Polar Desolvation and Position 226 of Pancreatic and Neutrophil Elastases Are Crucial to their Affinity for the Kunitz-Type Inhibitors ShPI-1 and ShPI-1/K13L

<div><p>The Kunitz-type protease inhibitor ShPI-1 inhibits human neutrophil elastase (HNE, <i>K</i>_<i>i</i> = 2.35·10⁻⁸ M) but does not interact with the porcine pancreatic elastase (PPE); whereas its P1 site variant, ShPI-1/K13L, inhibits both HNE and PPE (<i>K</i>_<i>i</i> = 1.3·10⁻⁹ M, and <i>K</i>_<i>i</i> = 1.2·10⁻⁸ M, respectively). By employing a combination of molecular modeling tools, e.g., structural alignment, molecular dynamics simulations and Molecular Mechanics Generalized-Born/Poisson-Boltzmann Surface Area free energy calculations, we showed that D226 of HNE plays a critical role in the interaction of this enzyme with ShPI-1 through the formation of a strong salt bridge and hydrogen bonds with K13 at the inhibitor’s P1 site, which compensate the unfavorable polar-desolvation penalty of the latter residue. Conversely, T226 of PPE is unable to establish strong interactions with K13, thereby precluding the insertion of K13 side-chain into the S1 subsite of this enzyme. An alternative conformation of K13 site-chain placed at the entrance of the S1 subsite of PPE, similar to that observed in the crystal structure of ShPI-1 in complex with chymotrypsin (PDB: 3T62), is also unfavorable due to the lack of stabilizing pair-wise interactions. In addition, our results suggest that the higher affinity of ShPI-1/K13L for both elastases mainly arises from the lower polar-desolvation penalty of L13 compared to that of K13, and not from stronger pair-wise interactions of the former residue with those of each enzyme. These results provide insights into the PPE and HNE inhibition and may contribute to the design of more potent and/or specific inhibitors toward one of these proteases.</p></div

Time Evolution of Instantaneous RMSD Values for Different Heavy Atom Sets of the Three Existing Complexes.

<p>(A)HNE:ShPI-1/K13L, (B) HNE:ShPI-1, (C) PPE:ShPI-1/K13L. RMSD values with respect to the initial (<i>t</i> = 0) structure were calculated for different heavy atom sets during the MD simulation of each complex. The dashed lines represent the <i>t</i>_<i>eq</i> value (5 ns) chosen from the analysis of instantaneous RMSD. The complex interface was defined by using a cutoff radius of 4 Å.</p

Linear Correlation between the <i>pr</i>EFED (Δ<i>G</i>_<i>sc</i>) and CAS (ΔΔ<i>G</i>) Results for Residues of the Studied Complexes.

<p>(A) All the selected residues (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137787#pone.0137787.s013" target="_blank">S8</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137787#pone.0137787.s014" target="_blank">S9</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137787#pone.0137787.s015" target="_blank">S10</a> Tables) were considered in the correlation analysis, including D226 and K13 of the HNE:ShPI-1 complex (blue and red dots, respectively). (B) Same as in (A) but excluding residues D226 and K13. The linear-fit equations and the Pearson and Spearman coefficients (<i>r</i>_<i>p</i> and <i>r</i>_<i>s</i>, respectively) are shown.</p

Time Evolution of Instantaneous Δ<i>G</i>_<i>eff</i> Values for the Four Complexes.

<p>(A) HNE:ShPI-1/K13L, (B) HNE:ShPI-1, (C) PPE:ShPI-1/K13L and (D) PPE:ShPI-1. Instantaneous Δ<i>G</i>_<i>eff</i> values (in blue) were calculated using the GB^OBC2 model (<i>igb</i> = 5). Histogram insets indicate the normal distribution of the Δ<i>G</i>_<i>eff</i> values. The Δ<i>G</i>_<i>eff</i> values corresponding to the PPE:ShPI-1<i>in</i> and PPE:ShPI-1<i>up</i> complexes are depicted in blue and orange, respectively. The black line indicates the accumulated mean value of Δ<i>G</i>_<i>eff</i> for each trajectory. The dashed line represents the <i>t</i>_<i>eq</i> value (5 ns) determined from the analysis of instantaneous RMSD and Δ<i>G</i>_<i>eff</i> values. Frames selected for Δ<i>G</i>_<i>eff</i> calculations in (D) are indicated by dashed rectangles.</p

S1:P1 Interfaces of the Two Alternative Conformations of the PPE:ShPI-1 Complex Sampled during the MD Simulation.

<p>(A) PPE:ShPI-1<i>in</i> and (B) PPE:ShPI-1<i>up</i> complex. Hydrogen bonds with occupancies ≥40% are represented as yellow dashed lines. Residues involved in hydrogen bond formation have been labeled in bold. Donor and acceptor atom names are labeled in bold and plain styles, respectively. Hydrogen atoms have been removed from the structures for clarity’s sake. The S1:P1 interfaces shown here correspond to the representative structures of the complexes.</p

Polar and non-Polar MM-GBSA Energy Components Associated with the Complex Formation in Solution.

<p>^aPolar energy components.</p><p>^bNon-polar energy components.</p><p>^cMean value ±standard deviation of the mean.</p><p>^dΔΔ<i>E</i> stands for the difference between the energy component values of the same column.</p><p>^eΔΔ<i>E</i>_<i>in</i> = Δ<i>E</i>(PPE:ShPI-1/K13L)- Δ<i>E</i>(PPE:ShPI-1<i>in</i>) and ΔΔ<i>E</i>_<i>up</i> = Δ<i>E</i>(PPE:ShPI-1/K13L)-Δ<i>E</i>(PPE:ShPI-1<i>up</i>)</p><p>Polar and non-Polar MM-GBSA Energy Components Associated with the Complex Formation in Solution.</p

Per-Residue Energy Contributions to the Formation of the Studied Complexes.

<p>Residues identified as warm/hot-spots by <i>pr</i>EFED were selected for CAS, except for Cys, Pro, Ala and Gly. The vertical dashed lines separate the enzyme’s residues from those of the inhibitor. X13 stands for L13 or K13 depending on the complex. ΔΔ<i>G</i> = Δ<i>G</i>_<i>eff</i>(native complex)-Δ<i>G</i>_<i>eff</i>(mutated complex), therefore, a negative ΔΔ<i>G</i> value indicates a favorable energy contribution of the mutated residue to the complex formation. Conversely, a positive ΔΔ<i>G</i> value indicates and unfavorable contribution of the mutated residue. Error bars were not included since standard errors were always lesser than 5% of the mean values. A structural representation of the warm-/hot-spot residues at the interface of each complex is shown. Residues were colored according to their energy contribution to the complex formation, see color-gradient bars.</p

Different Outcomes of the MD Simulations Performed for the PPE:ShPI-1 and HNE:ShPI-1 Complexes upon Slight Interface Disruption.

<p>The time profiles of RMSD values calculated for different atom sets as well as the distance between T226(CG2)/D226(OD1) of PPE/HNE and K13(NZ) of ShPI-1 as a function of time are shown for each complex. The structures of some frames extracted from each MD simulation are also depicted. Note that the disrupted starting structures of each complex (<i>t</i> = 0) were superimposed prior to the representation and that the initial position of ShPI-1 (gray) is shown only for comparison purposes. K13 of ShPI-1 and D226/T226 of HNE/PPE are depicted in stick representation.</p

Calculated and Experimental ΔΔ<i>G</i> Values and <i>K</i>_<i>i</i> Ratios.

<p>^a(A) ΔΔ<i>G</i>_<i>calc</i> = Δ<i>G</i>_<i>eff</i>(E:ShPI-1/K13L)-Δ<i>G</i>_<i>eff</i>(E:ShPI-1), where E stands for either PPE or HNE, (B) ΔΔ<i>G</i>_<i>calc</i> = Δ<i>G</i>_<i>eff</i>(HNE:I)-Δ<i>G</i>_<i>eff</i>(PPE:I), where I stands for either ShPI-1 or ShPI-1/K13L.</p><p>^b(A) ΔΔ<i>G</i>_<i>exp</i> = <i>RTlnK</i>_<i>i</i>(HNE:ShPI-1/K13L)/<i>K</i>_<i>i</i>(HNE:ShPI-1), or (B) ΔΔ<i>G</i>_<i>exp</i> = <i>RTlnK</i>_<i>i</i>(HNE:ShPI-1/K13L)/<i>K</i>_<i>i</i>(PPE:ShPI-1/K13L), where the <i>K</i>_<i>i</i> values were experimentally determined.</p><p>^c(A) <math><mrow><mrow><mrow><msubsup><mi>K</mi><mrow><mi>i</mi><mo>,</mo><mi>c</mi><mi>a</mi><mi>l</mi><mi>c</mi></mrow><mrow><mrow><mo>(</mo><mn>1</mn><mo>)</mo></mrow></mrow></msubsup></mrow><mo>/</mo><mrow><msubsup><mi>K</mi><mrow><mi>i</mi><mo>,</mo><mi>c</mi><mi>a</mi><mi>l</mi><mi>c</mi></mrow><mrow><mrow><mo>(</mo><mn>2</mn><mo>)</mo></mrow></mrow></msubsup></mrow></mrow><mo>=</mo><mrow><mrow><msubsup><mi>K</mi><mrow><mi>i</mi><mo>,</mo><mi>c</mi><mi>a</mi><mi>l</mi><mi>c</mi></mrow><mrow><mrow><mo>(</mo><mrow><mi>E</mi><mo>:</mo><mi>S</mi><mi>h</mi><mi>P</mi><mi>I</mi><mo>−</mo><mn>1</mn></mrow><mo>)</mo></mrow></mrow></msubsup></mrow><mo>/</mo><mrow><msubsup><mi>K</mi><mrow><mi>i</mi><mo>,</mo><mi>c</mi><mi>a</mi><mi>l</mi><mi>c</mi></mrow><mrow><mrow><mo>(</mo><mrow><mi>E</mi><mo>:</mo><mi>S</mi><mi>h</mi><mi>P</mi><mi>I</mi><mo>−</mo><mn>1</mn><mo>/</mo><mi>K</mi><mn>13</mn><mi>L</mi></mrow><mo>)</mo></mrow></mrow></msubsup></mrow></mrow><mo>=</mo>exp<mrow><mo>(</mo><mrow><mrow><mrow><mo>Δ</mo><mo>Δ</mo><mi>G</mi></mrow><mo>/</mo><mrow><mi>R</mi><mi>T</mi></mrow></mrow></mrow><mo>)</mo></mrow></mrow></math>, where E stands for either PPE or HNE, (B) <b><math><mrow><mrow><mrow><msubsup><mi>K</mi><mrow><mi>i</mi><mo>,</mo><mi>c</mi><mi>a</mi><mi>l</mi><mi>c</mi></mrow><mrow><mrow><mo>(</mo><mn>1</mn><mo>)</mo></mrow></mrow></msubsup></mrow><mo>/</mo><mrow><msubsup><mi>K</mi><mrow><mi>i</mi><mo>,</mo><mi>c</mi><mi>a</mi><mi>l</mi><mi>c</mi></mrow><mrow><mrow><mo>(</mo><mn>2</mn><mo>)</mo></mrow></mrow></msubsup></mrow></mrow><mo>=</mo><mrow><mrow><msubsup><mi>K</mi><mrow><mi>i</mi><mo>,</mo><mi>c</mi><mi>a</mi><mi>l</mi><mi>c</mi></mrow><mrow><mrow><mo>(</mo><mrow><mi>E</mi><mi>N</mi><mi>H</mi><mo>:</mo><mi>I</mi></mrow><mo>)</mo></mrow></mrow></msubsup></mrow><mo>/</mo><mrow><msubsup><mi>K</mi><mrow><mi>i</mi><mo>,</mo><mi>c</mi><mi>a</mi><mi>l</mi><mi>c</mi></mrow><mrow><mrow><mo>(</mo><mrow><mi>E</mi><mi>P</mi><mi>P</mi><mo>:</mo><mi>I</mi></mrow><mo>)</mo></mrow></mrow></msubsup></mrow></mrow><mo>=</mo>exp<mrow><mo>(</mo><mrow><mrow><mrow><mo>Δ</mo><mo>Δ</mo><mi>G</mi></mrow><mo>/</mo><mrow><mi>R</mi><mi>T</mi></mrow></mrow></mrow><mo>)</mo></mrow></mrow></math></b>, where I stands for either ShPI-1 or ShPI-1/K13L.</p><p>^dExperimental <i>K</i>_<i>i</i> values taken from Garcia-Fernandez <i>et al</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137787#pone.0137787.ref007" target="_blank">7</a>].</p><p>^eFor ΔΔ<i>G</i> calculations an average Δ<i>G</i>_<i>eff</i> value of PPE:ShPI-1 complex obtained from those of the two conformations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137787#pone.0137787.s012" target="_blank">S7 Table</a>) was employed.</p><p>^fThe GB model used for ΔΔ<i>G</i> calculation is indicated by the value of the <i>igb</i> variable between parentheses.</p><p>^gThe PB model used for for ΔΔ<i>G</i> calculation is indicated between parentheses, where pb1, pb2 and pb3 stand for the PB model using Tan and Luo, mbondi2 and mbondi3 atomic radii, respectively.</p><p>^hNo PPE:ShPI-1 complex formation has been experimentally detected, hence, ΔΔ<i>G</i>_<i>exp</i> value is expected to be a large and negative number, and the <i>K</i>_<i>i</i> ratio must tend to zero.</p><p>Calculated and Experimental ΔΔ<i>G</i> Values and <i>K</i>_<i>i</i> Ratios.</p

Time Profiles of RMSD Values and Structural Interface Representation for a Hypothetical PPE:ShPI-1 Complex.

<p>During the first ~43 ns a ‘in’ conformation of the PPE:ShPI-1 complex in which the side-chain of K13 lies within the S1 subsite of PPE was sampled (see structures at <i>t</i> = 0 and <i>t</i> = 20 ns). From <i>t</i>≈43 ns to <i>t</i>≈62 ns, a reorganization of the complex interface occurred in order to provide space for K13 exit (see structure at <i>t</i> = 60 ns). Finally, at <i>t</i>≈62 ns the side-chain of K13 changed to an ‘up’ conformation and remained in that conformation up to 125 ns (see structure at <i>t</i> = 125 ns).</p