New membrane protein structure reveals details of peptide transport
New research from MRC Fellow Dr Simon Newstead and colleagues has provided detailed structural and biochemical insight into how a family of membrane transporters, responsible for absorbing and retaining peptides in the human body, work at a molecular level.
Reported in The EMBO Journal (1) is the crystal structure of a bacterial peptide transporter accompanied by a detailed structural and mutagenesis analysis investigating the role of conserved residues between the bacterial and mammalian proteins.
The results that have emerged will improve our understanding of a key element of nutrition, namely how our bodies absorb digested protein, and in the long-term might be used by pharmaceutical companies to help them improve oral drug absorption and retention.
Cefadroxil is an oral antibiotic in the cephalosporin family of drugs. It is actively transported within the human body by both PepT1 and PepT2 and is active against many bacteria including S. aureus, S. pneumoniae and E. coli. The reported crystal structure is a significant step towards understanding how such antibiotics are efficiently absorbed and retained in the body (Click to enlarge)
Proteins are the main source of dietary nitrogen for humans and many other organisms. They are broken down in the gastrointestinal system into di- and tri-peptides which are efficiently absorbed into the cells lining the small intestine by a membrane transporter known as PepT1. A related transporter, PepT2, helps to retain peptides by reabsorbing them in the kidneys.
Aside from this nutritional angle, explains Dr Newstead, peptide transporters are of interest to pharmaceutical companies as they are also responsible for the uptake and retention of many commonly prescribed drug molecules. ‘A large number of different pharmaceutical classes including the beta-lactam antibiotics such as penicillin and cefadroxil have a similar backbone stereochemistry to di- and tri-peptides, so they are also recognised through the same membrane peptide transport proteins.’
Pharmaceutical companies have exploited this phenomenon by attaching peptide linkers to other drugs thereby increasing their oral bioavailability and retention. However, companies currently rely predominantly on cell-based approaches to find peptides that work and the process is largely trial and error. This is where Dr Newstead’s work will have an impact.
‘One of the aims which I hope we can move towards in the coming years,’ explains Dr Newstead, ‘is to use our structural data to create an accurate 3D pharmacophore model of the peptide binding site in PepT1 and PepT2 . This will allow us to rationally design peptide mimetic and peptide pro-drug candidates using molecular docking and simulation approaches before manufacturing the compounds for laboratory testing.’
The peptide transporters under investigation belong to a family known as proton dependent oligopeptide transporters, or POTs. Like many membrane transporters they work by utilising the proton electrochemical gradient, using it to drive the uptake of peptides across the cell membrane. Over the last few years, Dr Newstead’s work has focused on the bacterial homologues of PepT1 and PepT2.
‘We’ve been trying to get the X-ray crystal structures of the simpler bacterial proteins to learn about how these transporters recognise peptides and how they couple transport to the proton electrochemical gradient.’
In 2011, he and his colleagues at Imperial College and Diamond Light Source published the crystal structure of the first bacterial homologue for this family of transporters (2). The transporter had been captured in the occluded state, with a ligand trapped inside the binding site.
Although a significant advance in our understanding of how these proteins work, this was only a partial snapshot of the transport process, as these proteins undergo substantial structural changes as they shuttle peptides across the membrane. A typical transport cycle would involve the protein adopting an ‘outward open’ state - when both proton and peptide bind, triggering the adoption of an occluded state. This would be followed by a further conformational change to an ‘inward open’ state, whereupon the peptide and proton are released internally.
The next step, as Dr Newstead explains, was to try and solve the structures of the transporter in these different transport states, enabling comparisons to be made and thereby highlighting the essential structural features required for peptide binding and transport.
Recently published crystal structure of a POT family peptide transporter from Streptococcus thermophilus, PepTSt, shown in cartoon representation, viewed from above (left) and in the plane of the membrane (right) and coloured blue to red from the N-terminus. The transporter adopts an inward open conformation with the central peptide-binding site exposed to the intracellular side of the membrane. Image taken from (1) (Click to enlarge)
The recently published paper in EMBO (1) describes the group’s latest achievements. It reveals the structure of another bacterial homologue of PepT1 – this time in the ‘inward open’ state – allowing the group to make a comparison with their previous occluded structure.
A series of mutagenesis experiments on the binding site have in addition provided essential insight into the role of specific residues and whether they are likely to bind protons or recognise the peptide ligands.
‘We have tried to follow a logical path through the transporter and have come up with a broad model for how we think it is working,’ says Dr Newstead.
‘We see that there are salt-bridge interactions on the extracellular and intracellular side of the binding site, and these work in alternating fashion to stabilise the different states. Interestingly, electrostatic interactions involving conserved salt bridge pairs is emerging as an important mechanistic theme in many secondary active transport proteins and it is satisfying to observe similar interactions being used in this family of transporters.’
POT family transporters are part of a much larger transporter superfamily - the major facilitator superfamily (MFS), which represents the largest number of transporters in the human body. The structures described by Dr Newstead and colleagues are important because they are the first example of different states in the transport cycle from the same MFS transporter family and so provide valuable insight into how the wider MFS family may function at a molecular level.
Further structural work on membrane proteins in Dr Newstead’s lab will benefit from a grant recently awarded from the Royal Society. The International Exchanges Award, a prestigious travel bursary, will allow exchange of researchers between the Newstead lab and Professor So Iwata’s lab in Kyoto, Japan.
The International Exchanges grant will be used in developing monoclonal antibody fragments to improve the resolution of crystals for protein X-ray crystallography – particularly valuable for tackling the challenge of mammalian membrane proteins such as PepT1 and PepT2.