Protein-protein interactions: structurally conserved residues distinguish between binding sites and exposed protein surfaces

Abstract

Polar residue hot spots have been observed at protein-protein binding sites. Here we show that hot spots occur predominantly at the interfaces of macromolecular complexes, distinguishing binding sites from the remainder of the surface. Consequently, hot spots can be used to define binding epitopes. We further show a correspondence between energy hot spots and structurally conserved residues. The number of structurally conserved residues, particularly of high ranking energy hot spots, increases with the binding site contact size. This finding may suggest that effectively dispersing hot spots within a large contact area, rather than compactly clustering them, may be a strategy to sustain essential key interactions while still allowing certain protein flexibility at the interface. Thus, most conserved polar residues at the binding interfaces confer rigidity to minimize the entropic cost on binding, whereas surrounding residues form a flexible cushion. Furthermore, our finding that similar residue hot spots occur across different protein families suggests that affinity and specificity are not necessarily coupled: higher affinity does not directly imply greater specificity. Conservation of Trp on the protein surface indicates a highly likely binding site. To a lesser extent, conservation of Phe and Met also imply a binding site. For all three residues, there is a significant conservation in binding sites, whereas there is no conservation on the exposed surface. A hybrid strategy, mapping sequence alignment onto a single structure illustrates the possibility of binding site identification around these three residues.

Figure 1

(a) Correlation of new binding-site conservation propensities with experimental enrichment of hot spots. (b) Correlation of new binding-site conservation propensities with the previous work of Hu et al. (c) No correlation of exposed surface conservation propensity with experimental hot spots.

Figure 2

Propensity map of the residues to be in the binding-site contact layer versus exposed surface (a) and conservation propensities in the binding-site contact layer versus exposed surface (b).

Figure 3

Comparison of structural alignment and sequential alignment of 1bbbAB interfaces.

Figure 4

Correlations of the number of all conserved residues with the number of contact residues (a), the number of high ranking hot spot (Trp, Arg, Tyr, Leu+Ile, Asp, His, Pro, and Lys) residues with the number of contact residues (b), the number of all conserved polar residues with the number of contact residues (c), and the number of all conserved hydrophobic residues with the number of contact residues (d).

Figure 5

The conserved hot spots on the D chain of 1babAD interface. The hot spots are illustrated as sticks, and for the remaining residues only the surface is shown. Note that the carbonyl backbone atoms of the hot spots are exposed. There is a hydrogen bond between Trp-37 and Arg-40. This arrangement is consistent with the UDHB (under-dehydrated hybrogen bonds; see ref. 5).