Research ArticleBIOPHYSICS

Structure of human Vitronectin C-terminal domain and interaction with Yersinia pestis outer membrane protein Ail

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Science Advances  11 Sep 2019:
Vol. 5, no. 9, eaax5068
DOI: 10.1126/sciadv.aax5068
  • Fig. 1 Domain organization and NMR of Vn.

    All domain representations are scaled to sequence. The HX domain is colored by repeat unit (HX1, blue; HX2, green; HX3, yellow; HX4, red). (A) Vn starts at Asp20 and comprises an SMB domain (black), RGD sequence (white), HX domain (colored by HX repeat), and three variable regions VR1 to VR3 (gray). The six Cys in the C-terminal domain (pink lines) and sites for phosphorylation (orange circles), sulfation (green circles), N-glycosylation (blue hexagons), and protease cleavage (arrows) are shown. Vn-HX starts at Glu154. (B) Alignment of Vn HX1-HX4 sequence repeats (Clustal color scheme). VR2 and VR3 are omitted for clarity (slashes). Structural elements (bridge-β1-β2-β3-rim-α) of the HX repeats are depicted above the alignment. Black arrowheads mark intron positions. The HX consensus was derived for Vn and other HX proteins of known structure (fig. S2). (C) HX structural repeat units viewed in the frame of the exon structure (dashed wedges). VR2 and VR3 form insertions in exons 6 and 7. (D and E) Solution NMR 1H/15N spectra of 15N-labeled Vn-HX in 6 M urea (D) or refolded in nondenaturing buffer (E). (F) Size exclusion chromatography of folded Vn-HX and SDS–polyacrylamide gel electrophoresis (PAGE) analysis of the eluted fractions. Vn-HX elutes with an apparent Stokes diameter of ~42 Å.

  • Fig. 2 Structure and topology of Vn-HX.

    Colors denote structural repeat units HX1 (blue), HX2 (green), HX3 (yellow), and HX4 (red). Spheres denote metal (Na+, pink) or Cl (cyan) ions and O (red) or S (yellow) atoms. Broken lines denote gaps in the protein chain due to missing electron density. Dotted lines denote VR2 (yellow) and VR3 (red). (A and B) Side and top views of the structure, showing the positions of the two N-glycosylation sites (Asn242 and Asn169) and cysteines. (C) Schematic topology of the Vn C terminus showing β-strands (arrows), α-helices (cylinders), VR2 and VR3 residues deleted in Vn-HX (gray highlight), residues with missing electron density (red letters), exon breaks (black solid lines), Cys (white circles), disulfide bond (dashed line), and sites for phosphorylation (orange circles), N-glycosylation (blue hexagons), and protease cleavage (white squares). (D) Top and bottom views with surface colored by electrostatic potential from −10 kT/e (red) to +10 kT/e (blue). (E) Backbone structure of the channel for molecule A (left) and molecule B (right) of the asymmetric unit, showing key side chains (wheat). (F) SO42− binding site on the surface of Vn-HX. The positively charged surface groove is lined by Arg212, Arg229, and Arg241 at the HX2-HX3 repeat interface.

  • Fig. 3 Binding activity of Vn-HX.

    (A) Antibody (Ab) binding activity detected by Western dot blots of Vn and Vn-HX with anti-Vn antibodies directed to residues 141 to 154 (Abn), 209 to 258 (Abm), or 446 to 472 (Abc). The sequence of Vn-HX starts at Glu154 and does not react with Abn. (B) Top view of Vn-HX. Surface exposed epitopes for Abm (green) and Abc (ruby and pink) map to HX2 and HX4, respectively. The binding site for insulin-like growth factor II (4) maps to the surface-exposed outer rim of HX4 (pink). (C and D) Ail-mediated microbial binding activity assayed by co-sedimentation of Vn-HX (C) or full-length Vn (D) with Ail-expressing Y. pestis bacterial cells. Immunoblots were probed with anti-Vn, anti-Ail, or anti-FLAG antibodies. Y. pestis cell types were wild-type expressing native ail (wt); ail deletion mutant (Δail); and Δail complemented with plasmid expressing FLAG tag (Δail-pF), FLAG-tagged OmpX (Δail-pF-OmpX), or FLAG-tagged Ail (Δail-pF-Ail). Arrows mark positions of Vn, Vn-HX, Ail, FLAG-Ail, and FLAG-OmpX. (E) Vn-HX binds purified Ail. Plates preadsorbed with Vn-HX (5 μg/ml) (black circles), full-length Vn (5 μg/ml) (white circles), or gelatin (20 μg/ml) (white squares) were incubated overnight with increasing concentrations of purified refolded His-tagged Ail. Binding was detected with ELISA using a mouse anti-His antibody, by measuring light absorbance at 490 nm (A490). Each data point represents the mean ± SEM of three independent experiments. Binding curves with dissociation constants of Kd = 700 ± 50 nM (Vn-HX) and Kd = 400 ± 50 nM (Vn) were estimated by fitting (red lines; R2 = 0.99) the ELISA data relative to the maximum fraction of bound Ail (A490max) in each titration. No Ail binding is observed to plates preadsorbed with gelatin.

  • Table 1 Crystallographic data and refinement statistics.

    Data collection*
    Space groupP21
    Cell dimensions
    a, b, c (Å)40.56, 97.36, 49.16
    α, β, γ (°)90.00, 99.90, 90.00
    Resolution (Å)26.96–1.90 (1.94–1.90)*
    Rsym or Rmerge0.071 (0.326)
    II7.2 (2.0)
    Completeness (%)98.1 (96.1)
    Redundancy2.3 (2.3)
    Refinement
    Resolution (Å)24.21–1.90 (1.96–1.90)
    No. of reflections28,986 (2453)
    Rwork/Rfree0.1793/0.2102 (0.2481/0.2940)
    No. of atoms (nonhydrogen)
      Protein3012
      Ligand/ion30
      Water168
    B factors
      Protein44.24
      Ligand/ion57.02
      Water41.98
    Root mean square deviations
      Bond lengths (Å)0.005
      Bond angles (°)0.832

    *One crystal was used for the structure. Values in parentheses are for highest-resolution shell.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaax5068/DC1

      Fig. S1. Alignment of orthologous Vn sequences.

      Fig. S2. Alignment of the HX domains of human Vn, rabbit hemopexin, and human matrix metalloproteases.

      Fig. S3. HX repeat and intron/exon structures of human Vn, hemopexin N- and C-terminal domains, and matrix metalloproteases.

      Fig. S4. Structure, B factors, and surface electrostatics of Vn-HX.

      Fig. S5. Geometry of the ion-binding sites in the central channel of Vn-HX.

      Fig. S6. Conserved prolines stabilize outward facing aromatic side chains.

    • Supplementary Materials

      This PDF file includes:

      • Fig. S1. Alignment of orthologous Vn sequences.
      • Fig. S2. Alignment of the HX domains of human Vn, rabbit hemopexin, and human matrix metalloproteases.
      • Fig. S3. HX repeat and intron/exon structures of human Vn, hemopexin N- and C-terminal domains, and matrix metalloproteases.
      • Fig. S4. Structure, B factors, and surface electrostatics of Vn-HX.
      • Fig. S5. Geometry of the ion-binding sites in the central channel of Vn-HX.
      • Fig. S6. Conserved prolines stabilize outward facing aromatic side chains.

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