Role of the C-terminal modules of Klebsiella phage KP32 receptor-binding protein gp38 in protein and phage functionality

Despite significant progress in understanding phage biology and their clinical applications, the specificity of phages remains only poorly understood and a matter of empirical testing. Phage receptor-binding proteins (RBPs), which mediate the initial contact with bacterial cells and govern host recognition, possess a modular architecture. The N-terminal domains primarily serve a structural role, facilitating the attachment of the RBP (or RBP complex) to the virion. In contrast, the function of the C-terminal modules, and their interplay with the central enzymatic domain in impacting RBP and phage specificity, remains underexplored. This study investigates the receptor-binding protein KP32gp38 of Klebsiella phage KP32, which contains an unusual C-terminal combination of a carbohydrate-binding module (CBM) and a lectin-like (LEC) domain, two elements that are typically found separately rather than in tandem. We dissected the roles of these modules in trimerization, substrate binding, and specificity at both the protein and phage level. By deletions, fusions, and exchanges of the modules through both protein and phage engineering, we examined the impact of the domains on the specificity of the RBP and the host range of the phage. Protein fusions with GFP were tested for their ability to bind the bacterial capsule. To verify the influence of the domains on RBP trimerization, different variants were analysed with SEC - MALLS. The results revealed that the LEC domain is essential for trimerization, whereas the CBM domain is crucial for enzymatic activity and capsule binding. Engineered phages lacking these domains confirmed the necessity of both CBM and LEC for full functionality. This work underscores the versatility and evolutionary adaptation of CBM and LEC folds in phage RBPs, providing valuable insights into phage specificity mechanisms. Our findings offer a blueprint for understanding the molecular determinants of phage-host interactions, crucial for advancing phage-based antibacterial therapies.