Robert  K.  Nakamoto

Degree(s): PhD
Graduate School: Univ of Maryland
Primary Appointment: Professor, Molecular Physiology and Biological Physics

Director of the SCBB Graduate Program

Research Interests:
Structure-Function of Active Transporters

Office Address: PO Box 800886, 480 Ray C. Hunt Drive, Snyder Building Rm,    Office Phone: +1 434-982-0279, +1 434-924-5002   Fax Phone: +1 434-243-8271   Home Phone: +1 434-825-4583
Email Address: rkn3c@virginia.edu

Other Webpages: http://www.virginia.edu/bmg/faculty/nakamoto.html

Research Description

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. 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. In such ways, we 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 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 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.

Publications

Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation. Sekiya M, Hosokawa H, Nakanishi-Matsui M, Al-Shawi MK, Nakamoto RK, Futai M. J Biol Chem. 2010 Dec 31;285(53):42058-67. Epub 2010 Oct 25. PMID: 20974856

The mechanism of rotating proton pumping ATPases. Nakanishi-Matsui M, Sekiya M, Nakamoto RK, Futai M. Biochim Biophys Acta. 2010 Aug;1797(8):1343-52. Epub 2010 Feb 17. Review. PMID:20170625

Temperature dependence of single molecule rotation of the Escherichia coli ATP synthase F1 sector reveals the importance of gamma-beta subunit interactions in the catalytic dwell. Sekiya M, Nakamoto RK, Al-Shawi MK, Nakanishi-Matsui M, Futai M. J Biol Chem. 2009 Aug 14;284(33):22401-10. Epub 2009 Jun 5. PMID:19502237

Genetic selection system for improving recombinant membrane protein expression in E. coli. Massey-Gendel E, Zhao A, Boulting G, Kim HY, Balamotis MA, Seligman LM, Nakamoto RK, Bowie JU. Protein Sci. 2009 Feb;18(2):372-83. PMID: 19165721

A rotor-stator cross-link in the F1-ATPase blocks the rate-limiting step of rotational catalysis. Scanlon JA, Al-Shawi MK, Nakamoto RK. J Biol Chem. 2008 Sep 19;283(38):26228-40. Epub 2008 Jul 15. PMID:18628203

Special issue on transport ATPases. Inesi G, Nakamoto RK. Arch Biochem Biophys. 2008 Aug 1;476(1):1-2. Epub 2008 Jun 11. No abstract available.  PMID:18573231

The rotary mechanism of the ATP synthase. Nakamoto RK, Baylis Scanlon JA, Al-Shawi MK. Arch Biochem Biophys. 2008 Aug 1;476(1):43-50. Epub 2008 May 20. Review PMCID: PMC2581510

Determination of the partial reactions of rotational catalysis in F1-ATPase. Scanlon JA, Al-Shawi MK, Le NP, Nakamoto RK. Biochemistry. 2007 Jul 31;46(30):8785-97. Epub 2007 Jul 10. PMID: 17620014

Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli. Korepanova A, Gao FP, Hua Y, Qin H, Nakamoto RK, Cross TA. Protein Sci. 2005 Jan;14(1):148-58. PMID:15608119

Mitochondria in nonalcoholic fatty liver disease. Caldwell SH, Chang CY, Nakamoto RK, Krugner-Higby L. Clin Liver Dis. 2004 Aug;8(3):595-617, x. Review. PMID:15331066

Structure of a constitutively activated RhoA mutant (Q63L) at 1.55 A resolution. Longenecker K, Read P, Lin SK, Somlyo AP, Nakamoto RK, Derewenda ZS. Acta Crystallogr D Biol Crystallogr. 2003 May;59(Pt 5):876-80. Epub 2003 Apr 25. PMID: 12777804

LPP, a LIM protein highly expressed in smooth muscle. Gorenne I, Nakamoto RK, Phelps CP, Beckerle MC, Somlyo AV, Somlyo AP. Am J Physiol Cell Physiol. 2003 Sep;285(3):C674-85. Epub 2003 May 21. PMID:12760907

F0 cysteine, bCys21, in the Escherichia coli ATP synthase is involved in regulation of potassium uptake and molecular hydrogen production in anaerobic conditions. Mnatsakanyan N, Bagramyan K, Vassilian A, Nakamoto RK, Trchounian A. Biosci Rep. 2002 Jun-Aug;22(3-4):421-30. PMID: 12516783

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. Andrews SH, Peskova YB, Polar MK, Herlihy VB, Nakamoto RK. Biochemistry. 2001 Sep 4;40(35):10664-70. PMID: 11524011

Regulation by GDI of RhoA/Rho-kinase-induced Ca2+ sensitization of smooth muscle myosin II. Gong MC, Gorenne I, Read P, Jia T, Nakamoto RK, Somlyo AV, Somlyo AP. Am J Physiol Cell Physiol. 2001 Jul;281(1):C257-69. PMID:11401849

Molecular Features of Energy Coupling in the F(0)F(1) ATP Synthase. Nakamoto RK. News Physiol Sci. 1999 Feb;14:40-46. PMID:11390817

Site-specific phosphorylation and point mutations of telokin modulate its Ca2+-desensitizing effect in smooth muscle. Walker LA, MacDonald JA, Liu X, Nakamoto RK, Haystead TA, Somlyo AV, Somlyo AP. J Biol Chem. 2001 Jul 6;276(27):24519-24. Epub 2001 May 9. PMID: 11346659

Expression and purification of Rho/RhoGDI complexes. Read PW, Nakamoto RK. Methods Enzymol. 2000;325:15-25. No abstract available.  PMID:11036588

Catalytic control and coupling efficiency of the Escherichia coli FoF1 ATP synthase: influence of the Fo sector and epsilon subunit on the catalytic transition state. Peskova YB, Nakamoto RK. Biochemistry. 2000 Sep 26;39(38):11830-6. PMID:10995251

Phosphorylation of telokin by cyclic nucleotide kinases and the identification of in vivo phosphorylation sites in smooth muscle. MacDonald JA, Walker LA, Nakamoto RK, Gorenne I, Somlyo AV, Somlyo AP, Haystead TA. FEBS Lett. 2000 Aug 18;479(3):83-8. PMID:10981712

Molecular mechanisms of rotational catalysis in the F(0)F(1) ATP synthase. Nakamoto RK, Ketchum CJ, Kuo PH, Peskova YB, Al-Shawi MK. Biochim Biophys Acta. 2000 May 31;1458(2-3):289-99. Review. PMID:10838045

Intragenic and intergenic suppression of the Escherichia coli ATP synthase subunit a mutation of Gly-213 to Asn: functional interactions between residues in the proton transport site. Kuo PH, Nakamoto RK. Biochem J. 2000 May 1;347 Pt 3:797-805. PMID:10769185

Functional role of charged residues in the transmembrane segments of the yeast plasma membrane H+-ATPase. Petrov VV, Padmanabha KP, Nakamoto RK, Allen KE, Slayman CW. J Biol Chem. 2000 May 26;275(21):15709-16. PMID:10747929

Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein. Figler RA, Omote H, Nakamoto RK, Al-Shawi MK. Arch Biochem Biophys. 2000 Apr 1;376(1):34-46. PMID:10729188

Human RhoA/RhoGDI complex expressed in yeast: GTP exchange is sufficient for translocation of RhoA to liposomes. Read PW, Liu X, Longenecker K, Dipierro CG, Walker LA, Somlyo AV, Somlyo AP, Nakamoto RK. Protein Sci. 2000 Feb;9(2):376-86. PMID: 10716190

Escherichia coli ATP synthase alpha subunit Arg-376: the catalytic site arginine does not participate in the hydrolysis/synthesis reaction but is required for promotion to the steady state. Le NP, Omote H, Wada Y, Al-Shawi MK, Nakamoto RK, Futai M. Biochemistry. 2000 Mar 14;39(10):2778-83. PMID:10704230