Crystal Structures Reveal the Multi-Ligand Binding Mechanism of Staphylococcus aureus ClfB

Staphylococcus aureus (S. aureus) pathogenesis is a complex process involving a diverse array of extracellular and cell wall components. ClfB, an MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family surface protein, described as a fibrinogen-binding clumping factor, is a key determinant of S. aureus nasal colonization, but the molecular basis for ClfB-ligand recognition remains unknown. In this study, we solved the crystal structures of apo-ClfB and its complexes with fibrinogen α (Fg α) and cytokeratin 10 (CK10) peptides. Structural comparison revealed a conserved glycine-serine-rich (GSR) ClfB binding motif (GSSGXGXXG) within the ligands, which was also found in other human proteins such as Engrailed protein, TCF20 and Dermokine proteins. Interaction between Dermokine and ClfB was confirmed by subsequent binding assays. The crystal structure of ClfB complexed with a 15-residue peptide derived from Dermokine revealed the same peptide binding mode of ClfB as identified in the crystal structures of ClfB-Fg α and ClfB-CK10. The results presented here highlight the multi-ligand binding property of ClfB, which is very distinct from other characterized MSCRAMMs to-date. The adherence of multiple peptides carrying the GSR motif into the same pocket in ClfB is reminiscent of MHC molecules. Our results provide a template for the identification of other molecules targeted by S. aureus during its colonization and infection. We propose that other MSCRAMMs like ClfA and SdrG also possess multi-ligand binding properties.金黄色葡萄球菌（Staphylococcus aureus, S. aureus）的致病过程是一个复杂的生物学过程，涉及多种胞外组分与细胞壁成分。ClfB属于微生物表面识别黏附基质分子（Microbial Surface Components Recognizing Adhesive Matrix Molecules, MSCRAMM）家族的表面蛋白，被定义为纤维蛋白原结合型凝集因子，是金黄色葡萄球菌鼻腔定植的关键决定因素，然而ClfB与配体的识别分子基础至今仍未明确。本研究解析了无配体结合态ClfB（apo-ClfB）及其与纤维蛋白原α（fibrinogen α, Fg α）、细胞角蛋白10（cytokeratin 10, CK10）肽段的复合物晶体结构。结构比对分析显示，各类配体中均存在一段保守的富含甘氨酸-丝氨酸（Glycine-Serine-rich, GSR）的ClfB结合基序（GSSGXGXXG），该基序同样存在于Engrailed蛋白（Engrailed protein）、TCF20以及真皮蛋白（Dermokine）等多种人类蛋白质中。后续结合实验证实了真皮蛋白与ClfB之间存在相互作用。针对真皮蛋白来源的15肽段与ClfB的复合物晶体结构解析结果表明，ClfB的肽段结合模式与此前解析的ClfB-Fg α、ClfB-CK10复合物晶体结构中观察到的模式完全一致。本研究结果凸显了ClfB的多配体结合特性，这与目前已被表征的其他MSCRAMM家族成员存在显著差异。多个携带GSR基序的肽段结合至ClfB的同一结合口袋的现象，与主要组织相容性复合体（Major Histocompatibility Complex, MHC）分子的配体结合机制颇为相似。本研究结果为鉴定金黄色葡萄球菌在定植与感染过程中靶向的其他分子提供了结构模板。我们推测，包括ClfA与SdrG在内的其他MSCRAMM家族蛋白同样具备多配体结合特性。