Using DEER, Modelling and Simulations to Investigate the Dynamics of PepTSo, a Major Facilitator Superfamily Transporter of Biomedical Importance

hPepT1 is an Major Facilitator Superfamily (MFS) transporter expressed in the gastrointestinal tract and transports not only di- and tri-peptides across cell membranes but also a wide-range of hydrophilic drugs, including the beta-lactam antibiotics. Crystal structures of two homologous bacterial MFS transporters, PepTSo [1] and PepTSt [2], have recently been determined in two conformational states. In this study we examine the dynamics of PepTSo using Double Electron-Electron Resonance (DEER) measurements, modelling and computer simulation. We first generated models of the outward facing state using the repeat-swapping method [3]. The DEER residue pairs were carefully chosen so our results can also be compared to a previous study of LacY, another MFS transporter [4]. We also ran extensive molecular dynamics simulations during which PepTSo partially changes conformation.Taken together the results allow us to (i) examine the universality of MFS dynamics, (ii) tentatively assign some of the DEER peaks to known conformations and (iii) validate our outward-facing models of PepTSo. Comparison between the outward-facing model and inward-facing crystal structure suggest that kinking of the helices at conserved proline residues may be vital for conformational changes between the two states. Biochemical and DEER data reveal that mutating these proline residues abolishes transport activity, and significantly alters the protein dynamics.1. Newstead S, Drew D, Cameron AD et al (2011) EMBO J 30:4172. Solcan N, Kwok J, Fowler PW, Cameron AD, Drew D, Iwata S, Newstead S (2012) EMBO J 31:34113. Radestock S, Forrest LR (2011) J Mol Biol 407:6984. Smirnova I, Kasho V, Choe JY, et al (2007) PNAS 104:16504