Structure and mechanisms of sodium-pumping KR2 rhodopsin

High resolution structures reveal the mechanisms of sodium, potassium, and proton pumping by a light-driven microbial rhodopsin. Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+ pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics.

[1]  Alexei Vagin,et al.  Molecular replacement with MOLREP. , 2010, Acta crystallographica. Section D, Biological crystallography.

[2]  O. Nureki,et al.  Role of Asn112 in a Light-Driven Sodium Ion-Pumping Rhodopsin. , 2016, Biochemistry.

[3]  I. Kawamura,et al.  Solid-State Nuclear Magnetic Resonance Structural Study of the Retinal-Binding Pocket in Sodium Ion Pump Rhodopsin. , 2017, Biochemistry.

[4]  V. Gordeliy,et al.  Microbial Rhodopsins. , 2018, Sub-cellular biochemistry.

[5]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[6]  Tudor Savopol,et al.  Molecular basis of transmembrane signalling by sensory rhodopsin II–transducer complex , 2002, Nature.

[7]  A photosystem other than PS370 also mediates the negative phototaxis of Halobacterium halobium , 1985 .

[8]  Daniel J. Muller,et al.  Folding and assembly of proteorhodopsin. , 2008, Journal of molecular biology.

[9]  Michael Y. Galperin,et al.  Eukaryotic G protein-coupled receptors as descendants of prokaryotic sodium-translocating rhodopsins , 2015, Biology Direct.

[10]  Hyeon Joo,et al.  OPM database and PPM web server: resources for positioning of proteins in membranes , 2011, Nucleic Acids Res..

[11]  Randy J. Read,et al.  Phenix - a comprehensive python-based system for macromolecular structure solution , 2012 .

[12]  Y. Mukohata,et al.  Two possible roles of bacteriorhodopsin; a comparative study of strains of Halobacterium halobium differing in pigmentation. , 1977, Biochemical and biophysical research communications.

[13]  J. Spudich,et al.  Cross-protomer interaction with the photoactive site in oligomeric proteorhodopsin complexes. , 2013, Acta crystallographica. Section D, Biological crystallography.

[14]  E. Bamberg,et al.  Crystal structure of a light-driven sodium pump , 2015, Nature Structural &Molecular Biology.

[15]  B. Schobert,et al.  Halorhodopsin is a light-driven chloride pump. , 1982, The Journal of biological chemistry.

[16]  J. Spudich,et al.  In Vitro Demonstration of Dual Light-Driven Na⁺/H⁺ Pumping by a Microbial Rhodopsin. , 2015, Biophysical journal.

[17]  N. Pannu,et al.  REFMAC5 for the refinement of macromolecular crystal structures , 2011, Acta crystallographica. Section D, Biological crystallography.

[18]  Christoph Mueller-Dieckmann,et al.  In crystallo optical spectroscopy (icOS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF , 2015, Acta crystallographica. Section D, Biological crystallography.

[19]  E. Round,et al.  An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin , 2015, PloS one.

[20]  L. Brown,et al.  Recent advances in biophysical studies of rhodopsins - Oligomerization, folding, and structure. , 2017, Biochimica et biophysica acta. Proteins and proteomics.

[21]  H. Kandori,et al.  Kinetic Analysis of H(+)-Na(+) Selectivity in a Light-Driven Na(+)-Pumping Rhodopsin. , 2015, The journal of physical chemistry letters.

[22]  E. Bamberg,et al.  Structure of the light‐driven sodium pump KR2 and its implications for optogenetics , 2016, The FEBS journal.

[23]  N. Dencher,et al.  Two photosystems controlling behavioural responses of Halobacterium halobium , 1975, Nature.

[24]  R. Henderson,et al.  Three-dimensional model of purple membrane obtained by electron microscopy , 1975, Nature.

[25]  M. R. Hoque,et al.  Structural basis for Na+ transport mechanism by a light-driven Na+ pump , 2015, Nature.

[26]  F. Studier,et al.  Protein production by auto-induction in high density shaking cultures. , 2005, Protein expression and purification.

[27]  W. Stoeckenius,et al.  Reconstitution of purple membrane vesicles catalyzing light-driven proton uptake and adenosine triphosphate formation. , 1974, The Journal of biological chemistry.

[28]  O. Nureki,et al.  Mutant of a Light-Driven Sodium Ion Pump Can Transport Cesium Ions. , 2016, The journal of physical chemistry letters.

[29]  Shinya Tahara,et al.  Ultrafast photoreaction dynamics of a light-driven sodium-ion-pumping retinal protein from Krokinobacter eikastus revealed by femtosecond time-resolved absorption spectroscopy. , 2015, The journal of physical chemistry letters.

[30]  T. Uchihashi,et al.  Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy , 2018, Scientific Reports.

[31]  M. Murakami,et al.  Specific damage induced by X-ray radiation and structural changes in the primary photoreaction of bacteriorhodopsin. , 2002, Journal of molecular biology.

[32]  D. Oesterhelt,et al.  Rhodopsin-like protein from the purple membrane of Halobacterium halobium. , 1971, Nature: New biology.

[33]  A. Kaulen,et al.  M-decay in the bacteriorhodopsin photocycle: effect of cooperativity and pH. , 1995, Biophysical chemistry.

[34]  E. Round,et al.  Low-dose X-ray radiation induces structural alterations in proteins. , 2014, Acta crystallographica. Section D, Biological crystallography.

[35]  C. Bamann,et al.  Solid-state NMR analysis of the sodium pump Krokinobacter rhodopsin 2 and its H30A mutant. , 2019, Journal of structural biology.

[36]  N. Dencher,et al.  Bacteriorhodopsin monomers pump protons , 1979, FEBS letters.

[37]  A. Arseniev,et al.  Structural insights into the proton pumping by unusual proteorhodopsin from nonmarine bacteria , 2013, Proceedings of the National Academy of Sciences.

[38]  E. Bamberg,et al.  Inward H+ pump xenorhodopsin: Mechanism and alternative optogenetic approach , 2017, Science Advances.

[39]  Hideki Kandori,et al.  A light-driven sodium ion pump in marine bacteria , 2013, Nature Communications.

[40]  P. Hegemann,et al.  Electrical properties, substrate specificity and optogenetic potential of the engineered light-driven sodium pump eKR2 , 2018, Scientific Reports.

[41]  J. Rosenbusch,et al.  Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[42]  V. Cherezov,et al.  Crystallizing membrane proteins using lipidic mesophases , 2009, Nature Protocols.

[43]  E. Round,et al.  X-ray-radiation-induced changes in bacteriorhodopsin structure. , 2011, Journal of molecular biology.

[44]  H Luecke,et al.  Structure of bacteriorhodopsin at 1.55 A resolution. , 1999, Journal of molecular biology.

[45]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[46]  A. Baykov,et al.  Identification of the key determinant of the transport promiscuity in Na+-translocating rhodopsins. , 2018, Biochemical and biophysical research communications.

[47]  I. Kawamura,et al.  Long-distance perturbation on Schiff base-counterion interactions by His30 and the extracellular Na+-binding site in Krokinobacter rhodopsin 2. , 2018, Physical chemistry chemical physics : PCCP.

[48]  Bosco K. Ho,et al.  HOLLOW: Generating Accurate Representations of Channel and Interior Surfaces in Molecular Structures , 2008, BMC Structural Biology.