RESUMEN
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.
Asunto(s)
Candida glabrata , Proteínas Fúngicas , Lectinas , Secuencias de Aminoácidos , Células CACO-2 , Candida glabrata/química , Candida glabrata/genética , Candida glabrata/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Lectinas/química , Lectinas/genética , Lectinas/metabolismo , Dominios ProteicosRESUMEN
For host colonization, the human fungal pathogen Candida glabrata is known to utilize a large family of highly related surface-exposed cell wall proteins, the lectin-like epithelial adhesins (Epas). To reveal the structure-function relationships within the entire Epa family, we have performed a large scale functional analysis of the adhesion (A) domains of 17 Epa paralogs in combination with three-dimensional structural studies of selected members with cognate ligands. Our study shows that most EpaA domains exert lectin-like functions and together recognize a wide variety of glycans with terminal galactosides for conferring epithelial cell adhesion. We further identify several conserved and variable structural features within the diverse Epa ligand binding pockets, which affect affinity and specificity. These features rationalize why mere phylogenetic relationships within the Epa family are weak indicators for functional classification and explain how Epa-like adhesins have evolved in C. glabrata and related fungal species.
Asunto(s)
Candida glabrata/química , Moléculas de Adhesión Celular/química , Proteínas Fúngicas/química , Lectinas/química , Polisacáridos/química , Secuencia de Aminoácidos , Sitios de Unión , Evolución Biológica , Candida glabrata/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Variación Genética , Lectinas/genética , Lectinas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Polisacáridos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
The human pathogenic yeast Candida glabrata harbors more than 20 surface-exposed, epithelial adhesins (Epas) for host cell adhesion. The Epa family recognizes host glycans and discriminates between target tissues by their adhesin (A) domains, but a detailed structural basis for ligand-binding specificity of Epa proteins has been lacking so far. In this study, we provide high-resolution crystal structures of the Epa1A domain in complex with different carbohydrate ligands that reveal how host cell mucin-type O-glycans are recognized and allow a structure-guided classification of the Epa family into specific subtypes. Further detailed structural and functional characterization of subtype-switched Epa1 variants shows that specificity is governed by two inner loops, CBL1 and CBL2, involved in calcium binding as well as by three outer loops, L1, L2, and L3. In summary, our study provides the structural basis for promiscuity and specificity of Epa adhesins, which might further contribute to developing anti-adhesive antimycotics and combating Candida colonization.