Real-time identification of two substrate-binding intermediates for the light-driven sodium pump rhodopsin

Abstract

Membrane transport proteins undergo critical conformational changes during substrate uptake and release, as the substrate-binding site is believed to switch its accessibility from one side of the membrane to the other. Thus, at least two substrate-binding intermediates should appear during the process, that is, after uptake and before the release of the substrate. However, this view has not been verified for most transporters because of the difficulty in detecting short-lived intermediates. Here, we report real-time identification of these intermediates for the light-driven outward current-generating Na+-pump rhodopsin. We triggered the transport cycle of Na+-pump rhodopsin using a short laser pulse, and subsequent formation and decay of various intermediates was detected by time-resolved measurements of absorption changes. We used this method to analyze transport reactions and elucidated the sequential formation of the Na+-binding intermediates O1 and O2. Both intermediates exhibited red-shifted absorption spectra and generated transient equilibria with short-wavelength intermediates. The equilibria commonly shifted toward O1 and O2 with increasing Na+ concentration, indicating that Na+ is bound to these intermediates. However, these equilibria were formed independently; O1 reached equilibrium with preceding intermediates, indicating Na+ uptake on the cytoplasmic side. In contrast, O2 reached equilibrium with subsequent intermediates, indicating Na+ release on the extracellular side. Thus, there is an irreversible switch in “accessibility” during the O1 to O2 transition, which could represent one of the key processes governing unidirectional Na+ transport. Membrane transport proteins undergo critical conformational changes during substrate uptake and release, as the substrate-binding site is believed to switch its accessibility from one side of the membrane to the other. Thus, at least two substrate-binding intermediates should appear during the process, that is, after uptake and before the release of the substrate. However, this view has not been verified for most transporters because of the difficulty in detecting short-lived intermediates. Here, we report real-time identification of these intermediates for the light-driven outward current-generating Na+-pump rhodopsin. We triggered the transport cycle of Na+-pump rhodopsin using a short laser pulse, and subsequent formation and decay of various intermediates was detected by time-resolved measurements of absorption changes. We used this method to analyze transport reactions and elucidated the sequential formation of the Na+-binding intermediates O1 and O2. Both intermediates exhibited red-shifted absorption spectra and generated transient equilibria with short-wavelength intermediates. The equilibria commonly shifted toward O1 and O2 with increasing Na+ concentration, indicating that Na+ is bound to these intermediates. However, these equilibria were formed independently; O1 reached equilibrium with preceding intermediates, indicating Na+ uptake on the cytoplasmic side. In contrast, O2 reached equilibrium with subsequent intermediates, indicating Na+ release on the extracellular side. Thus, there is an irreversible switch in “accessibility” during the O1 to O2 transition, which could represent one of the key processes governing unidirectional Na+ transport. Membrane transport proteins play vital roles by transporting various substances across cell membranes. The function “transportation” seems to be simple but needs strategic conformational changes. In particular, during substrate uptake and release, essential changes should occur as proposed by the alternating access model (1Jardetzky O. Simple allosteric model for membrane pumps.Nature. 1966; 211: 969-970Crossref PubMed Scopus (827) Google Scholar). For substrate uptake, the protein lumen should be exposed only to the relevant side of the membrane. After substrate uptake, “accessibility switching” occurs so that the protein lumen is exposed to the other side of the membrane for substrate release. Thus, the transport cycle should contain at least two substrate-binding intermediates, both of which involve the substrate but have significantly different conformations. These consecutively appearing intermediates have been successfully identified by computational analyses for some transport proteins (2Moradi M. Enkavi G. Tajkhorshid E. Atomic-level characterization of transport cycle thermodynamics in the glycerol-3-phosphate:phosphate antiporter.Nat. Commun. 2015; 6: 8393Crossref PubMed Scopus (56) Google Scholar, 3Sauer D.B. Trebesch N. Marden J.J. Cocco N. Song J. Koide A. Koide S. Tajkhorshid E. Wang D.N. Structural basis for the reaction cycle of DASS dicarboxylate transporters.Elife. 2020; 9e61350Crossref PubMed Google Scholar). However, experimental identifications are not achieved for most proteins, reflecting the difficulty in detecting and characterizing short-lived intermediates. Here, we report the real-time identification of two substrate-binding intermediates for the light-driven Na+-pump rhodopsin (NaR). NaR belongs to the microbial rhodopsin family, a huge family of photoactive membrane proteins in unicellular microorganisms. Similar to visual rhodopsin in animal eyes, microbial rhodopsins consist of seven transmembrane helices and the chromophore retinal, which binds to a specific Lys residue via a protonated Schiff base (4Ernst O.P. Lodowski D.T. Elstner M. Hegemann P. Brown L.S. Kandori H. Microbial and animal rhodopsins: Structures, functions, and molecular mechanisms.Chem. Rev. 2014; 114: 126-163Crossref PubMed Scopus (582) Google Scholar, 5Govorunova E.G. Sineshchekov O.A. Li H. Spudich J.L. Microbial rhodopsins: Diversity, mechanisms, and optogenetic applications.Annu. Rev. Biochem. 2017; 86: 845-872Crossref PubMed Scopus (142) Google Scholar, 6Kandori H. Biophysics of rhodopsins and optogenetics.Biophys. Rev. 2020; 12: 355-361Crossref PubMed Scopus (15) Google Scholar). Upon light absorption, retinal isomerizes from the all-trans to the 13-cis state and distorts the protein conformation. This energized state of the protein is thermally relaxed to the original state via a variety of intermediates. During this cyclic reaction, called a photocycle, microbial rhodopsins perform various functions, such as ion pumps, ion channels, light sensors, and enzymes. The respective intermediates have their own “colors,” reflecting the differences in retinal configuration and its environment. This color difference is advantageous for analyzing photoreactions. When we use a laser pulse, a large amount of microbial rhodopsin simultaneously starts the photocycle. Thus, subsequent formations and decays of various intermediates can be detected by time-resolved measurements of the absorption changes. We adopted this method to analyze the NaR photocycle. NaR is the third ion pump rhodopsin widely spread in marine bacteria (7Jung K.H. New type of cation pumping microbial rhodopsins in marine bacteria.in: 244th ACS National Meeting & Exposition. 2012Google Scholar, 8Inoue K. Ono H. Abe-Yoshizumi R. Yoshizawa S. Ito H. Kogure K. Kandori H. A light-driven sodium ion pump in marine bacteria.Nat. Commun. 2013; 4: 1678Crossref PubMed Scopus (239) Google Scholar, 9Kandori H. Inoue K. Tsunoda S.P. Light-driven sodium-pumping rhodopsin: A new concept of active transport.Chem. Rev. 2018; 118: 10646-10658Crossref PubMed Scopus (37) Google Scholar). Unlike H+ and Cl− pump rhodopsins, unphotolyzed NaR does not involve the substrate Na+. After the photocycle start, NaR captures Na+ at the cytoplasmic (CP) side and releases it at the extracellular (EC) side, as illustrated in Figure 1A. The major intermediates that appeared in the NaR photocycle are shown in Figure 1B (8Inoue K. Ono H. Abe-Yoshizumi R. Yoshizawa S. Ito H. Kogure K. Kandori H. A light-driven sodium ion pump in marine bacteria.Nat. Commun. 2013; 4: 1678Crossref PubMed Scopus (239) Google Scholar, 10Inoue K. Konno M. Abe-Yoshizumi R. Kandori H. The role of the NDQ motif in sodium-pumping rhodopsins.Angew. Chem. Int. Ed. Engl. 2015; 54: 11536-11539Crossref PubMed Scopus (35) Google Scholar). Previously, we experimentally proved that Na+ uptake and release occur during the formation and decay of the O intermediate, which appears at the longer wavelength region in the latter half of the photocycle (11Murabe K. Tsukamoto T. Aizawa T. Demura M. Kikukawa T. Direct detection of the substrate uptake and release reactions of the light-driven sodium-pump rhodopsin.J. Am. Chem. Soc. 2020; 142: 16023-16030Crossref PubMed Scopus (4) Google Scholar). In that study, we demonstrated well-matched time courses of two flash-induced signals, viz., Na+ concentration change because of Na+ uptake and release by NaR and absorbance change reflecting O intermediate accumulation. However, as mentioned previously, at least two O intermediates should exist involving Na+ inside the protein. We previously suggested the formation of two O intermediates based on the absorbance changes but did not perform a definitive analysis (12Kajimoto K. Kikukawa T. Nakashima H. Yamaryo H. Saito Y. Fujisawa T. Demura M. Unno M. Transient resonance Raman spectroscopy of a light-driven sodium-ion-pump rhodopsin from Indibacter alkaliphilus.J. Phys. Chem. B. 2017; 121: 4431-4437Crossref PubMed Scopus (27) Google Scholar). In the present study, we examined the NaR photocycle in detail by flash-induced absorbance changes to unravel the O intermediate substrates. Our analysis revealed that the photocycle consists of seven intermediates, including two O intermediates, O1 and O2. These O intermediates appear sequentially in the longer wavelength region and independently attain transient equilibria with other intermediates, where O1 equilibrates with the preceding intermediates, but O2 does so with the subsequent intermediate. These results indicate that accessibility switching occurs during the O1 to O2 transition. For all experiments, we used a nanodisc containing NaR to increase the clarity of the sample. Figure 1C shows the flash-induced absorbance changes at three typical wavelengths. The Na+ concentration increased from traces 1 to 4, whose respective concentrations were 10, 30, 100, and 500 mM Na+, respectively. The negative deflection at 530 nm represents the original dark state's depletion, whereas the positive deflection at other wavelengths indicates intermediate formation. The first intermediate K appears at a very fast rate. Thus, our apparatus followed the photocycle after the completion of K formation. The traces for 600 nm start with positive values (at 0.01 ms), which reflect the presence of remaining K. Concomitant to its decay, the short-wavelength intermediate appears at 410 nm. This absorption change was previously assigned to the equilibrium of the L and M intermediates (8Inoue K. Ono H. Abe-Yoshizumi R. Yoshizawa S. Ito H. Kogure K. Kandori H. A light-driven sodium ion pump in marine bacteria.Nat. Commun. 2013; 4: 1678Crossref PubMed Scopus (239) Google Scholar, 12Kajimoto K. Kikukawa T. Nakashima H. Yamaryo H. Saito Y. Fujisawa T. Demura M. Unno M. Transient resonance Raman spectroscopy of a light-driven sodium-ion-pump rhodopsin from Indibacter alkaliphilus.J. Phys. Chem. B. 2017; 121: 4431-4437Crossref PubMed Scopus (27) Google Scholar, 13Hontani Y. Inoue K. Kloz M. Kato Y. Kandori H. Kennis J.T. The photochemistry of sodium ion pump rhodopsin observed by watermarked femto- to submillisecond stimulated Raman spectroscopy.Phys. Chem. Chem. Phys. 2016; 18: 24729-24736Crossref PubMed Google Scholar, 14Asido M. Eberhardt P. Kriebel C.N. Braun M. Glaubitz C. Wachtveitl J. Time-resolved IR spectroscopy reveals mechanistic details of ion transport in the sodium pump Krokinobacter eikastus rhodopsin 2.Phys. Chem. Chem. Phys. 2019; 21: 4461-4471Crossref PubMed Google Scholar). Finally, the red-shifted intermediate O appears at 600 nm and peaks at approximately 1 to 10 ms. As described previously, the substrate Na+ is captured and released during the formation and decay of O. Reflecting its Na+-binding state, O is not formed in the absence of Na+ (Fig. S2), whereas in the presence of Na+, O accumulation becomes prominent with increasing Na+ concentration (Fig. 1C). During Na+ uptake and release, O should make transient equilibria with its preceding and subsequent intermediates in a manner dependent on Na+ concentration. To clarify these aspects and the presence of O substrates, we analyzed the photocycle in detail. We performed global-fitting analyses for the datasets of the flash-induced absorbance changes (380–700 nm, 10 nm intervals) according to the sequential irreversible model: P0 → P1 → P2 → … → Pn → P0 (15Chizhov I. Chernavskii D.S. Engelhard M. Mueller K.H. Zubov B.V. Hess B. Spectrally silent transitions in the bacteriorhodopsin photocycle.Biophys. J. 1996; 71: 2329-2345Abstract Full Text PDF PubMed Scopus (138) Google Scholar). P0 denotes the original dark state, whereas Pi (i = 1, 2, … n) denotes the which are not intermediates. For rhodopsins, intermediates are to be with not only reactions but During the photocycle, these intermediates In this intermediates are not but the equilibrium state is these intermediates are in the Pi Thus, the sequential irreversible model is the model to the of intermediates in the photocycle and the of reactions details of analysis The of to the of in the function used for the Thus, we first performed analyses by the of to the of of the for the analyses are shown in Figure As shown the in at seven in the presence of Na+ concentration. The is large with for other microbial rhodopsins T. C. A. Tsukamoto T. M. Aizawa T. K. N. Demura M. the photocycle of with an and membrane of the 2015; PubMed Scopus Google Scholar, S. Kikukawa T. J. M. Aizawa T. K. N. Demura M. characterization of and its in the 2016; PubMed Scopus Google Scholar, T. Kikukawa T. Y. Aizawa T. S. N. Demura M. of a pumping 2019; PubMed Scopus Google Scholar, I. Engelhard M. and of the photocycle of from J. Full Text Full Text PDF PubMed Scopus Google Scholar). However, the of the seven is by Figure which indicates the of the decay time of and their changes with increasing Na+ concentration. For the decay time at mM Na+ were and for the respectively. Thus, we that the NaR photocycle seven The is which state to O accumulation. O can be substrates, O accumulation is described by two Thus, we the time courses of concentration changes of and the flash-induced absorbance change at 600 nm. The latter the absorbance change because of the formation and decay of O. As shown in Figure O are described by and at all Na+ Thus, both contain Na+-binding intermediates. we the absorption spectra of to the intermediates in state, in the and The results are in Figure As both the and contain red-shifted intermediates (Fig. 4, and in equilibrium with the wavelength intermediates. As the Na+ concentration is the equilibria commonly toward red-shifted intermediates. Thus, these intermediates Na+ and to the O intermediate. However, their values are The O in has at approximately nm, whereas O in has a of approximately nm. Thus, these intermediates should be assigned to different Na+-binding we O1 and respectively. For we the absorption spectra of the intermediates. Here, we the function to intermediate spectra The are in Figure and their are in As described we the photocycle shown in Figure which the the and the from this The indicates the sequential irreversible model used for the this the decay time at mM Na+ are The represents the photocycle The indicate the intermediates in the respective Pi The Na+ uptake and release are with the values for Na+. The O1 to O2 occurs and to the of accessibility indicates the of L and so their could not be In to the the state M in a This latter M is with which be formed from O2 reflecting Na+ the protonated Schiff base Figure the seven and (Fig. 4, and absorption of Na+ concentration. Thus, these contain only a intermediate. with a as shown in The first state, should to K. the state, has the as the dark Thus, is a of the dark state and should be as which has the protein as the dark a red-shifted intermediate. We this intermediate N. As in the preceding state, and O2 attain Thus, seems to have a whose Na+-binding site to the side. This be different from the subsequent and dark As mentioned previously, the dark state does not contain Na+. Thus, the of the photocycle, the site should from the The P2 and have and spectra (Fig. 4, and Thus, to contain the intermediates in the Here, we that K in the P2 and and longer shows of the remaining spectra after the of K. The remaining spectra are for and contain two intermediates L and M. For rhodopsins, the M intermediate is to contain the Schiff base it the absorption all intermediates. For M of this was experimentally N. M. Kandori H. Y. and a in the retinal chromophore transport by the pump Phys. Chem. B. 2019; PubMed Scopus Google Scholar). Similar to P2 and a appeared in at 10 mM Na+ (Fig. Thus, the equilibrium O1 and its preceding intermediates and which should be with the Na+ uptake reaction at the side. the at 10 mM was (Fig. and to that of in the state (Fig. Thus, represents the equilibrium at the side, where O2 is in equilibrium with the subsequent N. This equilibrium should be with the Na+ release The of this was by the of the Here, we first analyzed the and spectra at and 500 In both the spectra at two Na+ concentrations were the (Fig. 4, and Thus, the of the equilibria to be this the and spectra (Fig. 4, and we the absorbance values at of O1 and O2 respectively. These absorbance values are in Na+ concentration. The in absorption values at Na+ concentrations and Reflecting these the and spectra mM Na+ appear to contain O1 and O2 as the respective whereas at approximately nm, both spectra contain from intermediate. We assigned this intermediate as its in seems as We the spectra of and as shown in Here, we first the respective spectra at and 500 mM Na+. The two spectra were simultaneously using the where and represent the for the respective absorption and are two spectra in the The and are the to their respective As mentioned previously, O1 and O2 not appear simultaneously in the and Thus, was to for whereas was to for The the spectra (Fig. As described previously, P2 and commonly contain and M. Thus, the spectra of P2 and at all Na+ concentrations were simultaneously by the Here, we that is to that in we from the analysis of P1 (Fig. Thus, in the using 2, we and the three and The results are in the two in Figure The P2 spectra a Na+ concentration (Fig. As shown in the (Fig. and this the in the K and L and an increase in the M with increasing Na+ concentration. As mentioned previously, we performed time-resolved detection of Na+ concentration changes because of Na+ uptake and release by NaR (11Murabe K. Tsukamoto T. Aizawa T. Demura M. Kikukawa T. Direct detection of the substrate uptake and release reactions of the light-driven sodium-pump rhodopsin.J. Am. Chem. Soc. 2020; 142: 16023-16030Crossref PubMed Scopus (4) Google Scholar). we detected the change at only the formation and decay of indicating that uptake and release of Na+ should occur the formation of O. Thus, the Na+ in P2 from the of Na+ in the unphotolyzed The NaR is to Na+ I. K. A. E. T. of a light-driven sodium 2015; PubMed Scopus Google Scholar, K. I. A. R. T. S. A. G. E. and of sodium-pumping 2019; PubMed Google Scholar). This in the dark state is not essential but the protein and (8Inoue K. Ono H. Abe-Yoshizumi R. Yoshizawa S. Ito H. Kogure K. Kandori H. A light-driven sodium ion pump in marine bacteria.Nat. Commun. 2013; 4: 1678Crossref PubMed Scopus (239) Google Scholar, 13Hontani Y. Inoue K. Kloz M. Kato Y. Kandori H. Kennis J.T. The photochemistry of sodium ion pump rhodopsin observed by watermarked femto- to submillisecond stimulated Raman spectroscopy.Phys. Chem. Chem. Phys. 2016; 18: 24729-24736Crossref PubMed Google Scholar, 14Asido M. Eberhardt P. Kriebel C.N. Braun M. Glaubitz C. Wachtveitl J. Time-resolved IR spectroscopy reveals mechanistic details of ion transport in the sodium pump Krokinobacter eikastus rhodopsin 2.Phys. Chem. Chem. Phys. 2019; 21: 4461-4471Crossref PubMed Google Scholar, Inoue K. Abe-Yoshizumi R. Kato Y. Ono H. Konno M. S. T. H. Ito J. Yoshizawa S. K. M. T. basis for Na+ transport by a light-driven Na+ 2015; PubMed Scopus Google Scholar, A. Ito S. R. S. Inoue K. Kandori H. I. on Schiff by and the extracellular Na+-binding site in Krokinobacter rhodopsin 2.Phys. Chem. Chem. Phys. 2018; PubMed Google Scholar, A. M. Inoue K. Kandori H. Y. with the retinal chromophore ion in a light-driven sodium 2020; PubMed Scopus (4) Google Scholar). This Na+ the equilibria in the (8Inoue K. Ono H. Abe-Yoshizumi R. Yoshizawa S. Ito H. Kogure K. Kandori H. A light-driven sodium ion pump in marine bacteria.Nat. Commun. 2013; 4: 1678Crossref PubMed Scopus (239) Google Scholar, A. M. Inoue K. Kandori H. Y. with the retinal chromophore ion in a light-driven sodium 2020; PubMed Scopus (4) Google seems to be that observed in P2 (Fig. The spectra a (Fig. which the in the L and increase in the K (Fig. and These changes from the Na+ in the dark However, this seems to be Thus, there site to Na+. a site was suggested in a Y. Fujisawa T. Kikukawa T. A. H. Unno M. Raman spectroscopy of sodium-pump rhodopsin from of Na+ for active Na+ Chem. Chem. Phys. PubMed Google Scholar). As described previously, L and M appear simultaneously in P2 and Thus, are shown as in Figure reflecting the difficulty in the of their The and spectra at Na+ concentrations to be For we first to by the of and However, the of these three could not the spectra (Fig. results were by an of K (Fig. The results for all Na+ concentrations are in Figure where the of and was For the state, is at Na+ as mentioned Thus, we to the spectra by the of and N. As a were as in Figure In both analyses for and we only the of the respective intermediates (Fig. and because we all for the intermediates. As described previously, the spectra were with the of O1 and the preceding intermediates of and M. Thus, O1 is to the side and equilibrium with the preceding intermediates reflecting Na+ uptake at the side. the of O1 is increased by the of Na+ concentration, whereas the other as shown in Figure to the we to the of M in to O2 and N. However, the are O2 and N. Thus, is a of in O2 is to the side and equilibrium with the subsequent reflecting the release of Na+ at the side. changes (Fig. the for O2 is whereas the for is by the of Na+ concentration. As mentioned previously, the of M in seems M is the intermediate before the formation of and O1 is not in Thus, M should not appear in which should contain the intermediates after The of M is because this is with Na+ concentration (Fig. Thus, M in be a different intermediate from M in we use to the M in For M in a vital role is for Na+ the Schiff base the Schiff base is and seems to Na+ K. Konno M. Abe-Yoshizumi R. Kandori H. The role of the NDQ motif in sodium-pumping rhodopsins.Angew. Chem. Int. Ed. Engl. 2015; 54: 11536-11539Crossref PubMed Scopus (35) Google Scholar, Inoue K. Abe-Yoshizumi R. Kato Y. Ono H. Konno M. S. T. H. Ito J. Yoshizawa S. K. M. T. basis for Na+ transport by a light-driven Na+ 2015; PubMed Scopus Google Scholar). subsequent O1 and the Schiff base should be protonated and results in red-shifted the Schiff base should to the M. We that this be a side reaction after Na+ the protonated Schiff Na+ the of the Schiff base and Reflecting this is shown in Figure as an intermediate, which is not in the and formed from O2 as the The are to the respective Thus, we the of O1 and O2 for Na+. Here, we their (Fig. and using the where and the and Na+ concentration, respectively. The shown in Figure and L were at values of mM for O1 and mM for O2. For the substrate should be at the uptake side but at the release side. In contrast, NaR has a at the release side with the uptake side. This be by the irreversible from O1 to O2. As shown previously, these intermediates not appear in the state, indicating that equilibrium is not Thus, the O1 to O2 seems to be which Na+ in transport an across the membrane. The for the irreversible is an to be in In to O1 → the transitions after to be the state, equilibrium with O2. However, at the state, appears at Na+ concentration, that the Na+-binding site is from the Thus, the in have a different from the in This conformational change be a critical for Na+ at a in the two other irreversible → → and → NaR → the completion of the cycle and to Na+ transport. The molecular during these transitions should be in In this study, we analyzed the details of the NaR photocycle by using flash-induced absorbance changes. O1 and O2 were identified as Na+-binding intermediates. appear after Na+ uptake and before Na+ release. Thus, “accessibility switching” should occur during the O1 to O2 transition. To active the switch should involve strategic conformational whose details should be in

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