Structure determination of a major facilitator peptide transporter: Inward facing PepT(St) from Streptococcus thermophilus crystallized in space group P3(1)21

Major facilitator superfamily (MFS) peptide transporters (typically referred to as PepT, POT or PTR transporters) mediate the uptake of di- and tripeptides, and so play an important dietary role in many organisms. In recent years, a better understanding of the molecular basis for this process has emerged, which is in large part due to a steep increase in structural information. Yet, the conformational transitions underlying the transport mechanism are still not fully understood, and additional data is therefore needed. Here we report in detail the detergent screening, crystallization, experimental MIRAS phasing, and refinement of the peptide transporter PepT(St) from Streptococcus thermophilus. The space group is P3(1)21, and the protein is crystallized in a monomeric inward facing form. The binding site is likely to be somewhat occluded, as the lobe encompassing transmembrane helices 10 and 11 is markedly bent towards the central pore of the protein, but the extent of this potential occlusion could not be determined due to disorder at the apex of the lobe. Based on structural comparisons with the seven previously determined P2(1)2(1)2(1) and C222(1) structures of inward facing PepT(St), the structural flexibility as well as the conformational changes mediating transition between the inward open and inward facing occluded states are discussed. In conclusion, this report improves our understanding of the structure and conformational cycle of PepT(St), and can furthermore serve as a case study, which may aid in supporting future structure determinations of additional MFS transporters or other integral membrane proteins.