scholarly journals Specificity Residues Determine Binding Affinity for Two-Component Signal Transduction Systems

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Jonathan W. Willett ◽  
Nitija Tiwari ◽  
Susanne Müller ◽  
Katherine R. Hummels ◽  
Jon C. D. Houtman ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Myxococcus Xanthus ◽  
Titration Calorimetry ◽  
Signaling Proteins ◽  
Response Regulators ◽  
Histidine Kinases ◽  
Content Type ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACTTwo-component systems (TCS) comprise histidine kinases and their cognate response regulators and allow bacteria to sense and respond to a wide variety of signals. Histidine kinases (HKs) phosphorylate and dephosphorylate their cognate response regulators (RRs) in response to stimuli. In general, these reactions appear to be highly specific and require an appropriate association between the HK and RR proteins. TheMyxococcus xanthusgenome encodes one of the largest repertoires of signaling proteins in bacteria (685 open reading frames [ORFs]), including at least 127 HKs and at least 143 RRs. Of these, 27 arebona fideNtrC-family response regulators, 21 of which are encoded adjacent to their predicted cognate kinases. Using system-wide profiling methods, we determined that the HK-NtrC RR pairs display a kinetic preference during both phosphotransfer and phosphatase functions, thereby defining cognate signaling systems inM. xanthus. Isothermal titration calorimetry measurements indicated that cognate HK-RR pairs interact with dissociation constants (Kd) of approximately 1 µM, while noncognate pairs had no measurable binding. Lastly, a chimera generated between the histidine kinase, CrdS, and HK1190 revealed that residues conferring phosphotransfer and phosphatase specificity dictate binding affinity, thereby establishing discrete protein-protein interactions which prevent cross talk. The data indicate that binding affinity is a critical parameter governing system-wide signaling fidelity for bacterial signal transduction proteins.IMPORTANCEUsingin vitrophosphotransfer and phosphatase profiling assays and isothermal titration calorimetry, we have taken a system-wide approach to demonstrate specificity for a family of two-component signaling proteins inMyxococcus xanthus. Our results demonstrate that previously identified specificity residues dictate binding affinity and that phosphatase specificity follows phosphotransfer specificity for cognate HK-RR pairs. The data indicate that preferential binding affinity is the basis for signaling fidelity in bacterial two-component systems.

scholarly journals Blue Light Regulated Two-Component Systems: Enzymatic and Functional Analyses of Light-Oxygen-Voltage (LOV)-Histidine Kinases and Downstream Response Regulators

Biochemistry ◽  
2013 ◽  
Vol 52 (27) ◽  
pp. 4656-4666 ◽  
Author(s):  
Fernando Correa ◽  
Wen-Huang Ko ◽  
Victor Ocasio ◽  
Roberto A. Bogomolni ◽  
Kevin H. Gardner
Keyword(s):  
Blue Light ◽  
Response Regulators ◽  
Histidine Kinases ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Functional Analyses

scholarly journals Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Felipe Trajtenberg ◽  
Juan A Imelio ◽  
Matías R Machado ◽  
Nicole Larrieux ◽  
Marcelo A Marti ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Response Regulators ◽  
Histidine Kinases ◽  
Signaling Systems ◽  
Input Signals ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Conformational Rearrangements ◽  
Phosphoryl Groups

Two-component systems (TCS) are protein machineries that enable cells to respond to input signals. Histidine kinases (HK) are the sensory component, transferring information toward downstream response regulators (RR). HKs transfer phosphoryl groups to their specific RRs, but also dephosphorylate them, overall ensuring proper signaling. The mechanisms by which HKs discriminate between such disparate directions, are yet unknown. We now disclose crystal structures of the HK:RR complex DesK:DesR from Bacillus subtilis, comprising snapshots of the phosphotransfer and the dephosphorylation reactions. The HK dictates the reactional outcome through conformational rearrangements that include the reactive histidine. The phosphotransfer center is asymmetric, poised for dissociative nucleophilic substitution. The structural bases of HK phosphatase/phosphotransferase control are uncovered, and the unexpected discovery of a dissociative reactional center, sheds light on the evolution of TCS phosphotransfer reversibility. Our findings should be applicable to a broad range of signaling systems and instrumental in synthetic TCS rewiring.

scholarly journals Unequal twins: Unraveling the reaction mechanism of dimeric histidine kinases

2020 ◽  
Vol 295 (23) ◽  
pp. 8118-8119
Author(s):  
Wolfgang Gärtner
Keyword(s):  
Basic Function ◽  
Response Regulators ◽  
Histidine Kinases ◽  
Open Questions ◽  
Component Systems ◽  
Two Component ◽  
New Strategy ◽  
Two Component Systems ◽  
Future Work ◽  
Partner Proteins

Histidine kinases (HKs), together with their partner proteins, the response regulators (RRs), form the ubiquitous two-component systems that are global players in control and adjustment of microbial lifestyle. Although their basic function (i.e. the transfer of a phosphate group from the HK to its RR partner) is simple to articulate, deciphering the molecular details of this process has proven anything but simple, especially when quantitative aspects come into play. Bouillet et al. report a series of elegant and sophisticated experiments to quantitatively understand HK functions, clearing up several open questions and providing a new strategy for future work in the field.

scholarly journals Allosteric Activation of Bacterial Response Regulators: the Role of the Cognate Histidine Kinase Beyond Phosphorylation

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Felipe Trajtenberg ◽  
Daniela Albanesi ◽  
Natalia Ruétalo ◽  
Horacio Botti ◽  
Ariel E. Mechaly ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Histidine Kinase ◽  
Response Regulator ◽  
Activation Mechanism ◽  
Response Regulators ◽  
Content Type ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors. IMPORTANCE The ability to sense and respond to environmental variations is an essential property for cell survival. Two-component systems mediate key signaling pathways that allow bacteria to integrate extra- or intracellular signals. Here we focus on the DesK/DesR system, which acts as a molecular thermometer in B. subtilis, regulating the cell membrane’s fluidity. Using a combination of complementary approaches, including determination of the crystal structures of active and inactive forms of the response regulator DesR, we unveil novel molecular mechanisms of DesR’s activation switch. In particular, we show that the association of the cognate histidine kinase DesK triggers DesR activation beyond the transfer of the phosphoryl group. On the basis of sequence and structural analyses of other two-component systems, this activation mechanism appears to be used in a wide range of sensory systems, contributing a further level of specificity control among different signaling pathways.

scholarly journals Specificity of Subtilin-Mediated Activation of Histidine Kinase SpaK

2017 ◽  
Vol 83 (18) ◽  
Author(s):  
Christoph Geiger ◽  
Tobias Spieß ◽  
Sophie Marianne Korn ◽  
Peter Kötter ◽  
Karl-Dieter Entian
Keyword(s):  
Bacillus Subtilis ◽  
Histidine Kinase ◽  
Two Component System ◽  
Histidine Kinases ◽  
Component System ◽  
Content Type ◽  
Component Systems ◽  
Two Component ◽  
Specific Manner ◽  
Two Component Systems

ABSTRACT Autoinduction via two-component systems is a widespread regulatory mechanism that senses environmental and metabolic changes. Although the lantibiotics nisin and subtilin are closely related and share the same lanthionine ring structure, they autoinduce their biosynthesis in a highly specific manner. Subtilin activates only the two-component system SpaRK of Bacillus subtilis, whereas nisin activates solely the two-component system NisRK of Lactococcus lactis. To identify components that determine the specificity of subtilin autoinduction, several variants of the respective lantibiotics were analyzed for their autoinductive capacities. Here, we show that amino acid position 20 is crucial for SpaK activation, as an engineered nisin molecule with phenylalanine at position 20 (nisin N20F) was able to activate SpaK in a specific manner. In combination with the N-terminal tryptophan of subtilin (nisin I1W/N20F), SpaK autoinduction reached almost the level of subtilin-mediated autoinduction. Furthermore, the overall structure of subtilin is also important for its association with the histidine kinase. The destruction of the second lanthionine ring (subtilin C11A, ring B), as well as mutations that interfere with the flexibility of the hinge region located between lanthionine rings C and D (subtilin L21P/Q22P), abolished SpaK autoinduction. Although the C-terminal part of subtilin is needed for efficient SpaK autoinduction, the destruction of lanthionine rings D and E had no measurable impact. Based on these findings, a model for the interaction of subtilin with histidine kinase SpaK was established. IMPORTANCE Although two-component systems are important regulatory systems that sense environmental changes, very little information on the molecular mechanism of sensing or the interaction of the sensor with its respective kinase is available. The strong specificity of linear lantibiotics such as subtilin and nisin for their respective kinases provides an excellent model system to unravel the structural needs of these lantibiotics for activating histidine kinases in a specific manner. More than that, the biosyntheses of lantibiotics are autoinduced via two-component systems. Therefore, an understanding of their interactions with histidine kinases is needed for the biosynthesis of newly engineered peptide antibiotics. Using a Bacillus subtilis-based reporter system, we were able to identify the molecular constraints that are necessary for specific SpaK activation and to provide SpaK specificity to nisin with just two point mutations.

Two-Component Sensing and Regulation: How Do Histidine Kinases Talk with Response Regulators at the Molecular Level?

2019 ◽  
Vol 73 (1) ◽  
pp. 507-528 ◽  
Author(s):  
Alejandro Buschiazzo ◽  
Felipe Trajtenberg
Keyword(s):  
Molecular Level ◽  
Response Regulator ◽  
Response Regulators ◽  
Histidine Kinases ◽  
Universal Principles ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Internal Information ◽  
Living Organisms

Perceiving environmental and internal information and reacting in adaptive ways are essential attributes of living organisms. Two-component systems are relevant protein machineries from prokaryotes and lower eukaryotes that enable cells to sense and process signals. Implicating sensory histidine kinases and response regulator proteins, both components take advantage of protein phosphorylation and flexibility to switch conformations in a signal-dependent way. Dozens of two-component systems act simultaneously in any given cell, challenging our understanding about the means that ensure proper connectivity. This review dives into the molecular level, attempting to summarize an emerging picture of how histidine kinases and cognate response regulators achieve required efficiency, specificity, and directionality of signaling pathways, properties that rely on protein:protein interactions. α helices that carry information through long distances, the fine combination of loose and specific kinase/regulator interactions, and malleable reaction centers built when the two components meet emerge as relevant universal principles.

scholarly journals Involvement of Two-Component Signaling on Bacterial Motility and Biofilm Development

2017 ◽  
Vol 199 (18) ◽  
Author(s):  
Birgit M. Prüß
Keyword(s):  
Response Regulators ◽  
Signaling Mechanism ◽  
Content Type ◽  
Global Regulators ◽  
Phenotypic Changes ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
K 12 ◽  
Biofilm Development

ABSTRACT Two-component signaling is a specialized mechanism that bacteria use to respond to changes in their environment. Nonpathogenic strains of Escherichia coli K-12 harbor 30 histidine kinases and 32 response regulators, which form a network of regulation that integrates many other global regulators that do not follow the two-component signaling mechanism, as well as signals from central metabolism. The output of this network is a multitude of phenotypic changes in response to changes in the environment. Among these phenotypic changes, many two-component systems control motility and/or the formation of biofilm, sessile communities of bacteria that form on surfaces. Motility is the first reversible attachment phase of biofilm development, followed by a so-called swim or stick switch toward surface organelles that aid in the subsequent phases. In the mature biofilm, motility heterogeneity is generated by a combination of evolutionary and gene regulatory events.

scholarly journals Elucidation of Regulatory Modes for Five Two-Component Systems in Escherichia coli Reveals Novel Relationships

mSystems ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Kumari Sonal Choudhary ◽  
Julia A. Kleinmanns ◽  
Katherine Decker ◽  
Anand V. Sastry ◽  
Ye Gao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Target Gene ◽  
Target Genes ◽  
Rna Seq ◽  
Content Type ◽  
E Coli ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT Escherichia coli uses two-component systems (TCSs) to respond to environmental signals. TCSs affect gene expression and are parts of E. coli’s global transcriptional regulatory network (TRN). Here, we identified the regulons of five TCSs in E. coli MG1655: BaeSR and CpxAR, which were stimulated by ethanol stress; KdpDE and PhoRB, induced by limiting potassium and phosphate, respectively; and ZraSR, stimulated by zinc. We analyzed RNA-seq data using independent component analysis (ICA). ChIP-exo data were used to validate condition-specific target gene binding sites. Based on these data, we do the following: (i) identify the target genes for each TCS; (ii) show how the target genes are transcribed in response to stimulus; and (iii) reveal novel relationships between TCSs, which indicate noncognate inducers for various response regulators, such as BaeR to iron starvation, CpxR to phosphate limitation, and PhoB and ZraR to cell envelope stress. Our understanding of the TRN in E. coli is thus notably expanded. IMPORTANCE E. coli is a common commensal microbe found in the human gut microenvironment; however, some strains cause diseases like diarrhea, urinary tract infections, and meningitis. E. coli’s two-component systems (TCSs) modulate target gene expression, especially related to virulence, pathogenesis, and antimicrobial peptides, in response to environmental stimuli. Thus, it is of utmost importance to understand the transcriptional regulation of TCSs to infer bacterial environmental adaptation and disease pathogenicity. Utilizing a combinatorial approach integrating RNA sequencing (RNA-seq), independent component analysis, chromatin immunoprecipitation coupled with exonuclease treatment (ChIP-exo), and data mining, we suggest five different modes of TCS transcriptional regulation. Our data further highlight noncognate inducers of TCSs, which emphasizes the cross-regulatory nature of TCSs in E. coli and suggests that TCSs may have a role beyond their cognate functionalities. In summary, these results can lead to an understanding of the metabolic capabilities of bacteria and correctly predict complex phenotype under diverse conditions, especially when further incorporated with genome-scale metabolic models.

scholarly journals Conformational dynamics of the essential sensor histidine kinase WalK

2017 ◽  
Vol 73 (10) ◽  
pp. 793-803 ◽  
Author(s):  
Yongfei Cai ◽  
Mingyang Su ◽  
Ashfaq Ahmad ◽  
Xiaojie Hu ◽  
Jiayan Sang ◽  
...  
Keyword(s):  
Dynamic Properties ◽  
Conformational Dynamics ◽  
Bound State ◽  
Response Regulators ◽  
Histidine Kinases ◽  
Sequence Identity ◽  
Component Systems ◽  
Bacterial Signal Transduction ◽  
Two Component Systems ◽  
Intrinsic Dynamic

Two-component systems (TCSs) are key elements in bacterial signal transduction in response to environmental stresses. TCSs generally consist of sensor histidine kinases (SKs) and their cognate response regulators (RRs). Many SKs exhibit autokinase, phosphoryltransferase and phosphatase activities, which regulate RR activity through a phosphorylation and dephosphorylation cycle. However, how SKs perform different enzymatic activities is poorly understood. Here, several crystal structures of the minimal catalytic region of WalK, an essential SK fromLactobacillus plantarumthat shares 60% sequence identity with its homologue VicK fromStreptococcus mutans, are presented. WalK adopts an asymmetrical closed structure in the presence of ATP or ADP, in which one of the CA domains is positioned close to the DHp domain, thus leading both the β- and γ-phosphates of ATP/ADP to form hydrogen bonds to the ∊- but not the δ-nitrogen of the phosphorylatable histidine in the DHp domain. In addition, the DHp domain in the ATP/ADP-bound state has a 25.7° asymmetrical helical bending coordinated with the repositioning of the CA domain; these processes are mutually exclusive and alternate in response to helicity changes that are possibly regulated by upstream signals. In the absence of ATP or ADP, however, WalK adopts a completely symmetric open structure with its DHp domain centred between two outward-reaching CA domains. In summary, these structures of WalK reveal the intrinsic dynamic properties of an SK structure as a molecular basis for multifunctionality.

scholarly journals Molecular Characterization of Two-Component Systems of Helicobacter pylori

2000 ◽  
Vol 182 (8) ◽  
pp. 2068-2076 ◽  
Author(s):  
Dagmar Beier ◽  
Rainer Frank
Keyword(s):  
Helicobacter Pylori ◽  
Histidine Kinase ◽  
Response Regulator ◽  
Response Regulators ◽  
Reading Frame ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT Two-component systems are frequently involved in the adaptation of bacteria to changing environmental conditions at the level of transcriptional regulation. Here we report the characterization of members of the two-component systems of the gastric pathogenHelicobacter pylori deduced from the genome sequence of strain 26695. We demonstrate that the response regulators HP166, HP1043, and HP1021 have essential functions, as disruption of the corresponding genes is lethal for the bacteria, irrespective of the fact that HP1043 and HP1021 have nonconserved substitutions in crucial amino acids of their receiver domains. An analysis of the in vitro phosphorylation properties of the two-component proteins demonstrates that HP244-HP703 and HP165-HP166 are cognate histidine kinase-response regulator pairs. Furthermore, we provide evidence that the variability of the histidine kinase HP165 caused by a poly(C) tract of variable length close to the 3′ end of open reading frame 165/164 does not interfere with the kinase activity of the transmitter domain of HP165.

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