Structural and biophysical characterisation of the extra-cellular domains from the mammalian peptide transporters, PepT1 and PepT2
Keywords: extra-cellular domain
Peptide transport
structural biology

PepT1 and PepT2 are integral membrane proteins which couple the uptake of di- and tri-peptides to the proton electro-chemical gradient. PepT1 is predominantly expressed in the small intestine and is the main route through which dietary protein is absorbed. PepT2 shares 46% sequence identity with PepT1 and is expressed in the kidneys, lung and central nervous system. Many commonly prescribed drugs, such as penicillin are peptide mimetics, and PepT1 and PepT2 play a direct role in their transport and pharmacokinetic properties. Recent X-ray crystal structures and functional studies of the bacterial peptide transporters have provided significant insight into the likely mechanism by which such drugs are recognised by PepT1 and PepT2. The bacterial peptide transporters share approximately 30% sequence identity within their trans-membrane domains to the mammalian PepT1 and PepT2 transporters. However, a key structural difference exists; an additional 20 kDa extra-cellular loop is inserted between trans-membrane helices 9 and 10 in the animal peptide transporters, and this loop is absent in the bacterial homologues. It was not known, prior to this thesis, if this extra-cellular loop was structured and/or integral to the transport cycle, or whether it served an additional function to assist or regulate peptide transport.
To investigate the role of this domain, the crystal structures of the Mus musculus PepT1 and Rattus norvegicus PepT2 'loops' were determined to 2.10 and 2.06 Å resolution respectively. The structures indicated that the loop region in both PepT1 and PepT2 forms a bi-lobal, all β-sheet, self-contained extra-cellular domain (ECD). Despite low sequence similarity, the ECDs from PepT1 and PepT2 share a common architecture; two transthyretin-like folds connected by a flexible linker. Sequence and structural analyses have indicated that the lobe interface of MmPepTECD is maintained by two highly conserved salt bridges, whereas in RnPepT2ECD only one salt bridge is observed. Small-angle X-ray diffraction experiments indicated that the extra-cellular domains form a compact structural arrangement, although the lobe conformation of PepT2ECD was more dynamic than PepT1.
Using the X-ray crystal structures of the M. musculus and R. norvegicus ECDs, and the trans-membrane domain of PepTso from Shewanella oneidensis, the first structure-based homology models of H. sapiens PepT1 and PepT2 were constructed. The hybrid models indicated that the ECD sits on top of the transporters. Two-electrode voltage clamp studies then revealed that the ECDs do not play a part in the transport mechanism of the transporter, although PepT1ECD may play a role in transporter stability.
Surface plasmon resonance binding assays were performed between the ECDs and the intestinal proteases trypsin and α-chymotrypsin. An interaction between both ECDs and α-chymotrypsin was observed, although this interaction could not be saturated using this assay. Trypsin binding however, could be saturated for both MmPepT1ECD and RnPepT2ECD giving Kds of 90 ± 20 and 170 ± 30 μM. Physiologically the interaction would give trypsin a predisposition for the peptide transporter ECD; locating the protease in the vicinity of the transporter and aiding the presentation of substrate. The interaction between trypsin and PepT1ECD was explored further with a mutational analysis of potential binding residues. The results could not locate the binding site definitively, however, the work did highlight the probable binding face which is perhaps conserved between PepT1 and PepT2 ECD.