Structure determination of a major facilitator peptide transporter: Inward facing PepTSt from Streptococcus thermophilus crystallized in space group P3121

Abstract

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 PepTSt from Streptococcus thermophilus. The space group is P3121, 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 P212121 and C2221 structures of inward facing PepTSt, 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 PepTSt, 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.

Tripeptide binding in a proton‐dependent oligopeptide transporter

“…During the transport cycle, these domains move relative to each other to allow alternate access from the cytoplasmic and extracellular sides . Several X‐ray structures of bacterial POTs have been reported, either in the apo‐form , peptide bound state , or in complex with the phosphonodipeptide alafosfalin (Table S1). However, only three structures have hitherto been reported in which a tripeptide is bound.…”

“…Several X-ray structures of bacterial POTs have been reported, either in the apo-form [8][9][10][11][12][13][14][15][16], peptide bound state [17][18][19][20], or in complex with the phosphonodipeptide alafosfalin [21,22] (Table S1). However, only three structures have hitherto been reported in which a tripeptide is bound.…”

Lipid‐like Peptides can Stabilize Integral Membrane Proteins for Biophysical and Structural Studies

“…The use of detergents with all of their detrimental properties is one of the key limitations in structurals tudies of IMPs, because they are poor mimics of the lipid bilayer.N umerous detergents are available and have been tested to solubilize, purify,a nd crystallize IMPs. It is well known that short-chain detergents such as NM and NG are more denaturing than long-chain detergents such as DDM [44] (Figure S3), but at the same time, short-chain detergents are beneficial for crystallization because of their reduced micelle size. Recent molecular dynamics simulations suggested that this destabilizing effect could be explained by ad ecrease in a-helicity in the transmembrane regions (owing to as maller micelle size of the detergenta nd, consequently,e xposure of al argerf raction of the hydrophobic surface), suboptimal a-helicalp acking, and the interpenetration of detergentm olecules between TM-helices.…”

Conformational Transition Pathways in Major Facilitator Superfamily Transporters

“…15,16 A very important feature among POTs is substrate promiscuity 17 attributed to the binding site accommodating a range of peptides and peptide like molecules in multiple orientations. 18 Mammalian POTs are yet to be crystallized; however, several bacterial POT members have recently been crystallized including PepT so , 18,19 PepT st , 11,20 PepT so2 , 12 and GkPOT. 16 Particularly, the POT transporter from…”