All organisms carefully control the concentration of solutes within their cells, and are able to import required compounds or exclude cytotoxic ones. The protein machines that carry out these tasks are the primary active transporters, or pumps. These large, and often multiple subunit, integral membrane proteins utilize chemical energy usually from the hydrolysis of adenosine triphosphate or ATP, or the electrochemical energy stored in other ion gradients, to translocate solutes across a membrane against concentration gradients. The Nakamoto laboratory concentrates on three such transporters: the P-glycoprotein, a pump that has the ability to transport a broad range of compounds and confers multiple drug resistance to tumor cells; the ubiquitous FOF1 ATP synthase which uses the energy of an electrochemical gradient of protons to generate the vast majority of ATP; and the vitamin B12 transporter, BtuB of gram negative bacteria, which moves cyano-cobalamin across the outer membrane by a mechanism that is dependent upon the electrochemical gradient of protons across the inner cytoplasmic membrane.
The goal of Dr. Nakamoto’s lab is to understand the molecular mechanisms of these different transporters. It uses a variety of biochemical, biophysical and structural approaches combined with genetic and molecular biological approaches to probe the structure-function relationships. In such ways, it can obtain measurements of the structural dynamics that occur during the transport cycle. The dynamics are correlated to the kinetics of the partial reactions occurring during transport and the energetics of these transitions. The data are used to generate models which can then be computationally simulated. These approaches allow the Nakamoto lab to understand how the transporters use the energy derived from chemical reactions or from electrochemical gradients to couple to the mechanical movement of molecules from one side of a membrane to the other. With high resolution structural data as a guide, the lab uses site-directed mutagenesis to test its mechanistic models by altering single amino acids, or segments of the protein that carry out specific roles.
Not surprisingly, each of the transporters use vastly different molecular mechanisms. The P-glycoprotein binds substrate drugs from within the lipid bilayer and uses energy to rehydrate the transported compound on the exterior half of the membrane; the FOF1 transport and catalytic mechanisms are rotary motors which are coupled by a long coiled-coil structure akin to a drive shaft; and the BtuB outer membrane transporter interacts in a specific manner with the inner membrane protein TonB to activate the translocation of the large cyano-cobalamin molecule into the periplasmic space. In each case, the transporter mechanism is optimized for its specific physiological role.