31 research outputs found
A cyclic peptidic serine protease inhibitor:increasing affinity by increasing peptide flexibility
Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden
A Camelid derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior
A peptide segment that binds the active site of a serine protease in a substrate like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region H3 loop into the active site of the protease in a substrate like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30 40 of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra loop interaction network within the complementary determining region H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitor
Prostate cancer: 1.5 T endo-coil dynamic contrast-enhanced MRI and MR spectroscopy-correlation with prostate biopsy and prostatectomy histopathological data
Building Pedagogies. A historical study of teachers’ spatial work in new school architecture
Structural model of mupain-1 in complex with muPA.
<p>(A) Overall model of the complex between muPA (teal ribbon presentation) and mupain-1 (red stick representation). The enzyme is shown in teal cartoon presentation. (B) A zoom on the Lys<sup>143</sup> area of the model, showing distances from Lys<sup>143</sup> to the closest atoms in the peptide. (C) A zoom on the Lys<sup>41</sup> area of the model, showing the distances between Lys41 and peptide residues Tyr<sup>7</sup>and Asp<sup>9</sup>. (D) A zoom on the entrance to the S1 pocket of the model, with Val<sup>213</sup> at the entrance indicated; the enzyme is represented as teal surface. huPA-H99Y residues are labelled with black letters, peptide residues with dark red letters.</p
Surface plasmon resonance analysis of the binding of peptides to muPA or huPA-H99Y.
<p>The table shows the rate constants and the <i>K</i><sub>D</sub> values for the binding of the indicated peptides to muPA or huPA-H99Y at 25°C, pH 7.4. Means, standard deviations, and numbers of determinations are indicated. Examples of sensorgrams are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115872#pone.0115872.s003" target="_blank">S3 Fig</a>.</p><p>Surface plasmon resonance analysis of the binding of peptides to muPA or huPA-H99Y.</p
Inhibition of huPA-H99Y and huPA-H99Y exosite mutants by mupain-1 variants.
<p>The <i>K</i><sub>i</sub> values (in µM) for inhibition of the indicated enzymes by the indicated peptides at 37°C are shown as means ± S.D; the numbers of determinations are indicated in parentheses. *These values are reproduced from previous publications [Andersen et al., 2008; Hosseini et al., 2011] and shown here to facilitate comparison.</p><p>Inhibition of huPA-H99Y and huPA-H99Y exosite mutants by mupain-1 variants.</p
TOCSY (orange) and NOESY (green) of mupain-1-16.
<p>Assignments of the two isomeric forms is illustrated by connecting TOCSY H<sub>α</sub>(<i>i</i>)-H<sub>N</sub>(<i>i</i>) and NOESY H<sub>α</sub>(<i>i</i>)-H<sub>N</sub>(<i>i</i>+1) cross peaks with lines representing a shared reference commonly referred to as “backbone walks”.</p