamp binding sites: residues distinctive of. and of gamma/delta' that mutually interact in trans, centered on the catalytic base; several gamma/delta'-residues and six gamma/delta'-covariant residue pairs within the DNA binding N-termini of helices alpha 2 and alpha 3; and gamma/delta'-residues associated with the alpha 2 C-terminus and the clamp-binding loop. Most notable is a trans-acting./d' hydroxyl group that 99% of other AAA+ proteins lack. Mutation of this hydroxyl to a methyl group impedes clamp binding and opening, DNA binding, and ATP hydrolysis-implying a remarkably clamp-loader-specific function.