Robert K. Nakamoto
Professor of Physiology & Biological Physics
 and Biochemistry & Molecular Genetics
Ph.D., University of Maryland
Structural Biology of Membrane Proteins

 

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) to translocate solutes across a membrane against concentration gradients. Our 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.

Our goal is to understand the molecular mechanisms of these different transporters. We use a variety of biochemical, biophysical and structural approaches combined with genetic and molecular biological approaches to probe the structure-function relationships. We focus on the structural and chemical changes that occur during the transport cycle. Determining the kinetics of the partial reactions occurring during transport and the energetics of these transitions allows us 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, we use site-directed mutagenesis to test our mechanistic models by altering specific 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.


Selected References

Scanlon JA, Al-Shawi MK, Nakamoto RK. (2008) "A rotor-stator cross-link in the F1-ATPase blocks the rate-limiting step of rotational catalysis." J Biol Chem. Sep 283(38):26228-40. Epub 2008 Jul 15. [PubMed]

Nakamoto RK, Baylis Scanlon JA, Al-Shawi MK. (2008) "The rotary mechanism of the ATP synthase." Arch Biochem Biophys. Aug 476:43-50. Epub 2008 May 20. [PubMed]

Scanlon JA, Al-Shawi MK, Le NP, Nakamoto RK. (2007) "Determination of the partial reactions of rotational catalysis in F1-ATPase." Biochemistry. Jul 46(30):8785-97. Epub 2007 Jul 10. [PubMed]

Caldwell SH, Chang CY, Nakamoto RK, Krugner-Higby L. (2004) "Mitochondria in nonalcoholic fatty liver disease." Clin Liver Dis. 8:595-617, x. [PubMed]

Longenecker K, Read P, Lin SK, Somlyo AP, Nakamoto RK, Derewenda ZS. () "Structure of a constitutively activated RhoA mutant (Q63L) at 1.55 A resolution." [PubMed]

Gorenne I, Nakamoto RK, Phelps CP, Beckerle MC, Somlyo AV, Somlyo AP. (2003) "LPP, a LIM protein highly expressed in smooth muscle." Am J Physiol Cell Physiol. 285:C674-85. Epub 2003 May 21. [PubMed]

Andrews SH, Peskova YB, Polar MK, Herlihy VB, Nakamoto RK. (2001) "Conformation of the gamma subunit at the gamma-epsilon-c interface in the complete Escherichia coli F(1)-ATPase complex by site-directed spin labeling." Biochemistry. Sep 40(35):10664-70. [PubMed]

Gong MC, Gorenne I, Read P, Jia T, Nakamoto RK, Somlyo AV, Somlyo AP. (2001) "Regulation by GDI of RhoA/Rho-kinase-induced Ca2+ sensitization of smooth muscle myosin II." Am J Physiol Cell Physiol. 281:C257-69. [PubMed]

Nakamoto RK. (1999) "Molecular Features of Energy Coupling in the F(0)F(1) ATP Synthase." News Physiol Sci. 14:40-46. [PubMed]