Success selections of top ranked structures.

<p>Success selections of top ranked structures.</p

On the Molecular Basis of D-Bifunctional Protein Deficiency Type III

<div><p>Molecular basis of D-bifunctional protein (D-BP) deficiency was studied with wild type and five disease-causing variants of 3R-hydroxyacyl-CoA dehydrogenase fragment of the human MFE-2 (multifunctional enzyme type 2) protein. Complementation analysis <em>in vivo</em> in yeast and <em>in vitro</em> enzyme kinetic and stability determinants as well as <em>in silico</em> stability and structural fluctuation calculations were correlated with clinical data of known patients. Despite variations not affecting the catalytic residues, enzyme kinetic performance (K_m, V_max and k_cat) of the recombinant protein variants were compromised to a varying extent and this can be judged as the direct molecular cause for D-BP deficiency. Protein stability plays an additional role in producing non-functionality of MFE-2 in case structural variations affect cofactor or substrate binding sites. Structure-function considerations of the variant proteins matched well with the available data of the patients.</p> </div

Features of native recombinant <i>Hs</i>DH protein and its five clinically interesting patient mutation variants.

<p>The activities of human dehydrogenase and its variants were measured by detecting spectrophotometrically the formation of magnesium complex of 3R-hydroxydecanoyl-CoA at 303 nm from the 2E-decenoyl-CoA substrate in concentration range from 0.5 to 30 µM. Kinetic parameters are calculated by using GraFit 5.0 program (Erithacus Software). Main features characterizing the observed structural changes in the variant proteins and the patients are also given (and discussed in more details in the text).</p><p>n.d. = not determined (recording reliable and comparable data would have necessitated using a large surplus of NAD⁺).</p

Five biologically interesting variations located in the dehydrogenase dimer of human MFE-2.

<p>The two dehydrogenase monomers in the middle of the figure are colored in green and blue, amino acids T15, N158, E232, R248 and W249 are shaded grey and shown in stick presentations, NAD⁺ are colored in yellow and shown also in stick presentation. The rectangles indicate parts of the structure that are represented in larger details in small figures. The figures were done using the program PyMol (Schrödinger) and human 3R-hydroxyacyl-CoA dehydrogenase structure (PDB ID 1ZBQ; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053688#pone.0053688-Lukacik1" target="_blank">[7]</a>).</p

Growth of the BY4741 <i>Δfox2</i> cells on oleic acid transformed with pYE352::<i>ScMFE-2,</i> pYE352::<i>CTA1</i>, pYE352::<i>HsMFE-2</i>, and its five clinically interesting patient variants.

<p>The dilution series were done on YPD (0.2% glucose)- oleic acid (0.125%) plates and the BY4741 <i>Δfox2</i> cells were grown at +30°C for one week indicated by: (1) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>, (2) BY4741 <i>Δfox2</i>+ pYE352::<i>ScMFE-2</i>, (3) BY4741 <i>Δfox2</i> strain, (4) BY4741 <i>Δfox2</i>+ pYE352::<i>CTA1</i>, (5) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(T15A), (6) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(N158D), (7) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(E232K), (8) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(R248C), (9) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(W249G). When the yeast is able to utilize the oleic acid as a sole source of carbon, there will appear a clear zone around (samples 1, 2, 5, 6, 7, 8 & 9), but if the functional <i>fox2</i> gene is missing the environment remains opaque (samples 3 & 4).</p

Coomassie-stained SDS-PAGE gel of purified <i>Hs</i>DH recombinant protein and its five clinically interesting patient variants under reducing conditions.

<p>The first line represents Low molecular weight protein standard (Bio-Rad) and lines 2–7 protein samples purified by Ni-NTA and Superdex 200 gel filtration columns as follows: 2) wild type <i>Hs</i>DH, 3) T15A variant, 4) N158D variant, 5) E232K variant, 6) R248C variant and 7) W249G variant. The molecular masses of all the recombinant protein monomers (∼36 kDa) correspond to the monomer mass (36.14 kDa) estimated from the aa sequence of the wild type <i>Hs</i>DH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053688#pone.0053688-Gasteiger1" target="_blank">[29]</a>.</p

Virulence Regulation with Venus Flytrap Domains: Structure and Function of the Periplasmic Moiety of the Sensor-Kinase BvgS

<div><p>Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent <i>Bordetella pertussis</i>, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic <u>V</u>enus <u>f</u>ly<u>t</u>rap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in <i>Bordetella</i>. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.</p></div