Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

Abstract

Elastase-like enzymes are involved in important diseases such as acute pancreatitis, chronic inflammatory lung diseases, and cancer. Structural insights into their interaction with specific inhibitors will contribute to the development of novel anti-elastase compounds that resist rapid oxidation and proteolysis. Proteinaceous Kunitz-type inhibitors homologous to the bovine pancreatic trypsin inhibitor (BPTI) provide a suitable scaffold, but the structural aspects of their interaction with elastase-like enzymes have not been elucidated. Here, we increased the selectivity of ShPI-1, a versatile serine protease inhibitor from the sea anemone Stichodactyla helianthus with high biomedical and biotechnological potential, toward elastase-like enzymes by substitution of the P1 residue (Lys(13)) with leucine. The variant (rShPI-1/K13L) exhibits a novel anti-porcine pancreatic elastase (PPE) activity together with a significantly improved inhibition of human neuthrophil elastase and chymotrypsin. The crystal structure of the PPE·rShPI-1/K13L complex determined at 2.0 Å resolution provided the first details of the canonical interaction between a BPTI-Kunitz-type domain and elastase-like enzymes. In addition to the essential impact of the variant P1 residue for complex stability, the interface is improved by increased contributions of the primary and secondary binding loop as compared with similar trypsin and chymotrypsin complexes. A comparison of the interaction network with elastase complexes of canonical inhibitors from the chelonian in family supports a key role of the P3 site in ShPI-1 in directing its selectivity against pancreatic and neutrophil elastases. Our results provide the structural basis for site-specific mutagenesis to further improve the binding affinity and/or direct the selectivity of BPTI-Kunitz-type inhibitors toward elastase-like enzymes.

Keywords: BPTI-Kunitz type; crystal structure; protease inhibitor; protein complex; serine protease; site-directed mutagenesis.

FIGURE 1.

Structure of the rShPI-1/K13L·PPE complex (PDB code 3UOU). a, schematic model of the overall structure of the complex between PPE (green, surface representation) and rShPI-1/K13L (blue), showing the changed P1 residue Leu¹³ of the inhibitor in stick representation. The primary (P6-P5′ sites) and secondary binding loops are highlighted in red and orange, respectively, and the linking disulfide bridge, Cys¹²–Cys³⁶, is shown in yellow. b, close view of the entire complex interface centered on the S1 pocket of PPE, illustrating the formation of an antiparallel β-sheet by the inhibitor loops within the concave binding pocket of the enzyme that is typical for canonical inhibitors. The binding loops of rShPI-1/K13L (stick representation) are well defined by the 2F_o − F_c map (blue) countered at 1σ. The side chains of the catalytic triad residues His⁵⁷, Asp¹⁰², and Ser¹⁹⁵ as well as Ser¹⁸⁹ at the bottom of the S1 pocket of PPE are highlighted in stick representation. c, buried surface area (BSA) of rShPI-1/K13L residues (black) involved in the PPE interface compared with that of wild-type rShPI-1A (gray) bound to trypsin. The buried surface area represents a percentage of the total surface area that is buried after complex formation. The amino acid sequence of rShPI-1/K13L and the corresponding Pn sites are shown.

FIGURE 2.

Stereo view of the P1-S1 interaction at the rShPI-1/K13L·PPE complex interface compared with that in the trypsin complex of wild-type rShPI-1A (35). Enzyme residues involved in inhibitor contacts are shown as a line representation (PPE, green; trypsin, blue), and primary binding loop residues of rShPI-1/K13L (salmon) and wild-type rShPI-1A (blue) are displayed as sticks. Conserved residues are labeled in black, and non-conserved residues are in the same color as the corresponding enzyme. Hydrogen bonds are represented by black (PPE) and blue (trypsin) dashed lines. To simplify the figure, water molecules that are present in the trypsin complex (35) around the P1 position are not included. For details of the interactions in the rShPI-1/K13L·PPE complex, see Tables 3 and 4.

FIGURE 3.

Stereo view of the rShPI-1/K13L·PPE complex interface superposed with that of wild-type rShPI-1A in complex with trypsin (35) at the Pn (a) and Pn′ (b) site of the primary inhibitor binding loop. Enzyme residues involved in inhibitor contacts are shown in line representation (PPE, green; trypsin, blue; conserved residues His⁵⁷ and Gln¹⁹², black labels), and primary binding loop residues of rShPI-1/K13L (salmon) and wild-type rShPI-1A (blue) are displayed as sticks. The side chain at the P1 position of the inhibitor is included to enable a comparison with Figs. 1c and 2. Hydrogen bonds are represented by black (PPE) and blue (trypsin) dashed lines. Water molecules (W) are shown as red spheres and are labeled according to the PDB files in which they are assigned to the enzyme (e) or inhibitor (i) chains. Conformational differences between both complexes are restricted to position P3 at the Pn site of the primary binding loop. The P6 residue is only involved in the interface within the rShPI-1/K13L·PPE complex. For details of the interactions in the rShPI-1/K13L·PPE complex, see Tables 3 and 4.

FIGURE 4.

Stereo view of the rShPI-1/K13L·PPE complex interface maintained by the secondary binding loop superposed with that of wild-type rShPI-1A in complex with trypsin (35). Enzyme residues involved in inhibitor contacts are shown in line representation (PPE, green; trypsin, blue; conserved residue His⁵⁷, black). Secondary binding loop residues (Ile³²-Gly³⁷) of rShPI-1/K13L(orange) and wild-type rShPI-1A (light blue) are displayed as sticks, and the P3-P3′ segment of the primary binding loop of both inhibitors is shown schematically (rShPI-1/K13L, salmon; rShPI-1A, dark blue) with sticks at the P1 side chain. Hydrogen bonds are represented by black dashed lines. Water molecules (W) are shown as red spheres and are labeled according to the PDB file in which they are assigned to the enzyme (e) chain. For details of the interactions, see Tables 3 and 4.

FIGURE 5.

Structural superposition of elastases in complex with BPTI-Kunitz- and WAP-type inhibitors focused on the non-prime subsite of the primary binding loops of the inhibitors. a, interface between PPE (dark green, schematic representation) and rShPI-1/K13L (salmon, stick representation) around the P3 residue (Arg¹¹) at the Pn side of the primary binding loop (residues P6–P1). To highlight structural differences between elastases, the PPE structure is superposed with that of HNE (light green, PDB code 2Z7F), and PPE regions characterized by significant main chain deviations are highlighted in cyan. PPE residues interacting with the P3 residue of rShPI-1/K13L as well as the inhibitor side chains of Arg¹¹ (P3) and Leu¹³ (P1) are shown in stick representation. Hydrogen bonds are represented with black dashed lines. Water molecules (W) are shown as red spheres and are labeled according to the PDB file in which they are assigned to the inhibitor (i) chain. b, corresponding interface region in the PPE (dark green) and HNE (light green) complexes of the WAP inhibitors elafin (gray, PDB code 1FLE) and SLPI/D2 (yellow, PDB code 2Z7F), respectively, shown in schematically. The side chains of residues at their P5 sites, Tyr⁵⁸ in elafin and Leu²⁰ in SLPI/D2 (12), are highlighted as sticks.

FIGURE 6.

Superposition of the P1 side chain conformation of rShPI-1/K13L (salmon) in complex with PPE (green) compared with that of the wild-type inhibitor in complexes with trypsin (blue, PDB code 3MTQ) and chymotrypsin (yellow, PDB code 3T62). The inhibitor binding loops are shown schematically with the P1 residues in sticks. The insertion of Arg^217A in PPE triggers structural differences that prevent a stabilizing interaction of the basic P1 residue with Ser²¹⁷, as observed in the chymotrypsin complex (32). Here, the side chain of Lys¹³ adopts an up conformation, which is different from the down conformation in the trypsin complex (blue) and is stabilized by an H-bond with the oxygen atom of Ser²¹⁷. However, the insertion of Arg^217A in PPE (green) moves Ser²¹⁷ away from P1, suggesting that a similar stabilizing bond with basic residues at P1 is not possible.