DBP 7: Membrane Transporters
Membrane transporters perform the fundamental biological task of transporting or exchanging compounds across the cell membrane using a variety of energy sources. Several families of membrane transporters include known or putative drug targets. The study of mechanistically diverse membrane transporters is an important focus at the Center. Here, we describe and propose research on three biomedically relevant classes of transporters: neurotransmitter transporters, nutrient/drug transporters, and multidrug efflux transporters.
Neurotransmitter transporters are vital to the control of neurotransmission and, hence, are im- portant drug targets for treatment of a wide range of neurological conditions such as depression, anxiety and drug addiction. Nutrient and drug exporters from the major facilitator super-family (MFS) are present in all three kingdoms of life, and are particularly biomedical relevant for their role in conferring antibiotic resistance in bacteria. Multidrug efflux transporters such as MsbA and NorM, play crucial roles in mediating multidrug resistance (MDR) in both bacterial and cancer cells. The mechanism of drug/substrate transport in these transporters is largely unknown, impeding drug design efforts related to these proteins.
(a) Membrane transporters are responsible for active transport of substrates (squares) across the cell membrane, using energy provided by cotransport of ions (circles) or ATP hydrolysis (triangles). (b) The partially understood alternating access mechanism. Transporters undergo large conformational changes, switching between outward-facing (OF) and inward-facing (IF) states, going through several intermediates. Reconstructing this transport cycle requires the intervention of advanced computational methodologies.
The neurotransmitter sodium symporter (NSS) family includes known targets for antidepressants and is structurally represented by the bacterial leucine transporter (LeuT). LeuT also represents a fold observed in several other transporter families, the ubiquity of which suggests important structural and functional roles. Glutamate transporters (GlT) are neurotransmitter transporters that adopt an entirely different structural fold from LeuT, but perform a function similar to NSS, i.e., clearing the synaptic cleft of neurotransmitters following rounds of neurotransmission. Malfunction of GlT is associated with numerous diseases and pathological states including neurodegeneration, epilepsy, schizophrenia and traumatic brain injury. Crystal structures of a bacterial homologue, Gltph have been solved in two major transport cycle states, providing a rare opportunity to compare alternate conformational states for the same transporter and to identify the domains involved in structural transition.
The MFS is the largest superfamily of secondary active membrane transporters. Glycerol- 3-phosphate transporter (GlpT), a MFS member, facilitates import of glycerol-3- phosphate (G3P) using the downhill gradient of inorganic phosphate (Pi) from the cytoplasm to the periplasm. GlpT is also associated with antibiotic resistance. The crystal structure of GlpT is used as a structural model for several MFS proteins, including many that confer resistance to antibiotics and chemotherapeutics.
Members of the ATP-binding cassette (ABC) family are well-known multidrug efflux pumps, contributing to MDR in cancer cells or bacterial pathogens. ABC transporters couple ATP hydrolysis to translocation of diverse substrates and form one of the most abundant active transporter families. They play essential roles in the human body, such as in lipid homeostasis, ion homeostasis and adaptive immunity; and their malfunction results in diseases, e.g., cystic fibrosis and neonatal diabetes. MsbA is an ABC exporter with the rare distinction of having crystal structures known in three conformational states. The multidrug and toxic compound extrusion (MATE) family transporters play a crucial role in mediating MDR in both bacteria and mammals, modulating the efficacy of several pharmaceutical drugs. NorM is the only member of MATE family with a known crystal structure.
The transport cycle is described by a general mechanism, termed alternating access, wherein a transporter alternates between outward-facing (OF) and inward-facing (IF) states, going through several intermediate states, while interacting with several small molecules such as the substrate, ions, solvent, ATP, etc. The nature of the structural transitions involved, the coupling between various substrate/ion binding and translocation events, and their role in the transport mechanism, are still far from understood.
While experimental mutagenesis and structural studies have provided a wealth of information on transport function, characterizing the transport cycle requires a dynamical description, such as one from molecular dynamics (MD) simulations. Recently, MD simulations have been successfully applied to capture mechanistically relevant events in several membrane transport proteins. To address the challenges in describing the large conformational changes and long timescale events in membrane transport, development of novel methodologies and techniques, which will be undertaken by the Center, are required as described here.