Understanding the molecular determinants of specificity in proteinprotein interaction is an outstanding challenge of postgenome biology. The availability of large protein databases generated from sequences of hundreds of bacterial genomes enables various statistical approaches to this problem. In this context covariance-based methods have been used to identify correlation between amino acid positions in interacting proteins. However, these methods have an important shortcoming, in that they cannot distinguish between directly and indirectly correlated residues. We developed a method that combines covariance analysis with global inference analysis, adopted from use in statistical physics. Applied to a set of >2,500 representatives of the bacterial two-component signal transduction system, the combination of covariance with global inference successfully and robustly identified residue pairs that are proximal in space without resorting to ad hoc tuning parameters, both for heterointeractions between sensor kinase (SK) and response regulator (RR) proteins and for homointeractions between RR proteins. The spectacular success of this approach illustrates the effectiveness of the global inference approach in identifying direct interaction based on sequence information alone. We expect this method to be applicable soon to interaction surfaces between proteins present in only 1 copy per genome as the number of sequenced genomes continues to expand. Use of this method could significantly increase the potential targets for therapeutic intervention, shed light on the mechanism of protein-protein interaction, and establish the foundation for the accurate prediction of interacting protein partners.T he large majority of cellular functions are executed and controlled by interacting proteins. With up to several thousand types of proteins expressed in a typical bacterial cell at a given time, their concerted specific interactions regulate the interplay of biochemical processes that are the essence of life. Many protein interactions are transient, allowing proteins to mate with several partners or travel in cellular space to perform their functions. Understanding these transient interactions is one of the outstanding challenges of systems biology (reviewed in ref. 1). The characterization of the molecular details of the interface formed between known interacting proteins is a requirement for understanding the molecular determinants of protein-protein interaction, the knowledge of which may be important for a variety of applications including synthetic biology, e.g., designing new specific interaction between proteins (reviewed in ref.2), and pharmaceutics, e.g., protein interaction surfaces as drug targets (reviewed in ref.3).Experimental approaches to identify surfaces of interaction between proteins such as surface-scanning mutagenesis and cocrystal structure generation are arduous and/or serendipitous. Cocrystal structures provide the best molecular resolution but are particularly challenging to obtain for transient interactio...

SUMMARY The study of chemotaxis describes the cellular processes that control the movement of organisms toward favorable environments. In bacteria and archaea, motility is controlled by a two-component system involving a histidine kinase that senses the environment and a response regulator, a very common type of signal transduction in prokaryotes. Most insights into the processes involved have come from studies of Escherichia coli over the last three decades. However, in the last 10 years, with the sequencing of many prokaryotic genomes, it has become clear that E. coli represents a streamlined example of bacterial chemotaxis. While general features of excitation remain conserved among bacteria and archaea, specific features, such as adaptational processes and hydrolysis of the intracellular signal CheY-P, are quite diverse. The Bacillus subtilis chemotaxis system is considerably more complex and appears to be similar to the one that existed when the bacteria and archaea separated during evolution, so that understanding this mechanism should provide insight into the variety of mechanisms used today by the broad sweep of chemotactic bacteria and archaea. However, processes even beyond those used in E. coli and B. subtilis have been discovered in other organisms. This review emphasizes those used by B. subtilis and these other organisms but also gives an account of the mechanism in E. coli.

Two-component systems comprising sensor histidine kinases and response regulator proteins are among the most important players in bacterial and archaeal signal transduction and also occur in reduced numbers in some eukaryotic organisms. Given their importance to cellular survival, virulence and cellular development these systems are among the most scrutinized bacterial proteins. In recent years a flurry of bioinformatics, genetic, biochemical and structural studies have provided detailed insights into many of the molecular mechanisms that underlie the detection of signals and the generation of the appropriate response by two-component systems. Importantly, it has become clear that there is significant diversity in the mechanisms employed by individual systems. This review discusses the current knowledge on common themes and divergences from the paradigm of two-component system signaling. An emphasis is on the information gained by a flurry of recent structural and bioinformatics studies.

Bacteria use two-component signal transduction systems (TCS) extensively to sense and react to external stimuli. In these, a membrane-bound sensor histidine kinase (SK) autophosphorylates in response to an environmental stimulus and transfers the phosphoryl group to a transcription factor/response regulator (RR) that mediates the cellular response. The complex between these two proteins is ruled by transient interactions, which provides a challenge to experimental structure determination techniques. The functional and structural homolog of an SK/RR pair Spo0B/Spo0F, however, has been structurally resolved. Here, we describe a method capable of generating structural models of such transient protein complexes. By using existing structures of the individual proteins, our method combines bioinformatically derived contact residue information with molecular dynamics simulations. We find crystal resolution accuracy with existing crystallographic data when reconstituting the known system Spo0B/Spo0F. Using this approach, we introduce a complex structure of TM0853/TM0468 as an exemplary SK/RR TCS, consistent with all experimentally available data.direct coupling analysis ͉ signal transduction ͉ structure based simulations ͉ transient protein complexes ͉ two component system A protein's biological function is often dominated by transient interactions with other proteins with the resulting protein-protein interfaces being considered as targets for drug design (1). Experimental techniques like NMR or X-ray crystallography have proven tremendously successful in providing structural information but face problems when resolving transiently bound protein complexes. Structure prediction methods cannot readily close this gap, because database-driven methods like homology modeling (2) suffer from the lack of structural templates of protein complexes. Physics-based (3-5) approaches struggle with the accuracy of their force fields (6) and often have computationally prohibitive costs for these large complexes. Hybrid methods, which combine experimental data with structural information to generate predictions, have proven more successful (7). Going beyond approaches that rely on a single theoretical framework, the present study integrates information from genomic analysis into molecular dynamics simulation. A sequence-based genomic analysis investigates subtle statistical fluctuations accompanying the mutational patterns of coevolving proteins and suggests amino acids defining the interaction surface of the two proteins. This information is integrated into molecular dynamics simulations to predict the structure of the protein complex (Fig. 1).Because the accuracy of genomic analysis increases with available sequence information, two-component signal transduction systems (TCS), which are ubiquitous and highly amplified within sequenced bacterial genomes, are ideally suited for statistical analysis. In TCS, a sensor histidine kinase (SK) and a response regulator/transcription factor protein (RR) connect an input signal to an appropriate...

Predictive understanding of the myriads of signal transduction pathways in a cell is an outstanding challenge of systems biology. Such pathways are primarily mediated by specific but transient protein-protein interactions, which are difficult to study experimentally. In this study, we dissect the specificity of protein-protein interactions governing two-component signaling (TCS) systems ubiquitously used in bacteria. Exploiting the large number of sequenced bacterial genomes and an operon structure which packages many pairs of interacting TCS proteins together, we developed a computational approach to extract a molecular interaction code capturing the preferences of a small but critical number of directly interacting residue pairs. This code is found to reflect physical interaction mechanisms, with the strongest signal coming from charged amino acids. It is used to predict the specificity of TCS interaction: Our results compare favorably to most available experimental results, including the prediction of 7 (out of 8 known) interaction partners of orphan signaling proteins in Caulobacter crescentus. Surveying among the available bacterial genomes, our results suggest 15∼25% of the TCS proteins could participate in out-of-operon “crosstalks”. Additionally, we predict clusters of crosstalking candidates, expanding from the anecdotally known examples in model organisms. The tools and results presented here can be used to guide experimental studies towards a system-level understanding of two-component signaling.

Signal transduction proteins such as bacterial sensor histidine kinases, designed to transition between multiple conformations, are often ruled by unstable transient interactions making structural characterization of all functional states difficult. This study explored the inactive and signal-activated conformational states of the two catalytic domains of sensor histidine kinases, HisKA and HATPase. Direct coupling analyses, a global statistical inference approach, was applied to >13,000 such domains from protein databases to identify residue contacts between the two domains. These contacts guided structural assembly of the domains using MAGMA, an advanced molecular dynamics docking method. The active conformation structure generated by MAGMA simultaneously accommodated the sequence derived residue contacts and the ATP-catalytic histidine contact. The validity of this structure was confirmed biologically by mutation of contact positions in the Bacillus subtilis sensor histidine kinase KinA and by restoration of activity in an inactive KinA(HisKA):KinD(HATPase) hybrid protein.These data indicate that signals binding to sensor domains activate sensor histidine kinases by causing localized strain and unwinding at the end of the C-terminal helix of the HisKA domain. This destabilizes the contact positions of the inactive conformation of the two domains, identified by previous crystal structure analyses and by the sequence analysis described here, inducing the formation of the active conformation. This study reveals that structures of unstable transient complexes of interacting proteins and of protein domains are accessible by applying this combination of crossvalidating technologies.coevolution | signal transduction | two component system | protein structure prediction | biological physics P rotein-protein interaction is the essence of signal transduction and essential for protein function at all levels of life. Proteins are frequently required to undergo conformational transitions between multiple states characterized by unique transient residue interactions for each state. Attempts to describe features of these states depend upon techniques that investigate single molecules at sub-ms timescales (1), mechanically pull them (2), or quantify their dynamics (3). On the theoretical side, energy landscape theory provides a basis to understand protein folding and function (4), and atomically resolved simulations can describe transitions between multiple (native) conformations covering ms timescales on specialized supercomputers (5).Yet, despite all these advances, a full structural characterization of multiple conformations for a specific system remains a daunting task. Some states, active states in particular, are intrinsically short-lived and therefore difficult to stabilize for direct measurements or capture in a crystal lattice. By utilizing a strategy of integrating information from cross-disciplinary approaches, we show here that the shortcomings of individual techniques may be circumvented. By doing so, we introdu...

The YycFG two-component system is the only signal transduction system in Bacillus subtilis known to be essential for cell viability. This system is highly conserved in low-G؉C gram-positive bacteria, regulating important processes such as cell wall homeostasis, cell membrane integrity, and cell division. Four other genes, yycHIJK, are organized within the same operon with yycF and yycG in B. subtilis. Recently, it was shown that the product of one of these genes, the YycH protein, regulated the activity of this signal transduction system, whereas no function could be assigned to the other genes. Results presented here show that YycI and YycH proteins interact to control the activity of the YycG kinase. Strains carrying individual in-frame deletion of the yycI and yycH coding sequences were constructed and showed identical phenotypes, namely a 10-fold-elevated expression of the YycF-dependent gene yocH, growth defects, as well as a cell wall defect. Cell wall and growth defects were a direct result of overregulation of the YycF regulon, since a strain overexpressing YycF showed phenotypes similar to those of yycH and yycI deletion strains. Both YycI and YycH proteins are localized outside the cytoplasm and attached to the membrane by an N-terminal transmembrane sequence. Bacterial two-hybrid data showed that the YycH, YycI, and the kinase YycG form a ternary complex. The data suggest that YycH and YycI control the activity of YycG in the periplasm and that this control is crucial in regulating important cellular processes.

Two-component signal transduction systems consisting of a sensor histidine kinase and a response regulator/transcription factor interpret a multitude of environmental and cellular signals and coordinate the expression of a wide array of genes in bacteria. Signal recognition by sensor histidine kinases is the province of a sensor complex consisting of several protein domains that together serve to augment or attenuate the activity of the histidine kinase and thereby of gene expression. Recent investigations have shown the diverse strategies bacteria use to assemble protein domains into the sensor complexes to accomplish signaling. Structural studies of such domains are leading to an understanding of the mechanisms by which sensor complexes recognize signals and regulate kinase activity.