NMR assignments for the Sinorhizobium meliloti response regulator Sma0114

Response regulators are terminal ends of bacterial two-component systems that undergo extensive structural reorganization in response to phosphoryl transfer from their cognate histidine kinases. The response regulator encoded by the gene sma0114 of Sinorhizobium meliloti is a part of a unique class of two-component systems that employ HWE histidine kinases. The distinct features of Sma0114 include a PFxFATGY motif that houses the conserved threonine in the “Y–T coupling” conformational switch which mediates output response through downstream protein–protein interactions, and the replacement of the conserved phenylalanine/tyrosine in Y–T coupling by a leucine. Here we present ¹H, ¹⁵N, and ¹³C NMR assignments for Sma0114. We identify the secondary structure of the protein based on TALOS chemical shift analysis, ³J_HNHα coupling constants and hydrogen–deuterium exchange. The secondary structure determined by NMR is in good agreement with that predicted from the sequence. Both methods suggest that Sma0114 differs from standard CheY-like folds by missing the fourth α-helix. Our initial NMR characterization of Sma0114 paves the way to a full investigation of the structure and dynamics of this response regulator.     

Structure of the response regulator PhoP from Mycobacterium tuberculosis reveals a dimer through the receiver domain

The PhoP protein from Mycobacterium tuberculosis is a response regulator of the OmpR/PhoB subfamily, whose structure consists of an N-terminal receiver domain and a C-terminal DNA-binding domain. How the DNA-binding activities are regulated by phosphorylation of the receiver domain remains unclear due to a lack of structural information on the full-length proteins. Here we report the crystal structure of the full-length PhoP of M. tuberculosis. Unlike other known structures of full-length proteins of the same subfamily, PhoP forms a dimer through its receiver domain with the dimer interface involving α4-β5-α5, a common interface for activated receiver domain dimers. However, the switch residues, Thr99 and Tyr118, are in a conformation resembling those of nonactivated receiver domains. The Tyr118 side chain is involved in the dimer interface interactions. The receiver domain is tethered to the DNA-binding domain through a flexible linker and does not impose structural constraints on the DNA-binding domain. This structure suggests that phosphorylation likely facilitates/stabilizes receiver domain dimerization, bringing the DNA-binding domains to close proximity, thereby increasing their binding affinity for direct repeat DNA sequences.     

Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity

In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.     

Conservation of the human integrin-type beta-propeller domain in bacteria

Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca(2+)-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca(2+)-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and sometimes two tandem copies are found.     

SAS solution structures of the apo and Mg2+/BeF3(-)-bound receiver domain of DctD from Sinorhizobium meliloti

Two-component signal transduction is the predominant information processing mechanism in prokaryotes and is also present in single-cell eukaryotes and higher plants. A phosphorylation-based switch is commonly used to activate as many as 40 different types of output domains in more than 6000 two-component response regulators that can be identified in the sequence databases. Previous biochemical and crystallographic studies showed that phosphorylation of the two-component receiver domain of DctD causes a switch between alternative dimeric forms, but it was unclear from the crystal lattice of the activated protein precisely which of four possible dimeric configurations is the biologically relevant one [Park, S., et al. (2002) FASEB J. 16, 1964-1966]. Here we report solution structures of the apo and activated DctD receiver domain derived from small angle scattering data. The apo dimer closely resembles that seen in the crystal structure, and the solution data for the activated protein eliminate two of the possible four dimeric conformations seen in the crystal lattice and strongly implicate one as the biologically relevant structure. These results corroborate the previously proposed model for how receiver domains regulate their downstream AAA+ ATPase domains.     

A common switch in activation of the response regulators NtrC and PhoB: phosphorylation induces dimerization of the receiver modules.

During signal transduction, response regulators of two-component systems are phosphorylated in a conserved receiver module. Phosphorylation induces activation of the non-conserved output domain. We fused various domains of the response regulators NtrC, PhoB or CheB to the DNA binding domain of lambda repressor. Analysis of these hybrid proteins shows that the receiver modules of NtrC and PhoB are potential dimerization domains. In the unphosphorylated proteins, the ability of the receiver modules to dimerize is masked due to inhibition by their output domains. Inhibition can be relieved in two ways: phosphorylation of the receiver module or deletion of the output domain. In contrast, the receiver module of CheB lacks this ability for dimerization. We propose a model which groups response regulators into two classes. Common to both classes is the interaction between receiver and output domain in the unphosphorylated protein. In class I (e.g. NtrC and PhoB), this interaction leads to the inhibition of the receiver module. Phosphorylation relieves inhibition, thereby inducing activation via dimerization of the receiver modules. In class II (e.g. CheB), the interaction between receiver and output domain results in inhibition of the output domain. Phosphorylation relieves inhibition, thereby activating the output domain.     

Constitutive activation of two-component response regulators: characterization of VirG activation in Agrobacterium tumefaciens

Response regulators are the ultimate modulators in two-component signal transduction pathways. The N-terminal receiver domains generally accept phosphates from cognate histidine kinases to control output. VirG for example, the response regulator of the VirA/VirG two-component system in Agrobacterium tumefaciens, mediates the expression of virulence genes in response to plant host signals. Response regulators have a highly conserved structure and share a similar conformational activation upon phosphorylation, yet the sequence and structural features that determine or perturb the cooperative activation events are ill defined. Here we use VirG and the unique features of the Agrobacterium system to extend our understanding of the response regulator activation. Two previously isolated constitutive VirG mutants, VirGN54D and VirGI77V/D52E, provide the foundation for our studies. In vivo phosphorylation patterns establish that VirGN54D is able to accumulate phosphates from small-molecule phosphate donors, such as acetyl phosphate, while the VirGI77V/D52E allele carries conformational changes mimicking the active conformation. Further structural alterations on these two alleles begin to reveal the changes necessary for response regulator activation.     

Structural basis of transmembrane domain interactions in integrin signaling

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric αβ integrins is correlated to the association state of the single-pass α and β transmembrane domains. The association of integrin αIIbβ3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (αIIb) and tilted (β3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual αIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the β3 transmembrane helix, enabling αIIb(D723)β3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/β complex that overlap with the αβ transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.     

Identification and biochemical characterization of WbwB,a novel UDP-Gal: Neu5Ac-R α1,4-galactosyltransferase from the intestinal pathogen <Emphasis Type="Italic">Escherichia coli</Emphasis> serotype O104

The intestinal pathogen Escherichia coli serotype O104:H4 (ECO104) can cause bloody diarrhea and haemolytic uremic syndrome. The ECO104 O antigen has the unique repeating unit structure [4Galα1–4Neu5,7,9Ac₃α2–3Galβ1–3GalNAcβ1-], which includes the mammalian sialyl-T antigen as an internal structure. Previously, we identified WbwC from ECO104 as the β3Gal-transferase that synthesizes the T antigen, and showed that α3-sialyl-transferase WbwA transfers sialic acid to the T antigen. Here we identify the wbwB gene product as a unique α1,4-Gal-transferase WbwB that transfers Gal from UDP-Gal to the terminal sialic acid residue of Neu5Acα2–3Galβ1–3GalNAcα-diphosphate-lipid acceptor. NMR analysis of the WbwB enzyme reaction product indicated that Galα1-4Neu5Acα2–3Galβ1–3GalNAcα-diphosphate-lipid was synthesized. WbwB from ECO104 has a unique acceptor specificity for terminal sialic acid as well as the diphosphate group in the acceptor. The characterization studies showed that WbwB does not require divalent metal ion as a cofactor. Mutagenesis identified Lys243 within an RKR motif and both Glu315 and Glu323 of the fourth EX₇E motif as essential for the activity. WbwB is the final glycosyltransferase in the biosynthesis pathway of the ECO104 antigen repeating unit. This work contributes to knowledge of the biosynthesis of bacterial virulence factors.     

Crystal structure of a cyanobacterial phytochrome response regulator

The two-component signal transduction pathway widespread in prokaryotes, fungi, molds, and some plants involves an elaborate phosphorelay cascade. Rcp1 is the phosphate receiver module in a two-component system controlling the light response of cyanobacteria Synechocystis sp. via cyanobacterial phytochrome Cph1, which recognizes Rcp1 and transfers its phosphoryl group to an aspartate residue in response to light. Here we describe the crystal structure of Rcp1 refined to a crystallographic R-factor of 18.8% at a resolution of 1.9 A. The structure reveals a tightly associated homodimer with monomers comprised of doubly wound five-stranded parallel beta-sheets forming a single-domain protein homologous with the N-terminal activator domain of other response regulators (e.g., chemotaxis protein CheY). The three-dimensional structure of Rcp1 appears consistent with the conserved activation mechanism of phosphate receiver proteins, although in this case, the C-terminal half of its regulatory domain, which undergoes structural changes upon phosphorylation, contributes to the dimerization interface. The involvement of the residues undergoing phosphorylation-induced conformational changes at the dimeric interface suggests that dimerization of Rcp1 may be regulated by phosphorylation, which could affect the interaction of Rcp1 with downstream target molecules.     

A tale of two components: a novel kinase and a regulatory switch

Histidine protein kinases and response regulators form the basis of phosphotransfer signal transduction pathways. Commonly referred to as two-component systems, these modular and adaptable signaling schemes are prevalent in prokaryotes. Structures of the core domains of histidine kinases reveal a protein kinase fold different from that of the Ser/Thr/Tyr protein kinase family, but similar to that of other ATP binding domains. Recent structure determinations of phosphorylated response regulator domains indicate a conserved mechanism for the propagated conformational change that accompanies phosphorylation of an active site Asp residue. The altered molecular surface promotes specific protein-protein interactions that mediate the downstream response.     

Proteomic analysis of α4β1 integrin adhesion complexes reveals α-subunit-dependent protein recruitment

Integrin adhesion receptors mediate cell-cell and cell-extracellular matrix interactions, which control cell morphology and migration, differentiation, and tissue integrity. Integrins recruit multimolecular adhesion complexes to their cytoplasmic domains, which provide structural and mechanosensitive signaling connections between the extracellular and intracellular milieux. The different functions of specific integrin heterodimers, such as α4β1 and α5β1, have been attributed to distinct signal transduction mechanisms that are initiated by selective recruitment of adhesion complex components to integrin cytoplasmic tails. Here, we report the isolation of ligand-induced adhesion complexes associated with wild-type α4β1 integrin, an activated α4β1 variant in the absence of the α cytoplasmic domain (X4C0), and a chimeric α4β1 variant with α5 leg and cytoplasmic domains (α4Pα5L), and the cataloguing of their proteomes by MS. Using hierarchical clustering and interaction network analyses, we detail the differential recruitment of proteins and highlight enrichment patterns of proteins to distinct adhesion complexes. We identify previously unreported components of integrin adhesion complexes and observe receptor-specific enrichment of molecules with previously reported links to cell migration and cell signaling processes. Furthermore, we demonstrate colocalization of MYO18A with active integrin in migrating cells. These datasets provide a resource for future studies of integrin receptor-specific signaling events.     

Specificity of the BvgAS and EvgAS phosphorelay is mediated by the C-terminal HPt domains of the sensor proteins

Despite the presence of highly conserved signalling modules, significant cross-communication between different two-component systems has only rarely been observed. Domain swapping and the characterization of liberated signalling modules enabled us to characterize in vitro the protein domains that mediate specificity and are responsible for the high fidelity in the phosphorelay of the unorthodox Bvg and Evg two-component systems. Under equimolar conditions, significant in vitro phosphorylation of purified BvgA and EvgA proteins was only obtained by their histidine kinases, BvgS and EvgS respectively. One hybrid histidine kinase consisting of the BvgS transmitter and HPt domains and of the EvgS receiver domain (BvgS-TO-EvgS-R) was able to phosphorylate BvgA but not EvgA. In contrast, the hybrid protein consisting of the BvgS transmitter and the EvgS receiver and HPt domains (BvgS-T-EvgS-RO) was unable to phosphorylate BvgA but efficiently phosphorylated EvgA. These results demonstrate that the C-terminal HPt domains of the sensor proteins endow the unorthodox two-component systems with a high specificity for the corresponding regulator protein. In the case of the response regulators, the receiver but not the output domains contribute to the specific interaction with the histidine kinases, because a hybrid protein consisting of the EvgA receiver and the BvgA output domain could only be phosphorylated by the EvgS protein.     

Phosphorylation-Induced Activation of the Response Regulator VraR from Staphylococcus aureus: Insights from Hydrogen Exchange Mass Spectrometry

A two-component system consisting of the histidine kinase vancomycin-resistance-associated sensor and the response regulator vancomycin-resistance-associated regulator (VraR) allows Staphylococcus aureus to sense antibiotic-related cell wall stress and to mount a suitable response. An experimental structure of full-length VraR is not available yet, but previous work points to similarities between VraR and the well-characterized NarL. This work employs hydrogen exchange mass spectrometry to gain insights into the phosphorylation-induced activation of VraR, a process that primes the protein for dimerization and DNA binding. Whereas VraR is highly dynamic, phosphorylated VraR shows less extensive deuteration. This rigidification is most dramatic within the receiver domain, which carries the phosphorylation site D55. Alterations in the DNA-binding domain are much less pronounced. Changes in deuteration within the receiver domain are consistent with a Y-T coupling mechanism. In analogy to NarL, the activation of VraR is thought to involve separation and subsequent reorientation of the two domains, thereby allowing the α8-turn-α9 element to engage in DNA binding. The current work suggests that this structural transition is triggered by a reduction in the effective length of the linker through enhanced hydrogen bonding. In addition, separation of the two domains may be favored by the establishment of noncovalent protein-protein interactions and intradomain contacts at the expense of previously existing interdomain bonds. α9 appears to be packed against the receiver domain in nonactivated VraR. Support is presented for α1 as a dimerization interface in phosphorylated VraR, whereas protein-protein interactions for nonphosphorylated VraR are impeded by extensive disorder in this region.     

High-resolution solution structure of the beryllofluoride-activated NtrC receiver domain

Bacterial receiver domains mediate the cellular response to environmental changes through conformational changes induced by phosphorylation of a conserved aspartate residue. While the structures of several activated receiver domains have recently been determined, there is substantial variation in the conformational changes occurring upon activation. Here we present the high-resolution structure of the activated NtrC receiver domain (BeF(3)(-)-NtrC(r) complex) determined using NMR data, including residual dipolar couplings, yielding a family of structures with a backbone rmsd of 0.57 +/- 0.08 A, which is compared with the previous lower-resolution structure of the phosphorylated protein. Both phosphorylation and beryllofluoride addition induce a shift in register and an axial rotation of alpha-helix 4. In this high-resolution structure, we are able to observe a concerted change in the positions of Thr82 and Tyr101; this correlated change in two conserved residues (termed Y-T coupling) has been considered a general feature of the conformational change in receiver domains upon activation. In NtrC, this correlated side chain shift, leading to the helix reorientation, is distinctly different from the smaller reorganization seen in other activated receiver domains, and involves numerous other residues which do not participate in conformational changes seen in the other systems. Titration of the activated receiver domain with peptides from the NtrC ATPase domain provides direct evidence for interactions on the rearranged face of the receiver domain, which are likely to be responsible for enabling assembly into the active aggregate. Analysis of the active structure also suggests that His84 may play a role in controlling the phosphate hydrolysis rate.     

Activation of a cyanobacterial adenylate cyclase, CyaC, by autophosphorylation and a subsequent phosphotransfer reaction.

The CyaC protein, a cyanobacterial adenylate cyclase, has a unique primary structure composed of the catalytic domain of adenylate cyclase and the conserved domains of bacterial two-component regulatory systems, one transmitter domain and two receiver domains. In the present work, CyaC was produced in Escherichia coli as a histidine-tagged recombinant protein and purified to homogeneity. CyaC showed ability to autophosphorylate in vitro with the gamma-phosphate of [gamma-32P]ATP. CyaC derivatives were constructed by site-directed mutagenesis in which the highly conserved phosphorylation sites in the transmitter domain (His572) and receiver domains (Asp60 or Asp895) were replaced by glutamine and alanine residues, respectively. After autophosphorylation of the CyaC derivatives, the chemical stabilities of the phosphoryl groups bound to the derivatives were determined. It was found that His572 is the initial phosphorylation site and that the phosphoryl group once bound to His572 is transferred to Asp895. The enzyme activities of the CyaC derivatives defective in His572 or Asp895 were considerably reduced. Asp895 is phosphorylated by acetyl [32P]phosphate, a small phosphoryl molecule, but Asp60 is not. Acetyl phosphate stimulates adenylate cyclase activity only when Asp895 is intact. These results suggest that the phosphorylation of Asp895 is essential for the activation of adenylate cyclase and that Asp60 functions differently from Asp895 in regulating the enzyme activity.