Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin

Rajesh T Shenoy,Adrian Velazquez-Campoy,Jeak Ling Ding,Bow Ho,J Sivaraman,Saravanan Thangamani,Petri Kursula

doi:10.1371/journal.pone.0018838

Abstract

Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1∶2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.

Highlights

Serine proteases play an important immunomodulatory role in host-pathogen interactions
Each CrSPI-1 molecule interacted with two subtilisin molecules, i.e., domain 1 and domain 2 of CrSPI-1 interacted with two independent subtilisin molecules (Fig. 1)
In order to verify the hypothesis that the rigidity of the reactive site loop (RSL) protects against the proteolytic cleavage by subtilisin, we investigated the interaction of a peptide (VCTEEY) derived from the RSL region of domain 2 of CrSPI-1 using Isothermal Titration Calorimetric (ITC) experiments (Fig. 5)

Summary

Introduction

Serine proteases play an important immunomodulatory role in host-pathogen interactions. Invertebrates lack an adaptive immune system that recognizes and remembers specific pathogens [1]. As an evolutionarily conserved and ancient defense strategy, the innate immune system responds instantaneously to invading pathogens in a non-specific manner. The innate immune system in the horseshoe crab, Carcinoscorpius rotundicauda, comprises serine protease cascades that are similar to the blood coagulation, melanization and complement systems [2]. Horseshoe crab hemocytes contain granules filled with several serine protease zymogens. Clotting enzymes, in their precursor forms, are activated by a serine protease cascade that is triggered by bacterial endotoxins [3,4]. Several serine protease zymogens, including proclotting enzymes, Factor B and Factor C, are associated with the hemolymph coagulation system. The subsequent formation of the coagulation plug prevents further entry of pathogens [5,6]

Methods

Results

Conclusion