Robert K. Nakamoto


  • AB, University of California, Berkeley, CA
  • PhD, University of Maryland, Baltimore, MD
  • Postdoc, Yale University, New Haven, CT

Primary Appointment

  • Professor, Molecular Physiology and Biological Physics


Research Interest(s)

Structure-Function of Active Transporters

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.

List of Publications in Pubmed

Selected Publications

  • Margulieux K, Liebov B, Tirumala V, Singh A, Bushweller J, Nakamoto R, Hughes M. Bacillus anthracis Peptidoglycan Integrity Is Disrupted by the Chemokine CXCL10 through the FtsE/X Complex. Frontiers in microbiology. 2017;8 740. PMID: 28496437 | PMCID: PMC5406473
  • Verhalen B, Dastvan R, Thangapandian S, Peskova Y, Koteiche H, Nakamoto R, Tajkhorshid E, Mchaourab H. Energy transduction and alternating access of the mammalian ABC transporter P-glycoprotein. Nature. 2017;543(7647): 738-741. PMID: 28289287 | PMCID: PMC5558441
  • Margulieux K, Fox J, Nakamoto R, Hughes M. CXCL10 Acts as a Bifunctional Antimicrobial Molecule against Bacillus anthracis. mBio. 2016;7(3). PMID: 27165799 | PMCID: PMC4959661
  • Schutte K, Fisher D, Burdick M, Mehrad B, Mathers A, Mann B, Nakamoto R, Hughes M. Escherichia coli Pyruvate Dehydrogenase Complex Is an Important Component of CXCL10-Mediated Antimicrobial Activity. Infection and immunity. 2015;84(1): 320-8. PMID: 26553462 | PMCID: PMC4694015
  • Futai M, Nakanishi-Matsui M, Okamoto H, Sekiya M, Nakamoto R. Rotational catalysis in proton pumping ATPases: From E. coli F-ATPase to mammalian V-ATPase. Biochimica et biophysica acta. 2012;1817(10): 1711-21. PMID: 22459334
  • Sekiya M, Nakamoto R, Nakanishi-Matsui M, Futai M. Binding of phytopolyphenol piceatannol disrupts β/γ subunit interactions and rate-limiting step of steady-state rotational catalysis in Escherichia coli F1-ATPase. The Journal of biological chemistry. 2012;287(27): 22771-80. PMID: 22582396 | PMCID: PMC3391159
  • Sekiya M, Hosokawa H, Nakanishi-Matsui M, Al-Shawi M, Nakamoto R, Futai M. Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation. The Journal of biological chemistry. 2010;285(53): 42058-67. PMID: 20974856 | PMCID: PMC3009931
  • Massey-Gendel E, Zhao A, Boulting G, Kim H, Balamotis M, Seligman L, Nakamoto R, Bowie J. Genetic selection system for improving recombinant membrane protein expression in E. coli. Protein science : a publication of the Protein Society. 2009;18(2): 372-83. PMID: 19165721 | PMCID: PMC2708063
  • Sekiya M, Nakamoto R, Al-Shawi M, Nakanishi-Matsui M, Futai M. 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. The Journal of biological chemistry. 2009;284(33): 22401-10. PMID: 19502237 | PMCID: PMC2755962
  • Nakamoto R, Baylis Scanlon J, Al-Shawi M. The rotary mechanism of the ATP synthase. Archives of biochemistry and biophysics. 2008;476(1): 43-50. PMID: 18515057 | PMCID: PMC2581510
  • Scanlon J, Al-Shawi M, Nakamoto R. A rotor-stator cross-link in the F1-ATPase blocks the rate-limiting step of rotational catalysis. The Journal of biological chemistry. 2008;283(38): 26228-40. PMID: 18628203 | PMCID: PMC2533770
  • Scanlon J, Al-Shawi M, Le N, Nakamoto R. Determination of the partial reactions of rotational catalysis in F1-ATPase. Biochemistry. 2007;46(30): 8785-97. PMID: 17620014
  • Korepanova A, Gao F, Hua Y, Qin H, Nakamoto R, Cross T. Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli. Protein science : a publication of the Protein Society. 2004;14(1): 148-58. PMID: 15608119 | PMCID: 15608119
  • Gorenne I, Nakamoto R, Phelps C, Beckerle M, Somlyo A, Somlyo A. LPP, a LIM protein highly expressed in smooth muscle. American journal of physiology. Cell physiology. 2003;285(3): C674-85. PMID: 12760907 | PMCID: 12760907
  • Longenecker K, Read P, Lin S, Somlyo A, Nakamoto R, Derewenda Z. Structure of a constitutively activated RhoA mutant (Q63L) at 1.55 A resolution. Acta crystallographica. Section D, Biological crystallography. 2003;59 876-80. PMID: 12777804 | PMCID: 12777804
  • Andrews S, Peskova Y, Polar M, Herlihy V, Nakamoto R. 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. 2001;40(35): 10664-70. PMID: 11524011 | PMCID: 11524011
  • Figler R, Omote H, Nakamoto R, Al-Shawi M. Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein. Archives of biochemistry and biophysics. 2000;376(1): 34-46. PMID: 10729188 | PMCID: 10729188