Interaction fidelity in two-component signaling

Abstract

Two component signal transduction systems and phosphorelays have been adapted and amplified by bacteria to respond to a multitude of environmental, metabolic and cell cycle signals while maintaining essentially identical structures for the domains responsible for recognition and phosphotransfer between the sensor histidine kinase and the response regulator. Co-crystal structures of these domains have revealed the variable residues at the interaction surface of the two components responsible for interaction specificity in signal transfer. This information has formed the basis for the development and validation of statistical methods to identify interaction residues and surfaces from compiled databases of interacting proteins and holds forth the promise of determining structures of multi-protein complexes and signaling networks.

Figure 1

The Bacillus subtilis set of two component signaling pathways. The Gram-positive model organism B. subtilis reveals a set of 29 TCS signaling pathways, about the average for bacterial genomes. The modular SK proteins feature variable sets of signal detection domains (in blue) and most are transmembrane spanning proteins. Most RR proteins feature a DNA-binding transcription factor domain (green). The SK is defined by a structurally conserved autokinase catalytic core, which features a dimeric four-helix bundle HisKA (also referred to as DHp) domain (red rectangles), subject to phosphorylation by the ATP-binding (also referred to as CA) domain (red diamonds). The RR proteins are defined by a structurally conserved phosphoacceptor domain (red circle), which is subject to phosphorylation by the HisKA domain of the SK. Despite the conservation of the structural elements almost all pathways appear monogamously mated. A special case is the sporulation phosphorelay, in which five SK converge to phosphorylate a single RR Spo0F, which transfers its phosphoryl group to a second SK like protein Spo0B. The complex of Spo0B and Spo0F was the first structurally resolved example of the SK/RR interaction and has long served as a model to explain SK/RR fidelity. For clarity the chemotaxis pathway, which feature a structurally distinct kinase CheA and three RR proteins, CheY, CheB and CheV are excluded from the figure.

Figure 2

Structures of the SK/RR complex. (a) Top view and (b) side view of the three available representative structures of the SK/RR complex demonstrate identical structural orientation of RR phosphoacceptor domain (blue) and SK HisKA domain (red). The orientation of the ATPase domain differs in each case (green) The structure are from left to right the Spo0B/Spo0F complex from B. subtilis (PDBID 1f51), the HK853/RR468 complex (PDBID 3dge) and the ThkA/TrrA complex (PDBID 3a0r), both from T. maritima. (c) When overlaying the RR proteins Spo0F (blue) and RR468 (beige) the four-helix bundles of Spo0B (red) and HK853 (yellow) also overlay well, demonstrating the conservation of the structural organization of the complex between RR phosphoacceptor and SK HisKA domains. Significant contacts in the individual structures are made between the individual secondary structure elements as indicated and described in the text.

Figure 3

Co-variance analysis identifies specificity determining residues for the SK/RR interaction. (a) From multiple sequence alignments of all chromosomally adjacent SK/RR pairs the frequency f_i(A_i) and f_j(A_j) of individual amino acid choice A (which can be any of the 20 proteinogenic aminoacids or a gap) can be calculated at positions i in the SK HisKA domain and positions j in the RR phosphoacceptor (PA) domain. Mutual information (MI) calculation compares these individual frequencies to the co-frequency of amino acid choice f_i,j(A_i,A_j) for two residue positions. If two positions are unlinked, the co-frequency is equal to the product of the individual frequencies. This results in an MI_i,j close to 0. If two positions are linked, f_i,j(A_i,A_j) is larger than the product of the individual frequencies and MI_i,j is larger than 0. (b) Shown is the network of SK/RR position pairings with above background MI values (green and red). Some of the high MI values are due to direct correlations between two residue positions (red), whereas others show MI values that are inflated by correlation chains, due to the residue choice of neighboring residues (green) and are hence indirect. These different correlations can be disentangled when amending co-variance analysis by a statistical inference step [28]. Residue numbers are as in HK853 for the SK and as in RR468 for the RR for easy identification of residue pairings in the HK853/RR468 structure, but in contrast to the numbering used in [28]. (c) Mapped to the newly available HK853/RR468 co-crystal structure (PDBID 3dge) it is obvious that the directly correlated residue position pairings (red) are at the interaction surface, whereas the indirectly correlated ones are not. Both, indirect and direct correlations have been shown to contribute to fidelity of the SK/RR phosphotransfer reaction that occurs between the conserved His and Asp residues (blue).