Structural Biology of Transporters and Molecular Evolution of Probes
Skaggs School of Pharmacy and Pharmaceutical Sciences
Department of Pharmacology
Our research has been mostly focused on determining the x-ray structures of the four classes of multidrug resistance (MDR) transporters found in nature where the drug binding sites reside in the cell membrane. These crystal structures include a mammalian MDR ATP-Binding Cassette (ABC) transporter called P-glycoprotein (Pgp), three distinct structural conformations of a lipid ABC transporter called MsbA; the Small Multidrug Efflux (SMR) transporter EmrE with tetraphenylphosphonium (TPP); the Major Facilitator Superfamily (MFS) MDR transporter EmrD; and the Multi-Antimicrobial Toxin Extrusion (MATE) MDR transporter NorM. These drug efflux pumps confer resistance in the treatment of several bacterial infections, cancers, and also HIV. Taken together, the x-ray structures of these MDR transporters reveal a common theme in their molecular structural biology. For example, all these transporters have hydrophobic and aromatic side-chains in their poly-specific binding pockets. Our findings have also revealed that their molecular structures are all V-shaped with substrate-entry portals that open towards the lipid bilayer. The positions of these portals enable these transporters to extract hydrophobic substrates directly from the inner membrane leaflet. Upon structural rearrangement to an outward-facing conformation, they present the substrates to the outer membrane leaflet or to outside. The x-ray structures of multiple conformations of MDR ABC transporters have also demonstrated that they are indeed very flexible molecules that can accommodate the binding of large substrates. These structures provide a molecular structural framework for understanding poly-specific drug binding and the mechanics coupling ATP binding/hydrolysis with substrate transport.
The laboratory has a very high commitment to develop innovative techniques for overcoming the challenges of producing and crystallizing integral membrane proteins suitable for biophysical analysis. The lab is also a major component of NSF funded center called CROPS: Center for Research On Plant TransporterS. The focus of our center is to provide high-affinity binders and solve the x-ray structures of plant transporters relevant for food, and also human. We also have structure-function projects focused on drug transporters important for multidrug resistance and drug efficacy as well as transporters for parasites causing malaria. We are pioneering a new method for evolving molecular scaffolds (synthetic affinity maturation), which include antibodies funded by the NIH Eureka mechanism. We are also introducing and re-engineering oil transporters to secrete alkanes and other biofuel substrates partnership with the US Air Force Research Laboratory.
B.A. and M.S. in Biophysics (1993) University of Pennsylvania; Ph.D. in Molecular Biophysics (1996) University of Pennsylvania. Post doc in Chemistry (1996-1999) Caltech.
Awards and Honors:
PSI: Biology U54 Grant; Era of Hope Scholar (2004), Beckman Young Investigators (2001), Presidential Early Career Award for Scientists and Engineers (2000).
- Crystallized and determined molecular structures of four classes of MDR transporters.
- Methods development in membrane protein expression (including cell-free production of membrane proteins), purification, and crystallization.
- Demonstrated that MDR transporters have a V-like structure consist with their function moving hydrophobic substrates intercalated in the cell membrane.
- Development of a powerful molecular evolution platform applicable for antibody and biosensor discovery.
- Ward et al. (2013). Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain. Proc Natl Acad Sci U S A 110:13386-13391.
- Doshi et al. (2014). In vitro nanobody discovery for integral membrane protein targets. Sci Rep 4:6760
- Nicklish SC et al. (2016) Global marine pollutants inhibit P-glycoprotein: Environmental levels, inhibitory effects, and cocrystal structure. Sci. Adv. 2 (4): e1600001
- He X, Garza D, Nigam SK, Chang G (2016) Multispecific Organic Cation Transporter 1 (OCT1) from Bos taurus Has High Affinity and Slow Binding Kinetics towards Prostaglandin E2. PLoS One 11(4):e0152969.
- Doshi, R., et al. (2017) Functional characterization and discovery of modulators of SbMATE, the agronomically important aluminium tolerance transporter from Sorghum bicolor. Sci Rep 7(1): p. 17996.