Crystal structure of master biofilm regulator CsgD regulatory domain reveals an atypical receiver domain

The master regulator CsgD switches planktonic growth to biofilm formation by activating synthesis of curli fimbriae and cellulose in Enterobacteriaceae. CsgD was classified to be the LuxR response regulatory family, while its cognate sensor histidine kinase has not been identified yet. CsgD consists of a C‐terminal DNA binding domain and an N‐terminal regulatory domain that provokes the upstream signal transduction to further modulate its function. We provide the crystal structure of Salmonella Typhimurium CsgD regulatory domain, which reveals an atypical β5α5 response regulatory receiver domain folding with the α2 helix representing as a disorder loop compared to the LuxR/FixJ canonical response regulator, and the structure indicated a noteworthy α5 helix similar to the non‐canonical master regulator VpsT receiver domain α6. CsgD regulatory domain assembles with two dimerization interfaces mainly through α1 and α5, which has shown similarity to the c‐di‐GMP independent and stabilized dimerization interface of VpsT from Vibrio cholerae respectively. The potential phosphorylation site D59 is directly involved in the interaction of interfaces I and mutagenesis studies indicated that both dimerization interfaces could be crucial for CsgD activity. The structure reveals important molecular details for the dimerization assembly of CsgD and will shed new insight into its regulation mechanism.     

Solution structures of the inactive and BeF3-activated response regulator CheY2

The chemotactic signalling chain to the flagellar motor of Sinorhizobium meliloti features a new type of response regulator, CheY2. CheY2 activated by phosphorylation (CheY2-P) controls the rotary speed of the flagellar motor (instead of reversing the sense of rotation), and it is efficiently dephosphorylated by phospho-retrotransfer to the cognate kinase, CheA. Here, we report the NMR solution structures of the Mg(2+)-complex of inactive CheY2, and of activated CheY2-BeF(3), a stable analogue of CheY2-P, to an overall root mean square deviation of 0.042 nm and 0.027 nm, respectively. The 14 kDa CheY2 protein exhibits a characteristic open (alpha/beta)(5) conformation. Modification of CheY2 by BeF(3)(-) leads to large conformational changes of the protein, which are in the limits of error identical with those observed by phosphorylation of the active-centre residue Asp58. In BeF(3)-activated CheY2, the position of Thr88-OH favours the formation of a hydrogen bond with the active site, Asp58-BeF(3), similar to BeF(3)-activated CheY from Escherichia coli. In contrast to E.coli, this reorientation is not involved in a Tyr-Thr-coupling mechanism, that propagates the signal from the incoming phosphoryl group to the C-terminally located FliM-binding surface. Rather, a rearrangement of the Phe59 side-chain to interact with Ile86-Leu95-Val96 along with a displacement of alpha4 towards beta5 is stabilised in S.meliloti. The resulting, activation-induced, compact alpha4-beta5-alpha5 surface forms a unique binding domain suited for specific interaction with and signalling to a rotary motor that requires a gradual speed control. We propose that these new features of response regulator activation, compared to other two-component systems, are the key for the observed unique phosphorylation, dephosphorylation and motor control mechanisms in S.meliloti.     

Serine 83 in DosR,a response regulator from Mycobacterium tuberculosis, promotes its transition from an activated,phosphorylated state to an inactive,unphosphorylated state

A sensor kinase, DosS, and its corresponding response regulator, DosR, constitute a two component system for regulating gene expression under hypoxic conditions in Mycobacterium tuberculosis. Among response regulators in M. tuberculosis, NarL has high sequence similarity to DosR, and autophosphorylated DosS transfers its phosphate group not only to DosR but also to NarL. Phosphorylated DosR is more rapidly dephosphorylated than phosphorylated NarL. DosR and NarL differ with respect to the amino acids at positions T + 1 and T + 2 around the phosphorylation sites in the N-terminal phosphoacceptor domain; NarL has S83 and Y84, whereas DosR has A90 and H91. A DosR S83A mutant shows prolonged phosphorylation. Structural comparison with a histidinol phosphate phosphatase suggests that the hydroxyl group of DosR S83 could play a role in activating the water molecule involved in the triggering of autodephosphorylation.     

Structural and functional studies of the response regulator HupR

HupR is a response regulator that controls the synthesis of the membrane-bound [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus. The protein belongs to the NtrC subfamily of response regulators and is the second protein of a two-component system. We have crystallized the full-length protein HupR in the unphosphorylated state in two dimensions using the lipid monolayer technique. The 3D structure of negatively stained HupR was calculated to a resolution of approximately 23 A from tilted electron microscope images. HupR crystallizes as a dimer, and forms an elongated V-shaped structure with extended arms. The dimensions of the dimer are about 80 A length, 40 A width and 85 A thick. The HupR monomer consists of three domains, N-terminal receiver domain, central domain and C-terminal DNA-binding domain. We have fitted the known 3D structure of the central domain from NtrC1 Aquifex aeolicus protein into our 3D model; we propose that contact between the dimers is through the central domain. The N-terminal domain is in contact with the lipid monolayer and is situated on the top of the V-shaped structure. The central domain alone has been expressed and purified; it forms a pentamer in solution and lacks ATPase activity.     

Xanthomonas campestris sensor kinase HpaS co-opts the orphan response regulator VemR to form a branched two-component system that regulates motility

Xanthomonas campestris pv. campestris (Xcc) controls virulence and plant infection mechanisms via the activity of the sensor kinase and response regulator pair HpaS/hypersensitive response and pathogenicity G (HrpG). Detailed analysis of the regulatory role of HpaS has suggested the occurrence of further regulators besides HrpG. Here we used in vitro and in vivo approaches to identify the orphan response regulator VemR as another partner of HpaS and to characterize relevant interactions between components of this signalling system. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacts with VemR. Phos-tag SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of VemR in vivo. Mutation analysis reveals that HpaS and VemR contribute to the regulation of motility and this relationship appears to be epistatic. Additionally, we show that VemR control of Xcc motility is due in part to its ability to interact and bind to the flagellum rotor protein FliM. Taken together, the findings describe the unrecognized regulatory role of sensor kinase HpaS and orphan response regulator VemR in the control of motility in Xcc and contribute to the understanding of the complex regulatory mechanisms used by Xcc during plant infection.     

Phospho‐dependent signaling during the general stress response by the atypical response regulator and ClpXP adaptor RssB

In the model organism Escherichia coli and related species, the general stress response relies on tight regulation of the intracellular levels of the promoter specificity subunit RpoS. RpoS turnover is exclusively dependent on RssB, a two‐domain response regulator that functions as an adaptor that delivers RpoS to ClpXP for proteolysis. Here, we report crystal structures of the receiver domain of RssB both in its unphosphorylated form and bound to the phosphomimic BeF₃ ⁻. Surprisingly, we find only modest differences between these two structures, suggesting that truncating RssB may partially activate the receiver domain to a “meta‐active” state. Our structural and sequence analysis points to RssB proteins not conforming to either the Y–T coupling scheme for signaling seen in prototypical response regulators, such as CheY, or to the signaling model of the less understood FATGUY proteins.     

Structure and function of the NS1 protein of influenza A virus

The avian influenza A virus currently prevailing in Asia causes fatal pneumonia and multipleorgan failure in birds and humans.Despite intensive research,understanding of the characteristics of influenzaA virus that determine its virulence is incomplete.NS1A protein,a non-structural protein of influenza Avirus,was reported to contribute to its pathogenicity and virulence.NS1A protein is a multifunctionalprotein that plays a significant role in resisting the host antiviral response during the influenza infection.Thisreview briefly outlines the current knowledge on the structure and function of the NS1A protein.     

Acetylation of the chemotaxis response regulator CheY by acetyl-CoA synthetase purified from Escherichia coli

Acetylation of CheY, the excitatory response regulator of bacterial chemotaxis, by the enzyme acetyl-CoA synthetase (Acs) is involved in Escherichia coli chemotaxis, but its function is obscure. Here, we overproduced Acs from E.coli, purified it in quantities sufficient for biochemical work, and characterized both the enzyme and the CheY acetylation reaction that it catalyzes. Such characterization is essential for revealing the function of CheY acetylation in chemotaxis. The enzyme exhibited characteristics typical of prokaryotic Acs enzymes, and it could use either acetate or AcCoA as an acetyl donor for CheY acetylation. The Acs-catalyzed acetylation of CheY was reversible, an essential property for a regulatory process, and cooperative (Hill coefficient approximately 3). By Western blotting with specific anti-acetyl-lysine antibody we demonstrated that Acs undergoes autoacetylation, that CheY is acetylated to a small extent when isolated, and that the extent is elevated following in vitro acetylation. Exposing the intact protein to matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electro-spray mass spectrometry, we found that, in most cases, purified CheY is a mixture of species having zero to six acetyl groups per molecule, with non-acetylated CheY being the most abundant species. By proteolytic in-gel digestion of non-treated CheY followed by peptide fingerprinting, precursor ion scan, and tandem mass spectrometry, we found that the acetylation sites of CheY are clustered at the C terminus of the protein, with lysine residues 91, 92, 109, 119, 122 and 126 being the main acetylation sites. Following in vitro acetylation, the main change that seemed to occur was an incremental increase in the extent of acetylation of the same lysine residues. Thus, CheY is similar to many eukaryotic proteins involved in signaling, which undergo both phosphorylation and multiple acetylation, and in which the acetylation sites are restricted to a particular region.     

Evidence for activator and repressor functions of the response regulator MtrA from Corynebacterium glutamicum

Previous analysis of a Corynebacterium glutamicum Delta mtrAB mutant showed that the MtrAB two-component signal transduction system influences the expression of genes involved in cell wall metabolism or osmoregulation, but it remained unknown whether this influence is direct or indirect. In order to identify the direct target genes of the response regulator MtrA, chromatin immunoprecipitation as a genome-wide approach and DNA affinity chromatography as a gene-specific approach were used. The results indicate that mepA and nlpC, both encoding putative cell wall peptidases, are directly repressed by MtrA, whereas proP and betP, both encoding carriers for compatible solutes, are directly activated by MtrA.