Crystal Structures of a Plant Trypsin Inhibitor from Enterolobium contortisiliquum (EcTI) and of Its Complex with Bovine Trypsin

A serine protease inhibitor from Enterolobium contortisiliquum (EcTI) belongs to the Kunitz family of plant inhibitors, common in plant seeds. It was shown that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathway. We determined high-resolution crystal structures of free EcTI (at 1.75 angstrom) and complexed with bovine trypsin (at 2 angstrom). High quality of the resulting electron density maps and the redundancy of structural information indicated that the sequence of the crystallized isoform contained 176 residues and differed from the one published previously. the structure of the complex confirmed the standard inhibitory mechanism in which the reactive loop of the inhibitor is docked into trypsin active site with the side chains of Arg64 and Ile65 occupying the S1 and S1' pockets, respectively. the overall conformation of the reactive loop undergoes only minor adjustments upon binding to trypsin. Larger deviations are seen in the vicinity of Arg64, driven by the needs to satisfy specificity requirements. A comparison of the EcTI-trypsin complex with the complexes of related Kunitz inhibitors has shown that rigid body rotation of the inhibitors by as much as 15 degrees is required for accurate juxtaposition of the reactive loop with the active site while preserving its conformation. Modeling of the putative complexes of EcTI with several serine proteases and a comparison with equivalent models for other Kunitz inhibitors elucidated the structural basis for the fine differences in their specificity, providing tools that might allow modification of their potency towards the individual enzymes.United States Department of Energy, Office of Science, Office of Basic Energy SciencesCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)National Institutes of Health, National Cancer Institute, Center for Cancer ResearchNCI, Ctr Canc Res, Macromol Crystallog Lab, Frederick, MD 21701 USAUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilInst Butantan, Lab Bioquim & Biofis, Unidade Sequenciamento Prot & Peptideos, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilUnited States Department of Energy, Office of Science, Office of Basic Energy Sciences: W-31-109-Eng-38FAPESP: 09/53766-5Web of Scienc

Inhibitory properties of EcTI and the related inhibitors.

<p>n.i – no inhibition. The values in parentheses correspond to the standard deviations of the calculated data. Blank fields – data not collected or available.</p>*<p>Taken from Patil et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062252#pone.0062252-Patil1" target="_blank">[19]</a>.</p

The reactive loop of EcTI.

<p>(a,b) 2Fo-Fc electron density maps contoured at 1.2σ of the reactive loop area of (a) free EcTI and (b) its complex with trypsin. (c) A steroview of the hydrogen networks of the reactive loop region of a molecule of free EcTI. Blue dashed lines indicate the hydrogen bond network formed by the side chain of Asn13 and the interacting residues, and red dashed lines indicate the hydrogen bond network formed by the reactive loop region residues and their partners. Green spheres represent water molecules.</p

Data collection and structure refinement.

*<p>The highest resolution shell is shown in parentheses.</p>†<p><i>R</i>_merge = ∑_h∑_i|<i>I</i>_i–〈<i>I</i>〉|/∑_h∑_i<i>I</i>_i, where I_i is the observed intensity of the i-th measurement of reflection h, and 〈I〉 is the average intensity of that reflection obtained from multiple observations.</p>‡<p><i>R</i> = ∑||<i>F_o</i>|–|<i>F_c</i>||/∑<i>|F_o|,</i> where F_o and F_c are the observed and calculated structure factors, respectively, calculated for all data. <i>R</i>_free was defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062252#pone.0062252-Brnger1" target="_blank">[33]</a>.</p

EcTI and its complex with trypsin.

<p>(a) Ribbon representation of the overall three-dimensional structure of EcTI. β-strands labeled β1–12 are shown in red, and loops labeled L1–13 are in green. Loop L10 is only marked for consistency with the other related structures, since the main chain is broken in this region. (b) Pseudo-dimer of the two crystallographically independent molecules of EcTI in the asymmetric unit. The main contact loops are labeled. (c) Overall structure of the EcTI-trypsin complex shown as a cartoon diagram. EcTI is colored green, while trypsin is red, and the reactive loop is labeled.</p

Close-up of the interactions of EcTI and trypsin.

<p>(a) Steroview of the region of interactions, with residues belonging to trypsin shown as cyan sticks and the disulfide bond colored yellow. The residues of EcTI are shown as green sticks and the gray spheres represent water molecules. Hydrogen bonds are indicated by red dashed lines. (b) Interface of the EcTI-trypsin complex, with EcTI shown as a green ribbon with selected side chains in stick representation, whereas the surface of trypsin is colored according to charge (blue positive, red negative, gray uncharged).</p

Preparation of the EcTI-trypsin complex.

<p>Free EcTI and its complex with trypsin were run on the same Sephacryl S-100 HR column, with the same buffer. The two curves were overlaid, with the blue one representing EcTI-trypsin and the red one representing free EcTI.</p

Sequence alignment of EcTI and related serine protease inhibitors, partially structure-based (the structures of Acacia, DE5, and Bauhinia (rows 2–4) have not been determined).

<p>The top two sets of rows correspond to the long chain in EcTI, and the third set of rows represents the short chain. The secondary structure elements of EcTI are shown on top of the alignment in orange. Residues identical in all inhibitors are colored green and those identical in some, red. The reactive loop regions are highlighted in yellow, and a blue triangle indicates the position of P1 residue.</p

Structural basis for the differences in the inhibitory properties of Kunitz inhibitors towards different serine proteases.

<p>(a) Two possible areas of conflict between EcTI and Factor Xa. Factor Xa (gray) is superimposed on trypsin (green) complexed with EcTI (yellow). Complexes of STI and TKI are superimposed on the basis of their trypsin components (orange and magenta, respectively). (b) The third possible area of conflict between EcTI and Factor Xa. A modeled surface of Factor Xa (PDB ID: 2JKH) is gray. The colors for other molecules are as in (a). (c) Different orientation of the N terminus in EcTI (yellow) compared to STI (magenta) and TKI (orange).</p