Structure of the L Intermediate of Bacteriorhodopsin Bacteriorhodopsin (bR), a membrane protein found in the cell membrane of Halobacterium salinarum , functions as a li g ht - dri v en proton transport . I ts pol y peptide chain is folded into 7 transmembrane helices and retinal is bound to the ε - amino g roup of Ly s 216 via a protonated S chiff base lin k a g e ( F i g. 1 ) . T he photoisomeri z ation of the all - trans retinal into the 13 - cis , 15- anti confi g uration initiates a reaction c y cle in v ol v in g se v eral intermediates (bR 570 → K 590 → L 550 → M 410 → N 560 → O 630 → bR 570 ) . F or the elucidation of the proton pumpin g mechanism, it is important t o obtain structural information of the reaction intermediates . U sin g a 3D cr y stal belon g in g to the space g roup P622 , w e ha v e pre v iousl y determined the structures of the g round state (bR 570 ) and the K and the M intermediates at 2.3 - 2.6 Å resolutions [1 , 2]. O ur structural data sho w that, in the K intermediate, the retinal chromophore has a lar g el y t w isted 13- cis, 15- anti confi g uration . T he distortion in the retinal pol y ene chain is su gg ested to induce a v ertical mo v ement of heli x G upon the formation of the M intermediate, w hich is accompanied b y a lar g e rearran g ement in the h y dro g en - bondin g net w or k in the proton release channel . A n increasin g number of e x perimental data has sho w n that internal w ater molecules pla y an important role in re g ulatin g the p K a v alues of k e y residues in the path w a y of proton transport . T he current structural model of the unphotol y sed state (bR 570 ) sho w s that w ater molecules e x istin g in the acti v e site participate in stabili z in g the protonated S chiff base w ith a v er y hi g h p K a ( ~ ~ 13 ) and its counter ion (unprotonated A sp 85 ) w ith a v er y lo w p K a ( 2.5 ) . Recent FTI R studies of the L intermediate ha v e su gg ested that, before the primar y proton transfer, rearran g ements of internal w ater molecules ta k e place in such a manner that the y cause a si g nificant reduction in the p K a of the S chiff base and a concomitant increase in the p K a of A sp 85. H o w e v er, the structure of a k e y intermediate, i . e . , the L intermediate, has not y et been determined con v incin g l y , ma k in g it difficult to q uantitati v el y anal yz e the detailed mo v ements of internal w ater molecules . F or the structural in v esti g ation of the L intermediate [3] , w e carried out a q uantitati v e anal y sis for X- radiation dama g e and searched for optimum conditions for X- ra y measurements to minimi z e undesired effects of X- ra y- induced structural chan g es . A fter a careful structural anal y sis, w e constructed a structural model of the L intermediate at 2.4 Å resolution . Briefl y , the Fig. 1. Crystal structure of the ground-state of bacteriorhodopsin. Internal water molecules (cyan), the retinal-Lys216 chain (pink and light blue) and several key residues (red, blue and gray) are drawn. The green arrows indicate the pathway of proton translocation. P622 cr y stal w as illuminated w ith g reen li g ht at 160 K and subse q uentl y w ith red li g ht at 100K. T his y ielded a 1:4 mi x ture of the L intermediate and the g round state . D iffraction data from such cr y stals w ere collected usin g a lo w flu x of X- ra y s ( 2 × 10 15 photons / mm 2 per cr y stal), and their combined data w ere compared w ith those from unphotol yz ed cr y stals . F i g ure 2 sho w s a difference electron - densit y map bet w een the L intermediate and the g round state . T hese structural data, to g ether w ith our pre v ious data [1] , indicate that the retinal chromophore, w hich is lar g el y t w isted in the K intermediate, ta k es a more p l a n a r 1 3 - c i s , 1 5 - a n t i c o n f i g u r a t i o n i n t h e L intermediate . T his confi g urational chan g e, w hich is accompanied b y the reorientation of the S chiff base N-H bond to w ards the intracellular side, is coupled w ith a lar g e rotation of the side chain of an amino acid residue ( L eu 93 ) ma k in g contact w ith the C13 meth y l g roup of retinal . F ollo w in g these motions, a w ater molecule, at first h y dro g en - bonded to the S chiff base ~ ~ 26 Tsutomu Kouyama Department of Physics, Nagoya University E-mail: kouyama@bio.phys.nagoya-u.ac.jp Fig. 3. Movements of key water molecules (indicated by thin arrows) and conformational changes in Leu93, Trp182 and the retinal-Lys216 chain (indicated by thick arrows) during the bR 570 → K and K → L transitions. Atoms in bR 570 , K and L are drawn in different colors. References [1] Y. Matsui et al. : J . Mol. B iol. 324 (2002) 469 . [ 2 ] K. Take d a, Y. Matsui, N. Kamiya, S . Ad achi, H . O kumura an d T. Kouyama: submitte d to J . Mol. B iol. [ 3 ] T. Kouyama, T. Nishika w a, T. Tokuhisa an d H . O kumura: J . Mol. B iol. 335 (2004) 53 1. an d A sp 85 , is d ragge d to a space that is originally occupie d by L eu 93 . I n F ig. 3 , the movements of w ater molecules observe d in the b R 570 -to-K an d K-to- L transitions are summari z e d . A small d isplacement of W at 602 ( i.e., to w ar d s the C δ of L ys 2 1 6) is alrea d y initiate d in the primary photoreaction, w hich is accompanie d by the isomeri z ation of the retinal into a largely t w iste d 1 3 - cis , 1 5 - anti configuration. A large jump of W at 602 from the e x tracellular si d e of the S chiff base to the opposite si d e is assiste d by the ~ 90 rotation of the N- H bon d of the retinal S chiff base. No w , an interesting q uestion rises as to w hy the vertical movement of a key w ater molecule takes place prior to the primary proton transfer ( from the S chiff base to A sp 85) , w hich takes place in the L -to- M transition. S ince the retinal chromophore is accommo d ate d tightly in a hy d rophobic pocket, there is no channel for the passive movement of w ater across a hori z ontal plane passing through the chromophore. This plane can act as a structural barrier against the movement of w ater. Probably the back w ar d translocation of W at 602 w oul d not occur unless the cis-to-trans isomeri z ation of the retinal is initiate d by the e x citation of the L interme d iate. I n d ee d , d iffraction d ata from a crystal containing the M interme d iate sho w e d that this w ater molecule moves further to w ar d s the intracellular si d e in the L -to- M transition [ 3 ]. I t is very likely that the d etachment of this w ater molecule from the protonate d S chiff base causes a significant d ecrease in the pK a of the S chiff base, thereby facilitating the proton transfer to A sp 85 . O n the basis of these observations, w e argue that the vertical movement of a w ater molecule in the K-to- L transition is a key event d etermining the d irectionality of proton translocation in the protein. Fig. 2. Difference map between L and bR 570 , contoured at 3.5 σ (cyan, positive density; purple, negative density) and overlaid on the structural models of the ground state (green) and the L intermediate (carbon, oxygen and nitrogen atoms are drawn in orange, red and blue, respectively). Water molecules in L are represented by red (high occupancy) or gray spheres (low occupancy) . 27
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