Accelerated Molecular Dynamics and AlphaFold Uncover a Missing Conformational State of Transporter Protein OxlT

Abstract

Transporter proteins change their conformation to carry their substrate across the cell membrane. The conformational dynamics are vital to understanding the transport function. We have studied the oxalate transporter (OxlT), an oxalate:formate antiporter from Oxalobacter formigenes, significant in avoiding kidney stone formation. The atomic structure of OxlT has been recently solved in the outward-open and occluded states. However, the inward-open conformation is still missing, hindering a complete understanding of the transporter. Here, we performed an accelerated molecular dynamics simulation to sample the extensive conformational space of OxlT and successfully obtained the inward-open conformation where cytoplasmic substrate formate binding was preferred over oxalate binding. We also identified critical interactions for the inward-open conformation. The results were complemented by the highly accurate structure prediction by AlphaFold2. Although AlphaFold2 solely predicted OxlT in the outward-open conformation, mutation of the identified critical residues made it partly predict the inward-open conformation, identifying possible state-shifting mutations.

Predicting the alternative conformation of a known protein structure based on the distance map of AlphaFold2

“…These deep-learning-based methods also facilitated other related studies, such as utilizing predicted structures as AI-augmented inputs for molecular dynamics (MD) simulations. 7,8 Furthermore, the advent of AF2 has sparked renewed inspiration in predicting multi-state conformation structures, which is crucial for comprehending dynamic behaviors and exploring the conformation space from the structural biology perspective. [9][10][11][12] Nonetheless, determining multiple functional states of proteins, such as the apo and holo state of enzymes and the outward/inwardfacing states of transporter proteins, remains challenging both experimentally and computationally.…”