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Crystal structure of a fungal protease inhibitor from Antheraea mylitta
Sobhan Roy1, Penmatsa Aravind, Chaithanya Madhurantakam
1Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, India.
Abstract:
Indian tasar silk is produced by a wild insect called Antheraea mylitta. Insects do not have any antigen-antibody mediated immune system like vertebrates but they produce a wide variety of effector proteins and peptides possessing potent antifungal and antibacterial activity to combat microbial attack. Antheraea mylitta expresses a fungal protease inhibitor AmFPI-1, in the hemolymph that inhibits alkaline protease of Aspergillus oryzae for protection against fungal infection. AmFPI-1 is purified from the hemolymph, crystallized and the structure is solved using the single isomorphous replacement with anomalous scattering (SIRAS) method to a resolution of 2.1 A. AmFPI-1 is a single domain protein possessing a unique fold that consists of three helices and five beta strands stabilized by a network of six disulfide bonds. The reactive site of AmFPI-1 is located in the loop formed by residues 46-66, wherein Lys54 is the P(1) residue. Superimposition of the loop with reactive sites of other canonical protease inhibitors shows that reactive site conformation of AmFPI-1 is similar to them. The structure of AmFPI-1 provides a framework for the docking of a 1:1 complex between AmFPI-1 and alkaline protease. This study addresses the structural basis of AmFPI-1's specificity towards a fungal serine protease but not to mammalian trypsin and may help in designing specific inhibitors against fungal proteases.
Insights
Indian tasar silk moths produce AmFPI-1, a unique protein inhibitor. Its structure reveals how it specifically targets fungal proteases, offering insights for new antifungal drug design.
Area of Science:
- Insect immunology
- Structural biology
- Biochemistry
Background:
- Insects like the Indian tasar silk moth (Antheraea mylitta) lack a vertebrate immune system but possess antimicrobial proteins.
- Antheraea mylitta produces AmFPI-1, a hemolymph protein that inhibits fungal proteases, crucial for defense against infections.
Purpose of the Study:
- To determine the three-dimensional structure of AmFPI-1.
- To elucidate the structural basis for AmFPI-1's specificity against fungal proteases.
Main Methods:
- Purification of AmFPI-1 from Antheraea mylitta hemolymph.
- Crystallization and X-ray diffraction analysis using the SIRAS method.
- Structural determination to a resolution of 2.1 Å.
- In silico docking studies to model AmFPI-1-protease complex.
Main Results:
- The crystal structure of AmFPI-1 was solved, revealing a unique fold with three helices and five beta strands stabilized by six disulfide bonds.
- The reactive site, located in residues 46-66 with Lys54 as P(1), exhibits a conformation similar to other protease inhibitors.
- Docking analysis suggests a 1:1 complex formation between AmFPI-1 and Aspergillus oryzae alkaline protease.
Conclusions:
- The determined structure provides a detailed understanding of AmFPI-1's inhibitory mechanism against fungal serine proteases.
- AmFPI-1 displays specificity for fungal proteases over mammalian trypsin.
- This structural information can guide the rational design of novel, specific inhibitors targeting fungal proteases for therapeutic applications.
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