Structural transitions in the FixJ receiver domain

BACKGROUND: Two-component signal transduction pathways are sophisticated phosphorelay cascades widespread in prokaryotes and also found in fungi, molds and plants. FixL/FixJ is a prototypical system responsible for the regulation of nitrogen fixation in the symbiotic bacterium Sinorhizobium meliloti. In microaerobic conditions the membrane-bound kinase FixL uses ATP to transphosphorylate a histidine residue, and the response regulator FixJ transfers the phosphoryl group from the phosphohistidine to one of its own aspartate residues in a Mg(2+)-dependent mechanism. RESULTS: Seven X-ray structures of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) have been solved from two crystal forms soaked in different conditions. Three conformations of the protein were found. In the first case, the protein fold impairs metal binding in the active site and the structure reveals a receiver domain that is self-inhibited for catalysis. In the second conformation, the canonical geometry of the active site is attained, and subsequent metal binding to the protein induces minimal conformational changes. The third conformation illustrates a non-catalytic form of the protein where unwinding of the N terminus of helix alpha 1 has occurred. Interconversion of the canonical and self-inhibited conformations requires a large conformational change of the beta 3-alpha 3 loop region. CONCLUSIONS: These unphosphorylated structures of FixJN stress the importance of flexible peptide segments that delineate the active site. Their movements may act as molecular switches that define the functional status of the protein. Such observations are in line with structural and biochemical results obtained on other response regulator proteins and may illustrate general features that account for the specificity of protein-protein interactions.     

Styrene-catabolism regulation in Pseudomonas fluorescens ST: phosphorylation of StyR induces dimerization and cooperative DNA-binding

Styrene is an important chemical extensively used in the petrochemical and polymer industries. In Pseudomonas fluorescens ST, styrene metabolism is controlled by a two-component regulatory system, very uncommon in the degradation of aromatic compounds. The two-component regulatory proteins StyS and StyR regulate the expression of the styABCD operon, which codes for styrene degradation. StyS corresponds to the sensor kinase and StyR to the response regulator, which is essential for the activation of PstyA, the promoter of the catabolic operon. In two-component systems, the response regulator is phosphorylated by the cognate sensor kinase. Phosphorylation activates the response regulator, inducing DNA-binding. The mechanism underlying this activation has been reported only for a very few response regulators. Here, the effect of phosphorylation on the oligomeric state and on the DNA-binding properties of StyR has been investigated. Phosphorylation induces dimerization of StyR, the affinity of dimeric StyR for the target DNA is higher than that of the monomer, moreover dimeric StyR binding to the DNA target is cooperative. Furthermore, StyR oligomerization may be driven by the DNA target. This is the first direct demonstration that StyR response regulator binds to the PstyA promoter.     

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.     

The NMR structure of the Staphylococcus aureus response regulator VraR DNA binding domain reveals a dynamic relationship between it and its associated receiver domain

In Staphylococcus aureus, a two-component signaling system consisting of the histidine kinase VraS and the response regulator VraR stimulates gene expression in response to antibiotics that inhibit cell wall formation. With respect to understanding the mechanism of the VraSR response and precise interaction of VraR at promoter sites, the structure of the VraR DNA binding domain (DBD) was determined using NMR methods. The DBD demonstrates a four-helix configuration that is shared with the NarL/FixJ family of response regulators and is monomeric in solution. Unobservable amide resonances in VraR NMR spectra coincided with a set of DNA backbone contact sites predicted from a model of a VraR-DNA complex. This observation suggests that a degree of conformational sampling is required to achieve a high-affinity interaction with DNA. On the basis of chemical shift differences and line broadening, an amino-terminal 3 10 helix and a portion of helix H4 identify a continuous surface that may link the DBD to the receiver domain. The full-length VraR protein thermally denatured with a single transition, suggesting that the receiver domain and DBD were integrated and not simply tethered. Of note, the DBD alone denatured at a temperature that was 21 degrees C higher than that of the full-length protein. Thus, the DBD appears to be thermodynamically and structurally sensitive to state of the receiver domain.     

Vaccinia topoisomerase mutants illuminate conformational changes during closure of the protein clamp and assembly of a functional active site.

We present a mutational analysis of vaccinia topoisomerase that highlights the contributions of five residues in the catalytic domain (Phe-88 and Phe-101 in helix alpha1, Ser-204 in alpha5, and Lys-220 and Asn-228 in alpha6) to the DNA binding and transesterification steps. When augmented by structural information from exemplary type IB topoisomerases and tyrosine recombinases in different functional states, the results suggest how closure of the protein clamp around duplex DNA and assembly of a functional active site might be orchestrated by internal conformational changes in the catalytic domain. Lys-220 is a constituent of the active site, and a positive charge at this position is required for optimal DNA cleavage. Ser-204 and Asn-228 appear not to be directly involved in reaction chemistry at the scissile phosphodiester. We propose that (i) Asn-228 recruits the Tyr-274 nucleophile to the active site by forming a hydrogen bond to the main chain of the tyrosine-containing alpha8 helix and that (ii) contacts between Ser-204 and the DNA backbone upstream of the cleavage site trigger a separate conformational change required for active site assembly. Mutations of Phe-88 and Phe-101 affect DNA binding, most likely at the clamp closure step, which we posit to entail a distortion of helix alpha1.     

Solution structures and DNA binding properties of the N-terminal SAP domains of SUMO E3 ligases from Saccharomyces cerevisiae and Oryza sativa

SUMO E3 ligase of the Siz/PIAS family that promotes sumoylation of target proteins contains SAP motif in its N-terminal region. The SAP motif with a consensus sequence of 35 residues was first proposed to be as a new DNA binding motif found in diverse nuclear proteins involved in chromosomal organization. We have determined solution structures of the SAP domains of SUMO ligases Siz1 from yeast and rice by NMR spectroscopy, showing that the structure of the SAP domain (residues 2-105) of rice Siz1 is a four-helix bundle with an up-down-extended loop-down-up topology, whereas the SAP domain (residues 1-111) of yeast Siz1 is comprised of five helices where the fifth helix alpha5 causes a significant change in the alignment of the four-helix bundle characteristic to the SAP domains of the Siz/PIAS family. We have also demonstrated that both SAP domains have binding ability to an A/T-rich DNA, but that binding affinity of yeast Siz1 SAP is at least by an order of magnitude higher than that of rice Siz1 SAP. Our NMR titration experiments clearly showed that yeast Siz1 SAP uses alpha2-helix for DNA binding more effectively than rice Siz1 SAP, which would result from the dislocation of this helix due to the existence of the extra helix alpha5. In addition, based on the structures of the SAP domains determined here and registered in Protein Data Bank, general features of structures of the SAP domains are discussed in conjunction with equivocal nature of their DNA binding.     

The conserved Asn49 of maize glutathione S-transferase I modulates substrate binding, catalysis and intersubunit communication.

The functional and structural role of the conserved Asn49 of theta class maize glutathione S-transferase was investigated by site-directed mutagenesis. Asn49 is located in the type I beta turn formed by residues 49-52, and is involved in extensive hydrogen-bonding interactions between alpha helix 2 and the rest of the N-terminal domain. The substitution of Asn49 with Ala induces positive cooperativity for 1-chloro-2,4-dinitrobenzene (CDNB) binding as reflected by a Hill coefficient of 1.9 (S(0.5)CDNB = 0.43 mm). The positive cooperativity is also confirmed by following the isothermic binding of 1-hydroxyl-2,4-dinitrobenzene (HDNB) by UV-difference spectroscopy. In addition, the mutated enzyme exhibits: (a) an increase in the Km(GSH) value of about 6.5-fold, and decrease in kcat value of about fourfold; (b) viscosity-independent kinetic parameters; (c) lower thermostability, and (d) increased susceptibility to proteolytic attack by trypsin, when compared to the wild-type enzyme. It is concluded that Asn49 affects the rate-limiting step of the catalytic reaction, and contributes significantly to the structural and binding characteristics of both the glutathione binding site (G-site) and the electrophile substrate binding site (H-site) by affecting the structural integrity of a type I beta turn (comprising residues 49-52) and probably the flexibility of the highly mobile short 310 helical segment of alpha helix 2 (residues 35-46). These structural perturbations are probably transmitted, via Phe51 and Phe65, to alpha helix H3" of the adjacent subunit which contains key residues that interact with the electrophile substrate and contribute to the monomer-monomer contact region. This may accounts for the positive cooperativity observed.     

Structure of the DNA-binding domain of the response regulator PhoP from Mycobacterium tuberculosis

The PhoP-PhoR two-component signaling system from Mycobacterium tuberculosis is essential for the virulence of the tubercle bacillus. The response regulator, PhoP, regulates expression of over 110 genes. In order to elucidate the regulatory mechanism of PhoP, we determined the crystal structure of its DNA-binding domain (PhoPC). PhoPC exhibits a typical fold of the winged helix-turn-helix subfamily of response regulators. The structure starts with a four-stranded antiparallel beta-sheet, followed by a three-helical bundle of alpha-helices, and then a C-terminal beta-hairpin, which together with a short beta-strand between the first and second helices forms a three-stranded antiparallel beta-sheet. Structural elements are packed through a hydrophobic core, with the first helix providing a scaffold for the rest of the domain to pack. The second and third helices and the long, flexible loop between them form the helix-turn-helix motif, with the third helix being the recognition helix. The C-terminal beta-hairpin turn forms the wing motif. The molecular surfaces around the recognition helix and the wing residues show strong positive electrostatic potential, consistent with their roles in DNA binding and nucleotide sequence recognition. The crystal packing of PhoPC gives a hexamer ring, with neighboring molecules interacting in a head-to-tail fashion. This packing interface suggests that PhoPC could bind DNA in a tandem association. However, this mode of DNA binding is likely to be nonspecific because the recognition helix is partially blocked and would be prevented from inserting into the major groove of DNA. Detailed structural analysis and implications with respect to DNA binding are discussed.     

G protein-coupled receptor Kinase 2/G alpha q/11 interaction. A novel surface on a regulator of G protein signaling homology domain for binding G alpha subunits

G protein-coupled receptors (GPCRs) transduce cellular signals from hormones, neurotransmitters, light, and odorants by activating heterotrimeric guanine nucleotide-binding (G) proteins. For many GPCRs, short term regulation is initiated by agonist-dependent phosphorylation by GPCR kinases (GRKs), such as GRK2, resulting in G protein/receptor uncoupling. GRK2 also regulates signaling by binding G alpha(q/ll) and inhibiting G alpha(q) stimulation of the effector phospholipase C beta. The binding site for G alpha(q/ll) resides within the amino-terminal domain of GRK2, which is homologous to the regulator of G protein signaling (RGS) family of proteins. To map the Galpha(q/ll) binding site on GRK2, we carried out site-directed mutagenesis of the RGS homology (RH) domain and identified eight residues, which when mutated, alter binding to G alpha(q/ll). These mutations do not alter the ability of full-length GRK2 to phosphorylate rhodopsin, an activity that also requires the amino-terminal domain. Mutations causing G alpha(q/ll) binding defects impair recruitment to the plasma membrane by activated G alpha(q) and regulation of G alpha(q)-stimulated phospholipase C beta activity when introduced into full-length GRK2. Two different protein interaction sites have previously been identified on RH domains. The G alpha binding sites on RGS4 and RGS9, called the "A" site, is localized to the loops between helices alpha 3 and alpha 4, alpha 5 and alpha 6, and alpha 7 and alpha 8. The adenomatous polyposis coli (APC) binding site of axin involves residues on alpha helices 3, 4, and 5 (the "B" site) of its RH domain. We demonstrate that the G alpha(q/ll) binding site on the GRK2 RH domain is distinct from the "A" and "B" sites and maps primarily to the COOH terminus of its alpha 5 helix. We suggest that this novel protein interaction site on an RH domain be designated the "C" site.