Crystal Structure of a Charge Engineered Human Lysozyme Having Enhanced Bactericidal Activity

Avinash Gill,Karl E Griswold,Dean R Madden,Thomas C Scanlon,Daniel C Osipovitch,Claudine Mayer

doi:10.1371/journal.pone.0016788

Abstract

Human lysozyme is a key component of the innate immune system, and recombinant forms of the enzyme represent promising leads in the search for therapeutic agents able to treat drug-resistant infections. The wild type protein, however, fails to participate effectively in clearance of certain infections due to inherent functional limitations. For example, wild type lysozymes are subject to electrostatic sequestration and inactivation by anionic biopolymers in the infected airway. A charge engineered variant of human lysozyme has recently been shown to possess improved antibacterial activity in the presence of disease associated inhibitory molecules. Here, the 2.04 Å crystal structure of this variant is presented along with an analysis that provides molecular level insights into the origins of the protein's enhanced performance. The charge engineered variant's two mutated amino acids exhibit stabilizing interactions with adjacent native residues, and from a global perspective, the mutations cause no gross structural perturbations or loss of stability. Importantly, the two substitutions dramatically expand the negative electrostatic potential that, in the wild type enzyme, is restricted to a small region near the catalytic residues. The net result is a reduction in the overall strength of the engineered enzyme's electrostatic potential field, and it appears that the specific nature of this remodeled field underlies the variant's reduced susceptibility to inhibition by anionic biopolymers.

Highlights

Chronic pulmonary infections are a major cause of patient morbidity and mortality in diseases ranging from cystic fibrosis (CF) to chronic obstructive pulmonary disease (COPD) and pneumonias
Combinatorial libraries of charge engineered Human lysozyme (hLYS) variants were designed using bioinformatics and structural analysis, and approximately 150,000 mutated enzymes were screened for bacteriolytic activity in the presence of inhibitory alginate polyanion
The double mutant detailed in this manuscript is not the only functionally enhanced enzyme to be isolated in the course of large combinatorial library screens

Summary

Introduction

Chronic pulmonary infections are a major cause of patient morbidity and mortality in diseases ranging from cystic fibrosis (CF) to chronic obstructive pulmonary disease (COPD) and pneumonias. In CF, polymicrobial airway infections are established early, and by adulthood most patient airways are persistently colonized by the opportunistic pathogen Pseudomonas aeruginosa, the primary cause of patient mortality [1]. In addition to physiological factors that favor the persistent nature of CF infections, drug-resistance is a critical issue for both Gram-negative P. aeruginosa [3,4] and Gram-positive pathogens such as Staphylococcus aureus [5] and various streptococci [6]. To more effectively manage bacterial infections associated with CF and other diseases such as COPD and pneumonias, there is a critical need for generation antibiotics capable of treating drug-resistant pathogens. In one approach to new therapies, genetically engineered antimicrobial proteins are being developed based on knowledge of the mechanisms by which innate immune factors sometimes fail

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Conclusion