Influence of interdomain interfaces. Regulation of response regulator autophosphorylation through interdomain contacts.
Ontology highlight
ABSTRACT:
SUBMITTER:
PROVIDER: S-EPMC2952274 | biostudies-literature | 2010 Oct
REPOSITORIES: biostudies-literature
Publications
Influence of interdomain interfaces. Regulation of response regulator autophosphorylation through interdomain contacts.
The Journal of biological chemistry 20101001 42
Similar Datasets
Project description:DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the ?4-?5-?5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo.
| S-EPMC2952233 | biostudies-literature
Project description:Response regulator proteins within two-component signal transduction systems are activated by phosphorylation and can catalyze their own covalent phosphorylation using small molecule phosphodonors. To date, comprehensive kinetic characterization of response regulator autophosphorylation is limited to CheY, which follows a simple model of phosphodonor binding followed by phosphorylation. We characterized autophosphorylation of the response regulator PhoB, known to dimerize upon phosphorylation. In contrast to CheY, PhoB time traces exhibited an initial lag phase and gave apparent pseudo-first order rate constants that increased with protein concentration. Furthermore, plots of the apparent autophosphorylation rate constant versus phosphodonor concentration were sigmoidal, as were PhoB binding isotherms for the phosphoryl group analog BeF3(-). Successful mathematical modeling of the kinetic data necessitated inclusion of the formation of a PhoB heterodimer (one phosphorylated and one unphosphorylated monomer) with an enhanced rate of phosphorylation. Specifically, dimerization constants for the PhoB heterodimer and homodimer (two phosphorylated monomers) were similar, but the rate constant for heterodimer phosphorylation was ~10-fold higher than for the monomer. In a test of the model, disruption of the known PhoB(N) dimerization interface by mutation led to markedly slower and noncooperative autophosphorylation kinetics. Furthermore, phosphotransfer from the sensor kinase PhoR was enhanced by dimer formation. Phosphorylation-mediated dimerization allows many response regulators to bind to tandem DNA-binding sites and regulate transcription. Our data challenge the notion that response regulator dimers primarily form between two phosphorylated monomers and raise the possibility that response regulator heterodimers containing one phosphoryl group may participate in gene regulation.
| S-EPMC3724633 | biostudies-literature
Project description:Escherichia coli RfaH activates gene expression by tethering the elongating RNA polymerase to the ribosome. This bridging action requires a complete refolding of the RfaH C-terminal domain (CTD) from an α-helical hairpin, which binds to the N-terminal domain (NTD) in the free protein, to a β-barrel, which interacts with the ribosomal protein S10 following RfaH recruitment to its target operons. The CTD forms a β-barrel when expressed alone or proteolytically separated from the NTD, indicating that the α-helical state is trapped by the NTD, perhaps co-translationally. Alternatively, the interdomain contacts may be sufficient to drive the formation of the α-helical form. Here, we use functional and NMR analyses to show that the denatured RfaH refolds into the native state and that RfaH in which the order of the domains is reversed is fully functional in vitro and in vivo. Our results indicate that all information necessary to determine its fold is encoded within RfaH itself, whereas accessory factors or sequential folding of NTD and CTD during translation are dispensable. These findings suggest that universally conserved RfaH homologs may change folds to accommodate diverse interaction partners and that context-dependent protein refolding may be widespread in nature.
| S-EPMC3905879 | biostudies-literature
Project description:Specific binding between proteins plays a crucial role in molecular functions and biological processes. Protein binding interfaces and their atomic contacts are typically defined by simple criteria, such as distance-based definitions that only use some threshold of spatial distance in previous studies. These definitions neglect the nearby atomic organization of contact atoms, and thus detect predominant contacts which are interrupted by other atoms. It is questionable whether such kinds of interrupted contacts are as important as other contacts in protein binding. To tackle this challenge, we propose a new definition called beta (β) atomic contacts. Our definition, founded on the β-skeletons in computational geometry, requires that there is no other atom in the contact spheres defined by two contact atoms; this sphere is similar to the van der Waals spheres of atoms. The statistical analysis on a large dataset shows that β contacts are only a small fraction of conventional distance-based contacts. To empirically quantify the importance of β contacts, we design βACV, an SVM classifier with β contacts as input, to classify homodimers from crystal packing. We found that our βACV is able to achieve the state-of-the-art classification performance superior to SVM classifiers with distance-based contacts as input. Our βACV also outperforms several existing methods when being evaluated on several datasets in previous works. The promising empirical performance suggests that β contacts can truly identify critical specific contacts in protein binding interfaces. β contacts thus provide a new model for more precise description of atomic organization in protein quaternary structures than distance-based contacts.
| S-EPMC3632532 | biostudies-literature
Project description:Capturing the functionally relevant forms of dynamic, multidomain proteins is extremely challenging. Bruton's tyrosine kinase (BTK), a kinase essential for B and mast cell function, has stubbornly resisted crystallization in its full-length form. Here, nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry show that BTK adopts a closed conformation in dynamic equilibrium with open, active conformations. BTK lacks the phosphotyrosine regulatory tail of the SRC kinases, yet nevertheless achieves a phosphotyrosine-independent C-terminal latch. The unique proline-rich region is an internal "on" switch pushing the autoinhibited kinase toward its active state. Newly identified autoinhibitory contacts in the BTK pleckstrin homology domain are sensitive to phospholipid binding, which induces large-scale allosteric changes. The multiplicity of these regulatory contacts suggests a clear mechanism for gradual or "analog" kinase activation as opposed to a binary "on/off" switch. The findings illustrate how previously modeled information for recalcitrant full-length proteins can be expanded and validated with a convergent multidisciplinary experimental approach.
| S-EPMC5629114 | biostudies-literature
Project description:The serralysin family of bacterial metalloproteases is associated with virulence in multiple modes of infection. These extracellular proteases are members of the Repeats-in-ToXin (RTX) family of toxins and virulence factors, which mediated virulence in E. coli, B. pertussis, and P. aeruginosa, as well as other animal and plant pathogens. The serralysin proteases are structurally dynamic and their folding is regulated by calcium binding to a C-terminal domain that defines the RTX family of proteins. Previous studies have suggested that interactions between N-terminal sequences and this C-terminal domain are important for the high thermal and chemical stabilities of the RTX proteases. Extending from this, stabilization of these interactions in the native structure may lead to hyperstabilization of the folded protein. To test this hypothesis, cysteine pairs were introduced into the N-terminal helix and the RTX domain and protease folding and activity were assessed. Under stringent pH and temperature conditions, the disulfide-bonded mutant showed increased protease activity and stability. This activity was dependent on the redox environment of the refolding reaction and could be blocked by selective modification of the cysteine residues before protease refolding. These data demonstrate that the thermal and chemical stability of these proteases is, in part, mediated by binding between the RTX domain and the N-terminal helix and demonstrate that stabilization of this interaction can further stabilize the active protease, leading to additional pH and thermal tolerance.
| S-EPMC4574703 | biostudies-literature
Project description:A discrimination method between biologically relevant interfaces and artificial crystal-packing contacts in crystal structures was constructed. The method evaluates protein-protein interfaces in terms of complementarities for hydrophobicity, electrostatic potential and shape on the protein surfaces, and chooses the most probable biological interfaces among all possible contacts in the crystal. The method uses a discriminator named as "COMP", which is a linear combination of the complementarities for the above three surface features and does not correlate with the contact area. The discrimination of homo-dimer interfaces from symmetry-related crystal-packing contacts based on the COMP value achieved the modest success rate. Subsequent detailed review of the discrimination results raised the success rate to about 88.8%. In addition, our discrimination method yielded some clues for understanding the interaction patterns in several examples in the PDB. Thus, the COMP discriminator can also be used as an indicator of the "biological-ness" of protein-protein interfaces.
| S-EPMC3169932 | biostudies-literature
Project description:A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts.
| S-EPMC8986090 | biostudies-literature
Project description:Understanding how an adaptive integrated interface between lubricant additives and solid contacts works will enable improving the wear and friction of moving engine components. This work represents the comprehensive characterization of compositional and structural orientation at the sliding interface from the perspective of surface/interface tribochemistry. The integrated interface of a lubricant additive-solid resulting from the friction testing of Graphite-like carbon (GLC) and PVD-CrN coated rings sliding against cast iron under boundary lubrication was studied. The results indicate that in the case of the CrN/cast iron pair the antiwear and friction behavior were very strongly dependent upon lubricant. In contrast, the tribology of the GLC surface showed a much lower dependence on lubrication. In order to identify the compounds and their distribution across the interface, x-ray microanalysis phase mapping was innovatively applied and the principle of hard and soft acids and bases (HSAB) to understand the behaviour. Phase mapping clearly showed the hierarchical interface of the zinc-iron polyphosphate tribofilm for various sliding pairs and different sliding durations. This interface structure formed between lubricant additives and the sliding surfaces adapts to the sliding conditions - the term adaptive interface. The current results help explain the tribology of these sliding components in engine.
| S-EPMC5577201 | biostudies-literature
Project description:RfaH is a virulence factor from Escherichia coli whose C-terminal domain (CTD) undergoes a dramatic α-to-β conformational transformation. The CTD in its α-helical fold is stabilized by interactions with the N-terminal domain (NTD), masking an RNA polymerase binding site until a specific recruitment site is encountered. Domain dissociation is triggered upon binding to DNA, allowing the NTD to interact with RNA polymerase to facilitate transcription while the CTD refolds into the β-barrel conformation that interacts with the ribosome to activate translation. However, structural details of this transformation process in the context of the full protein remain to be elucidated. Here, we explore the mechanism of the α-to-β conformational transition of RfaH in the full-length protein using a dual-basin structure-based model. Our simulations capture several features described experimentally, such as the requirement of disruption of interdomain contacts to trigger the α-to-β transformation, confirms the roles of previously indicated residues E48 and R138, and suggests a new important role for F130, in the stability of the interdomain interaction. These native basins are connected through an intermediate state that builds up upon binding to the NTD and shares features from both folds, in agreement with previous in silico studies of the isolated CTD. We also examine the effect of RNA polymerase binding on the stabilization of the β fold. Our study shows that native-biased models are appropriate for interrogating the detailed mechanisms of structural rearrangements during the dramatic transformation process of RfaH.
| S-EPMC4521827 | biostudies-literature