Co-evolving motions at protein-protein interfaces of two-component signaling systems identified by covariance analysis

Abstract

Short-lived protein interactions determine signal transduction specificity among genetically amplified, structurally identical two-component signaling systems. Interacting protein pairs evolve recognition precision by varying residues at specific positions in the interaction surface consistent with constraints of charge, size, and chemical properties. Such positions can be detected by covariance analyses of two-component protein databases. Here, covariance is shown to identify a cluster of co-evolving dynamic residues in two-component proteins. NMR dynamics and structural studies of both wild-type and mutant proteins in this cluster suggest that motions serve to precisely arrange the site of phosphoryl transfer within the complex.

FIGURE 1

Two-component signaling best friend network. (A) Residues with significant covariance scores (i.e., S_i,j > 0.26) are mapped onto exemplary structures of a HisKA domain and a RR (see the text). Clusters 1–5 are colored cyan; cluster 6 is colored red or orange, and the phosphotransfer residues H260 and D54 are colored yellow. (B) Best friend network mapped onto a HisKA–RR cocrystal structure (Spo0B–Spo0F; see the text). Residues involved in the phosphotransfer reaction and metal ion orientation are colored yellow.

FIGURE 2

Lowest-energy NMR structures of Spo0F mutants and their role in active site conformation. Superpositions of wild-type Spo0F (light blue) with (A) L66A, (B) I90A, and (C) H101A mutations (light green). Residues K56, Y84, L87, and I90 (marked by their Cα atoms) were picked up in the covariance analysis as being important for SK interaction specificity: Spo0F in yellow and mutant proteins in orange. (D) Active site of Spo0B–Spo0F phosphotransfer shown in close-up.