Structural insights into VirB-DNA complexes reveal mechanism of transcriptional activation of virulence genes

VirB activates transcription of virulence genes in Shigella flexneri by alleviating heat-stable nucleoid-structuring protein-mediated promoter repression. VirB is unrelated to the conventional transcriptional regulators, but homologous to the plasmid partitioning proteins. We determined the crystal structures of VirB HTH domain bound by the cis-acting site containing the inverted repeat, revealing that the VirB-DNA complex is related to ParB-ParS-like complexes, presenting an example that a ParB-like protein acts exclusively in transcriptional regulation. The HTH domain of VirB docks DNA major groove and provides multiple contacts to backbone and bases, in which the only specific base readout is mediated by R167. VirB only recognizes one half site of the inverted repeats containing the most matches to the consensus for VirB binding. The binding of VirB induces DNA conformational changes and introduces a bend at an invariant A-tract segment in the cis-acting site, suggesting a role of DNA remodeling. VirB exhibits positive cooperativity in DNA binding that is contributed by the C-terminal domain facilitating VirB oligomerization. The isolated HTH domain only confers partial DNA specificity. Additional determinants for sequence specificity may reside in N- or C-terminal domains. Collectively, our findings support and extend a previously proposed model for relieving heat-stable nucleoid-structuring protein-mediated repression by Vir

Where is crystallography going?

Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and cryo-crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments. In addition, X-ray free-electron lasers offer to capture fragments of reciprocal space without radiation damage, and open up the subpicosecond regime of protein dynamics and activity. But electrons have recently stolen the limelight: so is X-ray crystallography in rude health, or will imaging methods, especially single-particle electron microscopy, render it obsolete for the most interesting biology, whilst electron diffraction enables structure determination from even the smallest crystals? We will lay out some information to help you decide

Structural characterization of the ribonuclease H-like type ASKHA superfamily kinase MK0840 from Methanopyrus kandleri

Murein recycling is a process in which microorganisms recover peptidoglycan-degradation products in order to utilize them in cell wall biosynthesis or basic metabolic pathways. Methanogens such as Methanopyrus kandleri contain pseudomurein, which differs from bacterial murein in its composition and branching. Here, four crystal structures of the putative sugar kinase MK0840 from M. kandleri in apo and nucleotide-bound states are reported. MK0840 shows high similarity to bacterial anhydro-N-acetylmuramic acid kinase, which is involved in murein recycling. The structure shares a common fold with panthothenate kinase and the 2-hydroxyglutaryl-CoA dehydratase component A, both of which are members of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases. Local conformational changes in the nucleotide-binding site between the apo and holo forms are observed upon nucleotide binding. Further insight is given into domain movements and putative active-site residues are identified

Structural insights into VirB-DNA complexes reveal mechanism of transcriptional activation of virulence genes

VirB activates transcription of virulence genes in Shigella flexneri by alleviating heat-stable nucleoid-structuring protein-mediated promoter repression. VirB is unrelated to the conventional transcriptional regulators, but homologous to the plasmid partitioning proteins. We determined the crystal structures of VirB HTH domain bound by the cis-acting site containing the inverted repeat, revealing that the VirB-DNA complex is related to ParB-ParS-like complexes, presenting an example that a ParB-like protein acts exclusively in transcriptional regulation. The HTH domain of VirB docks DNA major groove and provides multiple contacts to backbone and bases, in which the only specific base readout is mediated by R167. VirB only recognizes one half site of the inverted repeats containing the most matches to the consensus for VirB binding. The binding of VirB induces DNA conformational changes and introduces a bend at an invariant A-tract segment in the cis-acting site, suggesting a role of DNA remodeling. VirB exhibits positive cooperativity in DNA binding that is contributed by the C-terminal domain facilitating VirB oligomerization. The isolated HTH domain only confers partial DNA specificity. Additional determinants for sequence specificity may reside in N- or C-terminal domains. Collectively, our findings support and extend a previously proposed model for relieving heat-stable nucleoid-structuring protein-mediated repression by Vir

Crystal structure of Ca²⁺/H⁺ antiporter protein YfkE reveals the mechanisms of Ca²⁺ efflux and its pH regulation

Ca2+ efflux by Ca2+ cation antiporter (CaCA) proteins is important for maintenance of Ca2+ homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na+/Ca2+ exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca2+ efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca2+/H+ antiporter protein YfkE from Bacillus subtilis at 3.1-Å resolution. YfkE forms a homotrimer, confirmed by disulfide crosslinking. The protonated state of YfkE exhibits an inward-facing conformation with a large hydrophilic cavity opening to the cytoplasm in each protomer and ending in the middle of the membrane at the Ca2+-binding site. A hydrophobic “seal” closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca2+/H+ exchange pathway. In the Ca2+-binding site, two essential glutamate residues exhibit different conformations compared with their counterparts in NCX_Mj, whereas several amino acid substitutions occlude the Na+-binding sites. The structural differences between the inward-facing YfkE and the outward-facing NCX_Mj suggest that the conformational transition is triggered by the rotation of the kink angles of transmembrane helices 2 and 7 and is mediated by large conformational changes in their adjacent transmembrane helices 1 and 6. Our structural and mutational analyses not only establish structural bases for mechanisms of Ca2+/H+ exchange and its pH regulation but also shed light on the evolutionary adaptation to different energy modes in the CaCA protein family

The two conformations of <i>Af</i>AmzAs substrate-binding site.

<p>Superposition of NHis-<i>Af</i>AmzA (yellow) and nat-<i>Af</i>AmzA (green) substrate binding site. Changes in the position of side chains in the bulge edge segment (Met78, Phe80,82), side chains and the main chain of the S1′-wall forming segment (Phe136, Asn138) and in the position of the catalytic water molecule (H₂O_cat) are indicated by the arrows.</p

Structure of <i>Af</i>AmzA.

<p>Overall structure of <i>Af</i>AmzA in cartoon representation. The N-terminal domain (NTD) is colored in slate, the active site helix α2 in orange and the C-terminal domain (CTD) in green. The N- and C-termini, the edge strand β4 (cyan), the backing helix α1, the S-loop (yellow), the bulge edge segment (red), the S1′-wall forming segment (magenta) and the specificity loop (purple) are labeled. The residues involved in zinc ion binding, the catalytic base and the structurally important methionine are shown as sticks and the zinc ions as spheres.</p