Structure and function of fungal adhesins

GPI连接的细胞壁蛋白家族被称为真菌粘附素，在萌芽酵母酿酒酵母、人类致病酵母光滑假丝酵母和工业相关的毕赤酵母中具有独特的表面粘附性。到目前为止，大多数这些引起表面歧视和黏附的蛋白质的详细特征在很大程度上是未知的。在上一次资助期间，我们以原子分辨率测定了酿酒酵母Flo5和FLO11以及光滑葡萄球菌粘附素Epa1的粘附域的晶体结构。在下一个资助阶段，我们建议在体外和体内阐明酿酒酵母、光滑葡萄球菌和巴斯德酵母菌进一步的碳酸结合粘附素的结构和功能特性。具体地说，我们将分析光滑链霉菌EPA和PWP家族中选定的成员的N末端配体结合结构域，以及巴斯德链霉菌中与Flo5相关的粘附素。重组粘附素结构域将通过糖链微阵列分析筛选特定的配体结合，并通过X射线结晶学分析进行结晶和结构测定。不同的粘附素配体结合域的体内性质将进一步通过以酿酒酵母为异源系统的功能分析来研究。我们还将以酿酒酵母Flo5为模型，确定真菌粘附素中心重复B结构域的生化性质，其结构和功能尚不清楚。

A family of GPI-linked cell wall proteins, known as fungal adhesins, confers unique surface adhesion properties in the budding yeast Saccharomyces cerevisiae, the human pathogenic yeast Candida glabrata and the industrially relevant yeast Pichia pastoris. The detailed features of most of these proteins conferring surface discrimination and adherence were so far largely unknown. During the last funding period, we determined crystal structures of the adhesion domains of the S. cerevisiae floccu-lins Flo5 and Flo11 as well as of the C. glabrata adhesin Epa1 at atomic resolutions. For the next funding period, we propose to elucidate the structural and functional properties of further carbohy-drate-binding adhesins from S. cerevisiae, C. glabrata and P. pastoris in vitro and in vivo. Specifically, we will analyze N-terminal ligand-binding domains of selected members from the C. glabrata Epa and Pwp families and of Flo5-related putative adhesins from P. pastoris. Respective recombinant adhesin domains will be screened for specific ligand binding by glycan microarray analysis and subjected to crystallization and structure determination by X-ray crystallographic analysis. The in vivo properties of the different adhesin ligand-binding domains will further be investigated by functional analysis using S. cerevisiae as a heterologous system. We will also determine the biochemical properties of the central, repetitive B domains of fungal adhesins, whose structures and functions are not known in detail yet, by using the S. cerevisiae flocculin Flo5 as a model.