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

Abstract

Transporter proteins change their conformations to carry their substrate across the cell membrane. The conformational dynamics is 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 inwardopen conformation is still missing, hindering a complete understanding of the transporter.Here, we performed a Gaussian accelerated molecular dynamics simulation to sample the extensive conformational space of OxlT and successfully predicted 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 an AlphaFold2 structure prediction. 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.

Integration of AlphaFold with Molecular Dynamics for Efficient Conformational Sampling of Transporter Protein NarK

“…The alternative method for MSA construction showed the same bias toward the inward-open state (Figure S1). Here, conformations of the transporter were characterized by the distance of the periplasmic and cytoplasmic gates located above and below the substrate binding site (Figure A). , The structure with the highest ⟨pLDDT⟩ greater than 90, which is the average pLDDT score over all residues, has Cα-RMSD of 0.71 Å from an experimental NarK structure in the inward-open state . The substrate-binding site also shows a proper side-chain conformation consistent with the crystal structure (Figure C).…”

“…The predicted outward-open conformation was then compared to the known inward-open conformation to clarify specific interactions for the outward-open state. We analyzed inter-residue contact changes between N-terminal and C-terminal domains upon transition from the inward-open to the outward-open conformation, as in our previous study on other transporter proteins Figure A shows inter-residue contacts formed (broken) upon the transition, shown in blue (red) lines.…”

“…We further assume critical residue pairs contributing to the conformational state are strongly coevolved through the evolution of amino-acid sequence. , Thus, we consider evolutionary information in addition to the structural contact changes. To this end, we applied the direct coupling analysis to the MSA used in the AF2 structure prediction.…”

“…On the one hand, MD simulations can provide the conformational dynamics of transporter proteins in atomic details at a high computational cost. Although there is a limitation in the time scale of MD simulations, rare-event sampling methods using external forces, − boosting potentials, , string method, Markov state model (MSM), transition path sampling (TPS) , can overcome the problem. In particular, MD simulations can provide paths between stable states, including transition states that determine reaction rates and mechanisms.…”

“…It has been demonstrated that, while AF2 has learned a scoring function, or “energy function”, that is optimal around native conformations, such evolutionary information is vital to finding appropriate native conformations in the vast conformational space . MSA subsampling, clustering, masking, and coevolution-informed mutations have been developed to predict alternative or multiple conformational states.…”