Thermodynamic Characterization of Conformational Landscape in Proton-Coupled Oligopeptide Transporters
In this project, Blue Waters resources will be used to study the conformational landscape of a bacterial membrane transporter involved in peptide transport. Living cells rely on continuous exchange of molecules across cellular membranes for their normal function. Proton-coupled oligopeptide transporters (POTs) couple the inwardly directed proton flow to the transport of small peptides and peptide-like molecules. POTs, which belong to the major facilitator superfamily (MFS) of membrane transporters, are a model system for MFS superfamily, which is the largest family of secondary active transporters.
An important common feature of POTs is their substrate promiscuity. Human POTs PepT1 and PepT2, for instance, recognize several important families of peptide-like drug compounds in addition to natural dipeptides and tripeptides. It has been shown that POTs could improve the bioavailability of molecules attached to amino acids or dipeptides. PepT1 and PepT2 can uptake these so-called pro-drugs and may represent an important tool in the development of more effective medications. However, efficient design of these pro-drugs require reliable structural information on various functional states of POTs. Fortunately, recent structural studies have resulted in several crystal structures of prokaryotic POT members. High degree of sequence conservation within the transmembrane helices between pro- and eukaryotic POT members encourages the study of bacterial POTs, which could shed light on the transport mechanism of human PepT1 and PepT2.
While crystal structures provide a unique opportunity for establishing a structural basis for the mechanism of POTs, it is important to note that all these crystal structures are in the inward-facing (or occluded) state of these transporters. However, to function as molecular pumps, membrane transporters are known to alternate between distinct inward- and outward-facing states. Unfortunately, the large conformational changes involved are not known and despite much effort, the detailed description of the transport mechanism has remained elusive.
Molecular level description of large-scale conformational changes of proteins has posed major challenges to both experimental and computational techniques. The timescales involved in active membrane transport (typically on the order of milliseconds to seconds) are inaccessible to the conventional all-atom MD. The scope of all-atom MD is thus limited to the local conformational changes of individual states. Large-scale conformational changes, on the other hand, are often modeled using simplified representations such as coarse-graining, which does not preserve the atomic/chemical details.
Recognizing the limitations of conventional MD and coarse-grained models, we have recently developed a novel methodology based on rigorous statistical mechanics based sampling techniques to study large-scale conformational changes of proteins. The methodology is based on a combination of several distinct ensemble-based MD techniques to reconstruct the entire transport cycle of membrane transporters without compromising the atomic/chemical details.
Our computational techniques employ loosely-coupled multiple-copy MD simulations, which efficiently take advantage of petascale computing. Such algorithms require hundreds of parallel systems, each consisting of hundreds of thousands of atoms. These systems are simulated simultaneously and exchange information periodically in order to improve the sampling within an ensemble based enhanced sampling scheme. With the help of Blue Waters resources, our simulations provide a rigorous method for characterizing large-scale conformational changes of proteins and their coupling to chemical events. The structural and energetic information obtained from these simulations will provide an unprecedented level of details on the transport mechanism of POT transporters and open opportunities for investigating large-scale conformational changes of other MFS transporters.