Synthesis of histidine kinase inhibitors and their biological properties.

Infections caused by multidrug-resistant bacteria represent a significant and ever-increasing cause of morbidity and mortality. There is thus an urgent need to develop efficient and well-tolerated antibacterials targeting unique cellular processes. Numerous studies have led to the identification of new biological targets to fight bacterial resistance. Two-component signal transduction systems are widely employed by bacteria to translate external and cellular signals into a cellular response. They are ubiquitous in bacteria, absent in the animal kingdom and are integrated into various virulence pathways. Several chemical series, including isothiazolidones, imidazolium salts, benzoxazines, salicylanilides, thiophenes, thiazolidiones, benzimidazoles, and other derivatives deduced by different approaches have been reported in the literature to have histidine kinase (HK) inhibitory activity. In this review, we report on the design and the synthesis of these HKs inhibitors and their potential to serve as antibacterial agents.

Evolutionary couplings of amino acid residues reveal structure and function of bacterial signaling proteins.

The genomic era along with major advances in high-throughput sequencing technology has led to a rapid expansion of the genomic and consequently the protein sequence space. Bacterial extracytoplasmic function sigma factors have emerged as an important group of signaling proteins in bacteria involved in many regulatory decisions, most notably the adaptation to cell envelope stress. Their wide prevalence and amplification among bacterial genomes has led to sub-group classification and the realization of diverse signaling mechanisms. Mathematical frameworks have been developed to utilize extensive protein sequence alignments to extract co-evolutionary signals of interaction. This has proven useful in a number of different biological fields, including de novo structure prediction, protein-protein partner identification and the elucidation of alternative protein conformations for signal proteins, to name a few. The mathematical tools, commonly referred to under the name 'Direct Coupling Analysis' have now been applied to deduce molecular mechanisms of activation for sub-groups of extracytoplasmic sigma factors adding to previous successes on bacterial two-component signaling proteins. The amplification of signal transduction protein genes in bacterial genomes made them the first to be amenable to this approach but the sequences are available now to aid the molecular microbiologist, no matter their protein pathway of interest.

A multi-scale coevolutionary approach to predict interactions between protein domains.

Interacting proteins and protein domains coevolve on multiple scales, from their correlated presence across species, to correlations in amino-acid usage. Genomic databases provide rapidly growing data for variability in genomic protein content and in protein sequences, calling for computational predictions of unknown interactions. We first introduce the concept of direct phyletic couplings, based on global statistical models of phylogenetic profiles. They strongly increase the accuracy of predicting pairs of related protein domains beyond simpler correlation-based approaches like phylogenetic profiling (80% vs. 30-50% positives out of the 1000 highest-scoring pairs). Combined with the direct coupling analysis of inter-protein residue-residue coevolution, we provide multi-scale evidence for direct but unknown interaction between protein families. An in-depth discussion shows these to be biologically sensible and directly experimentally testable. Negative phyletic couplings highlight alternative solutions for the same functionality, including documented cases of convergent evolution. Thereby our work proves the strong potential of global statistical modeling approaches to genome-wide coevolutionary analysis, far beyond the established use for individual protein complexes and domain-domain interactions.

Large-scale identification of coevolution signals across homo-oligomeric protein interfaces by direct coupling analysis.

Proteins have evolved to perform diverse cellular functions, from serving as reaction catalysts to coordinating cellular propagation and development. Frequently, proteins do not exert their full potential as monomers but rather undergo concerted interactions as either homo-oligomers or with other proteins as hetero-oligomers. The experimental study of such protein complexes and interactions has been arduous. Theoretical structure prediction methods are an attractive alternative. Here, we investigate homo-oligomeric interfaces by tracing residue coevolution via the global statistical direct coupling analysis (DCA). DCA can accurately infer spatial adjacencies between residues. These adjacencies can be included as constraints in structure prediction techniques to predict high-resolution models. By taking advantage of the ongoing exponential growth of sequence databases, we go significantly beyond anecdotal cases of a few protein families and apply DCA to a systematic large-scale study of nearly 2,000 Pfam protein families with sufficient sequence information and structurally resolved homo-oligomeric interfaces. We find that large interfaces are commonly identified by DCA. We further demonstrate that DCA can differentiate between subfamilies with different binding modes within one large Pfam family. Sequence-derived contact information for the subfamilies proves sufficient to assemble accurate structural models of the diverse protein-oligomers. Thus, we provide an approach to investigate oligomerization for arbitrary protein families leading to structural models complementary to often-difficult experimental methods. Combined with ever more abundant sequential data, we anticipate that this study will be instrumental to allow the structural description of many heteroprotein complexes in the future.

Structural basis of histidine kinase autophosphorylation deduced by integrating genomics, molecular dynamics, and mutagenesis.

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 cross-validating technologies.

Statistical analyses of protein sequence alignments identify structures and mechanisms in signal activation of sensor histidine kinases.

Statistical analyses of genome sequence-derived protein sequence data can identify amino acid residues that interact between proteins or between domains of a protein. These statistical methods are based on evolution-directed amino acid variation responding to structural and functional constraints in proteins. The identified residues form a basis for determining structure and folding of proteins as well as inferring mechanisms of protein function. When applied to two-component systems, several research groups have shown they can be used to identify the amino acid interactions between response regulators and histidine kinases and the specificity therein. Recently, statistical studies between the HisKA and HATPase-ATP-binding domains of histidine kinases identified amino acid interactions for both the inactive and the active catalytic states of such kinases. The identified interactions generated a model structure for the domain conformation of the active state. This conformation requires an unwinding of a portion of the C-terminal helix of the HisKA domain that destroys the inactive state residue contacts and suggests how signal-binding determines the equilibrium between the inactive and active states of histidine kinases. The rapidly accumulating protein sequence databases from genome, metagenome and microbiome studies are an important resource for functional and structural understanding of proteins and protein complexes in microbes.

Augmentation of immune response to vaccinations through osteopathic manipulative treatment: a study of procedure.

CONTEXT: Anecdotal evidence suggested that osteopathic manipulative treatment (OMT) may have imparted survivability to patients in osteopathic hospitals during the 1918 influenza pandemic. In addition, previous OMT research publications throughout the past century have shown evidence of increased lymphatic movement, resulting in improved immunologic function qualitatively and quantitatively. OBJECTIVES: The following is a description of a proposed protocol to evaluate OMT effects on antibody generation in the peripheral circulation in response to a vaccine and its possible use in the augmentation of various vaccines. This protocol will serve as a template for OMT vaccination studies, and by adhering to the gold standard of randomized controlled trials (RCTs), future studies utilizing this outline may contribute to the much-needed advancement of the scientific literature in this field. METHODS: This manuscript intends to describe a protocol that will demonstrate increased antibody titers to a vaccine through OMT utilized in previous historical studies. Confirmation data will follow this manuscript validating the protocol. Study participants will be divided into groups with and without OMT with lymphatic pumps. Each group will receive the corresponding vaccine and have antibody titers measured against the specific vaccine pathogen drawn at determined intervals. RESULTS: These results will be statistically evaluated. Our demonstration of a rational scientific OMT vaccine antibody augmentation will serve as the standard for such investigation that will be reported in the future. These vaccines could include COVID-19 mRNA, influenza, shingles, rabies, and various others. The antibody response to vaccines is the resulting conclusion of its administration. Osteopathic manipulative medicine (OMM) lymphatic pumps have, in the past through anecdotal reports and smaller pilot studies, shown effectiveness on peripheral immune augmentation to vaccines. CONCLUSIONS: This described protocol will be the template for more extensive scientific studies supporting osteopathic medicine's benefit on vaccine response. The initial vaccine studies will include the COVID-19 mRNA, influenza, shingles, and rabies vaccines.

Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide.

Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F∼P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F∼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.

A role for the essential YycG sensor histidine kinase in sensing cell division.

The YycG sensor histidine kinase co-ordinates cell wall remodelling with cell division in Gram-positive bacteria by controlling the transcription of genes for autolysins and their inhibitors. Bacillus subtilis YycG senses cell division and is enzymatically activated by associating with the divisome at the division septum. Here it is shown that the cytoplasmic PAS domain of this multi-domain transmembrane kinase is a determining factor translocating the kinase to the division septum. Furthermore, translocation to the division septum, per se, is insufficient to activate YycG, indicating that specific interactions and/or ligands produced there are required to stimulate kinase activity. N-terminal truncations of YycG lose negative regulation of their activity inferring that this regulation is accomplished through its transmembrane and extramembrane domains interacting with the membrane associated YycH and YycI proteins that do not localize to the divisome. The data indicate that YycG activity in non-dividing cells is suppressed by its interaction with YycH and YycI and its activation is co-ordinated to cell division in dividing cells by specific interactions that occur within the divisome.

High-resolution protein complexes from integrating genomic information with molecular simulation.

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.

Identification of direct residue contacts in protein-protein interaction by message passing.

Understanding the molecular determinants of specificity in protein-protein 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.

An essential sensor histidine kinase controlled by transmembrane helix interactions with its auxiliary proteins.

Two-component signal transduction systems with membrane-embedded sensor histidine kinases are believed to recognize environmental signals and transduce this information over the cellular membrane to influence the activity of a transcription factor to which they are mated. The YycG sensor kinase of Bacillus subtilis, containing two transmembrane helices, is subject to a complicated activity-control circuit involving two other proteins with N-terminal transmembrane helices, YycH and YycI. Truncation studies of YycH and YycI demonstrated that the individual transmembrane helices of these proteins are sufficient to adjust YycG activity, indicating that this control is achieved at the membrane level. A replica exchange molecular dynamics computational approach generated in silico structural models of the transmembrane helix complex that informed mutagenesis studies of the YycI transmembrane helix supporting the accuracy of the in silico model. The results predict that signal recognition by any of the extracellular domains of the sensor histidine kinase YycG or the associated proteins YycH and YycI is transmitted across the cellular membrane by subtle alterations in the positions of the helices within the transmembrane complex of the three proteins.

Extracytoplasmic PAS-like domains are common in signal transduction proteins.

We present the crystal structure of the extracytoplasmic domain of the Bacillus subtilis PhoR sensor histidine kinase, part of a two-component system involved in adaptation to low environmental phosphate concentrations. In addition to the PhoR structure, we predict that the majority of the extracytoplasmic domains of B. subtilis sensor kinases will adopt a fold similar to the ubiquitous PAS domain.

Sensor complexes regulating two-component signal transduction.

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.

A bacterial Goldilocks mechanism.

Bacillus subtilis can measure the activity of the enzymes that remodel the cell wall to ensure that the levels of activity are 'just right'.

A sensor histidine kinase co-ordinates cell wall architecture with cell division in Bacillus subtilis.

The concerted interconnection between processes driving DNA synthesis, division septum formation and cell wall synthesis and remodelling in rapidly growing bacteria requires precise co-ordination by signalling mechanisms that are, for the most part, unknown. The YycG (sensor histidine kinase)-YycF (response regulator/transcription factor) two-component system of Bacillus subtilis controls the synthesis of enzymes and their inhibitors that function in cell wall remodelling and cell separation. Here it is shown that the YycG sensor histidine kinase is a component of the division septum in growing cells. RT-PCR quantification of YycF approximately PO(4)-regulated gene transcription, in wild type and FtsZ-depleted, septum-less cells, indicated that YycG kinase activity on YycF is dependent on YycG localization to a division septum. The data support a model in which the YycG sensor kinase perceives information at the division septum and regulates the reciprocal synthesis of autolysins and autolysin inhibitors to co-ordinate growth and division with cell wall restructuring.

Diversity in chemotaxis mechanisms among the bacteria and archaea.

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.

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

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.

Inter-residue, inter-protein and inter-family coevolution: bridging the scales.

Interacting proteins coevolve at multiple but interconnected scales, from the residue-residue over the protein-protein up to the family-family level. The recent accumulation of enormous amounts of sequence data allows for the development of novel, data-driven computational approaches. Notably, these approaches can bridge scales within a single statistical framework. Although being currently applied mostly to isolated problems on single scales, their immense potential for an evolutionary informed, structural systems biology is steadily emerging.

The essential YycFG two-component system of Bacillus subtilis.

The YycFG two-component system, highly conserved in the low G+C gram positives, is essential for cell viability in most organisms in which it has been studied. The system is organized within an operon that includes at least one but often three to four other genes. Products of two of these genes, yycH and yycI, have been shown to have a regulatory role on this two-component system. Immunofluorescent studies identified YycG kinase localization at the cell division sites consistent with its role in regulating cell divisional processes. The essential nature and operon organization of this system commanded special requirements in studying this system genetically. This chapter presents methods utilized in identifying the regulatory circuit that controls the activity of the YycG kinase in Bacillus subtilis. Most aspects of our approaches are applicable to other two-component systems in B. subtilis and the gram positives. Some are limited to essential systems, such as the YycFG system.