Linker-mediated domain separation enhances cold adaptation in cellulases.

Cold-adapted cellulases have the potential to reduce energy demands in industrial processes by enabling efficient cellulose saccharification at lower temperatures. However, the structural basis of cold adaptation in bidomain cellulases remains poorly understood. Our prior studies on bidomain amylases proposed a "linker spacer effect"-a greater magnitude of domain separation is associated with improved cold adaptation. Here, we investigated this effect in the bidomain cellulase Cel5G and its linker-modified variants through molecular dynamics simulations. Domain separation, quantified by domain separation index (DSI), positively correlates with catalytic turnover number at 10°C, meaning that Cel5G variants with a greater DSI demonstrates a higher cold activity. Structural analyses show that disulfide-bonded loops are pivotal to maintain a high population of extended conformations that involve a greater DSI and reduced interdomain hydrogen bonding (H-bonding). Besides serving as a spacer, linkers in bidomain enzymes can also modulate the active site by altering the protein's conformational dynamics, fine-tuning the frequency of H-bond interactions to influence catalytic residues' capability of binding or reacting to the substrate. Overall, this study enhances our structural and dynamics-based understanding of cold adaptation of bidomain enzymes, guiding the development of new strategies for engineering cold-adapted enzymes.