Functional reprogramming of Candida glabrata epithelial adhesins: the role of conserved and variable structural motifs in ligand binding

Daniel Hoffmann,Rike Diderrich,Viktoria Reithofer,Sabrina Friederichs,Michael Kock,Lars-Oliver Essen,Hans-Ulrich Mösch

doi:10.1074/jbc.ra120.013968

Abstract

For host-cell interaction, the human fungal pathogen Candida glabrata harbors a large family of more than 20 cell wall-attached epithelial adhesins (Epas). Epa family members are lectins with binding pockets containing several conserved and variable structural hot spots, which were implicated in mediating functional diversity. In this study, we have performed an elaborate structure-based mutational analysis of numerous Epa paralogs to generally determine the role of diverse structural hot spots in conferring host cell binding and ligand binding specificity. Our study reveals that several conserved structural motifs contribute to efficient host cell binding. Moreover, our directed motif exchange experiments reveal that the variable loop CBL2 is key for programming ligand binding specificity, albeit with limited predictability. In contrast, we find that the variable loop L1 affects host cell binding without significantly influencing the specificity of ligand binding. Our data strongly suggest that variation of numerous structural hot spots in the ligand binding pocket of Epa proteins is a main driver of their functional diversification and evolution.

Highlights

Human-pathogenic fungi often possess large repertoires of cell wall-associated adhesins for successful host recognition, invasion, and colonization [1,2,3,4]
In this study, we have performed a comprehensive structurebased mutational analysis of both conserved and variable motifs of functionally diverse members of the family of epithelial adhesins of C. glabrata to contribute to a deeper understanding of the molecular evolution of protein families with large numbers of closely related members and to provide a better structural basis for the engineering of synthetic lectins with novel properties
With respect to the conserved tryptophan in loop L3, we have previously shown that this aromatic residue is crucial for efficient binding of terminal galactose moieties in vitro and for efficient host cell binding in vivo [12]

Summary

Introduction

Human-pathogenic fungi often possess large repertoires of cell wall-associated adhesins for successful host recognition, invasion, and colonization [1,2,3,4]. Mutated EpaA variants were functionally characterized by in vivo studies using an S. cerevisiae expression system and human epithelial cells and complementary in vitro analyses employing glycan array assays and crystal structure analysis.

Results

Conclusion