類結構生物學sturcture biology explore

1、516Halorhodopsin: light-driven ion pumping made simple?Lars-Oliver EssenH. salinarum 11,12,13. Tetragonal 2D crystals formed spontaneously in vivo when halorhodopsin was homo- logously overproduced in the recombinant H. salinarum strain D2. Unlike the naturally occurring 2D crystals of bacteriorhodo

2、psin, which constitute the so-called purple membrane, the alternating inside-out and outside-in arrangement of halorhodopsin tetramers suggested that these 2D crystals were physiologically irrelevant artefacts of the overexpression conditions. Nevertheless, low-resolution data of up to 5 resolution

3、proved the bacteriorhodopsin- like architecture of halorhodopsin.Halorhodopsin, a light-driven halide pump, is the second archaeal rhodopsin involved in ion pumping to be studied at high resolution by X-ray crystallography. Like its cousin bacteriorhodopsin, halorhodopsin couples vectorial ion trans

4、port to the isomerisation state of a covalently linked retinal. Given the similarity and interconvertability of these two ion pumps, a unified mechanism for ion translocation by archaeal rhodopsins is now emerging.AddressesDepartment of Chemistry, Philipps University, Hans-Meerwein-Strasse,D-35032 M

5、arburg, Germany;: essenchemie.uni-marburg.deCurrent Opinion in Structural Biology 2002, 12:516522ructure of H. salinarum halorhodopsin0959-440X/02/$ see front matter 2002 Elsevier Sciencebe crystallised in a lipidic cubic phase 14,15. In these 3D crystals, halorhodopsin assembled into trimers and th

6、e crystal packing resembled the purple membrane organisation of trimeric bacteriorhodopsin (Figure 1). Despite this coincidence, the quaternary structure is not conserved because the monomers in the halorhodopsin trimer are tilted by 11 relative to the monomers in the bacterio- rhodopsin trimer 10.I

7、ntroductionTwo functions can be assigned to the protein family of archaeal rhodopsins 1: signalling by photoreception, as exemplified by sensory rhodopsins (reviewed by Spudich and Luecke; this issue, pp 540546); and light-driven ion pumping through lipid bilayers, as catalysed by bacterio- rhodopsi

8、ns and halorhodopsins. A plethora of studies on these heptahelical membrane proteins has focused on the following issues 13: how is photoisomerisation and reisomerisation of a covalently bound retinal chromophore coupled to unidirectional ion or signal transport; what conformational changes in the p

9、rotein moiety accompany the photocycle; and, finally, is there a common mechanism rationalising the different functions?Why does halorhodopsin crystallise as either a tetramer or a trimer, even though the protein used for the 2D and 3D crystallisations was isolated from the same H. salinarum strain?

10、 The X-ray structure shows that the halorhodopsin trimer included a lipidic plug consisting of the fatty acid palmitate (Figure 2). This surprising association of halorhodopsin with palmitate was first shown byBecause of its stability and abundance in haloarchaea, the proton pump bacteriorhodopsin w

11、as until recently the favourite subject for biophysical and structural studies, which culminated in a series of X-ray crystallographic snapshots of photocycle intermediates (reviewed in 4). Halorhodopsins were discovered and coined in 1980 5, and, shortly afterwards, recognised as inwardly directed

12、Cl pumps 6. The ion specificity and directionality were surprising, as halorhodopsins share significant sequence identities of 2535% with bacteriorhodopsins 7. Many subsequent studies on the halorhodopsins of Halobacterium salinarum and Natronomonas pharaonis addressed the binding and transport of C

13、l, other halides and nitrate, and their influence on photocycle kinetics, the chromophore and the primary photoreaction (for in-depth reviews, see 8,9). Here, I examine what the recently determined X-ray structure of halorhodopsin 10 implies, together with other biophysical data, regarding a common

14、mechanism for ion translocation by halorhodopsins and bacteriorhodopsins.Corcelli. 16, who also confirmed its noncovalentmode of binding 17. Previously, similar specific bindingof a lipid was demonstrated for trimeric bacteriorhodopsin, which encircled a patch of the haloarchaeal glycolipid S-TGA-1

15、18. However, the binding of palmitate by halorhodopsin and its physiological role are rather enig- matic, because pathways for the biosynthesis of palmitate and other unbranched fatty acids are likely to be missing in haloarchaea. Because of their chemoorganotrophic lifestyle and the ubiquitous pres

16、ence of palmitate, haloarchaea probably take up these fatty acids, as they do othernutrients, from the environment. Tome sequence ofH. salinarum strain NRC-1 19 corroborated this notion,as tome comprised several genes usually occurringin the b-oxidation pathway of fatty acids.The evidence that palmi

17、tate affects the function of halorhodopsin is only indirect; upon removal of palmitate, changes were noted for the photocycle kinetics and the pKa of the protonated Schiff base, at which the retinal is linked to the protein moiety 16. Palmitate might modulate halide pumping by H. salinarum halorhodo

18、psin analogously to the archaeal lipids that modify the cooperativity of proton pumping by bacteriorhodopsin. This could beMultiple quaternary arrangements for halorhodopsin?Cryo-electron microscopy on 2D crystals yieldedthefirst structural information about halorhodopsin fromHalorhodopsin Essen517F

19、igure 1Comparison of the packing of (a) halorhodopsin trimers in 3D crystals and (b) bacteriorhodopsin trimers in 2D purple membrane sheets. The monomers, as viewed from the extracellular side, are shown as ribbon mlipids (palmitate for halorhodopsin, S-TGA-1 for bacteriorhodopsin) are highlighted a

20、s space-filling mspecies that fill the space between the trimers are representedfeasible, because the X-ray structure 10 shows that the protonated Schiff base and the Cl next to it are 11 and 7 from the carboxylate of palmitate, respectively 10. Furthermore, halorhodopsin trimers in the native cell

21、membrane were implied by hexagonal 2D crystals, which were observed in the membranes of H. salinarum strain D2 by electron microscopy 20.Local softening of a helices: common mechanics for archaeal rhodopsins?The heptahelical transmembrane region is highly con- served among ion- and signal-translocat

22、ing rhodopsins. Distortions from the regular a-helical pattern occur only at regions that are thought to undergo conformational changes during the photocycle. The a-helical segment V239F245 in helix G (bacteriorhodopsin/sensory rhodopsin II: V213F219/L202F208) comprises the conserved lysine residue

23、to which retinal is bound as a protonated Schiff base (K242). In bacteriorhodopsin, photoisomerisation of retinal triggers the reorientation of the peptide planes preceding and following this lysine 21. Likewise, the a-helical segment A178W183 (bacterio- rhodopsin/sensory rhodopsin II: V151F156/Y140

24、M145) at the cytosolic end of helix E presumably acts as a hinge, supporting the swivel motions of the cytosolic part of helix F during the formation of late photocycle intermediates 22. From this, one might conclude that the mechanics of large-scale conformational changes, such as the tilting of he

25、lix F or changes of the hydrogen-bonding patterns nearthe transport site, involve a highly conserved way of local helix softening.In contrast to other archaeal rhodopsins, the extracellular BC loop of halorhodopsin is longer and covers most of the extracellular surface. Enhanced conformational flexi

26、bility of this loop was confirmed by atomic force microscopy on tetragonal 2D crystals 23. Whether this flexibility plays a role during the photocycle is not known. However, muta- tion of H95 at the tip of the BC loop caused a significant decrease in pumping activity, as well as some general destabi

27、lisation 24.How many chloride-binding sites?Much confusion arose about the precise number and definition of Cl-binding sites in halorhodopsins, as previously determined by spectroscopic and kinetic studies. The X-ray structure of unphotolysed halorhodopsin 10 defined onlyone site, the transport site

28、 (Figure 3). Its affiis about10 mM and 2.5 mM for halorhodopsins from H. salinarumand N. pharaonis 25, respectively. In the X-ray structure,the Cl is only 3.8 from the protonated Schiff base 10, indicating a strong electrostatic interaction, as extrapolated previously from FT-IR data 26,27 and chang

29、es in the visible spectrum upon halide binding 28,29. Other features of the transport site were not predicted.Firstly, for geometrical reasons, the location of Cl in the transport site was incompatible with a strong hydrogen(a)(b)67 62 Current Opinion in Structural Biology518 Membranesthe cytosol in

30、 the unphotolysed state 10. Here, the light-triggered movement of Cl into this cytoplasmic release site was postulated to trigger conformational changes in the cytoplasmic half that are akin to conforma- tional changes seen in bacteriorhodopsin 22 for establishing free exchange with the cytosolic su

31、rface. InN. pharaonis halorhodopsin, the transport site lowered itsFigure 2Cl affifrom 2.5 mM to 1.1 M after photoexcitation25, whereas the affiof the cytoplasmic release sitewas mostly unchanged (5.7 M; 33). Furthermore, the transport activity of halorhodopsin is inhibited by high concentrations of

32、 Cl or other halides 25,33. Interestingly,Okunoextracellular surface, that was not observed in the current X-ray structure and other kinetic studies 27.The order of ion pumping by halorhodopsin How might the existence of at least two halide-binding sites in halorhodopsin correlate with its halide-de

33、pendent photocycle? Subnanosecond formation of the K state (lmax 600 nm) upon illumination of the unphotolysed HR state (lmax 580 nm) is accompanied by photoiso- merisation of the retinal from all-trans to 13-cis, 15-anti, and by a flip of the NH dipole of the protonated Schiff base (Figure 4). Evid

34、ence for such a dipole flip comes from small negative electrogenic charge shifts upon K formation, as observed in halorhodopsin 34 and other archaeal rhodopsins. Apparently, the majority of the 23.2 kcal/mol still left in the K intermediate after photoexcitation 35 is stored in a twisted chromophore

35、 conformation, as the X-ray structures of K-like intermediates of bacterio- rhodopsin and sensory rhodopsin 36,37 revealed only minor conformational changes in the protein moiety.The binding of palmitate in halorhodopsin trimers. The approximate location of the lipid bilayer, as suggested by the loc

36、ations of1-monoolein molecules in the X-ray structure (sticks), is shown in blue.For clarity, one of the halorhodopsin monomers is omitted from the trimer.bond towards the Schiff base nitrogen. Secondly, the anion was still partially hydrated by a cluster of three water molecules and forms a hydroge

37、n bond to the hydroxyl of the conserved serine S115. Although R108 is essential for pumping activity 30, direct binding of its guanidinium group to Cl 26 was ruled out because of intervening water molecules. Thirdly, a cluster of aliphatic hydrogens around the Cl appeared to contribute significantly

38、 to halide binding 10. Lastly, the only a-helical distortion unique to halorhodopsin comprises the 310-like helical segment L110A113. This stretch includes T111, which is the corresponding residue of the primary proton acceptor in bacteriorhodopsin, D85. However, relative to the latter residue in ba

39、cteriorhodopsin, T111 is pulled 1.8 out of the Cl-occupied transport site of halorhodopsin due to the helical distortion. The additional space provided to the Cl might become lost upon Cl translocation, when T111 moves back into the transport site, with concomitant restoration of the a-helical hydro

40、gen-bonding pattern. Interestingly, the hydroxyl of T111 is superfluous for Cl binding, as shown by the 3D structure and activity studies of the T111V mutant 31. Only retardation of Cl reup- take was found for the T111V mutant, the structural basis of which is not clear 31. According to stopped-flow

41、 exper- iments on N. pharaonis halorhodopsin, Cl uptake itself is mostly ruled by passive diffusion through an extracellular pathway during later stages of the photocycle 32.The transition to an L state (lmax 520 nm) is reversible (DGKL 1.8 kcal/mol; 35) and presumably reflects the movement of the h

42、alide from its transport site to the cyto- plasmic release site 10,27. Time-resolved FT-IR data27 suggested that the transport preference for Br over Cl derives from the tighter binding of Br to the cytoplasmic release site. In structural terms, this translocation event might be some form of ion dra

43、gging, whereby the Cl fol- lows the flipped dipole of the NH Schiff base bond 10 and forms a strong hydrogen bond with the protonated Schiff base 26. The opening of an extracellular pathway from the cytoplasmic release site towards the protein surface was attributed either to an L2O transition 38 or

44、 to a spectroscopically silent transition between two L-like intermediates, L1 and L2 (25,34,39; Figure 4).The precise order of the late photocycle events is still ambiguous and differs between halorhodopsins fromH. salinarum and N. pharaonis. For example, in the former, an O state (lmax 600 nm) is

45、not detectable, presumably for kinetic reasons 40, although such a bathochromically shifted O intermediate should correspond to halorhodopsin after Cl release and reisomerisation of the chromophore.Cl release towards the cytosol was previously assigned toThe X-ray structure suggested a second, trans

46、ient halide- binding site that borders the cytoplasmic side of the Schiff base (Figure 3), but is completely occluded from access toPalmitateLipid bilayerCurrent Opinion in Structural BiologyHalorhodopsin Essen519Figure 3Comparison of the transport and cytoplasmic release sites in (a) halorhodopsin

47、and(b) bacteriorhodopsin. In both molecules, a cluster of three water molecules (red spheres) fills the transport site between the protonated Schiff base of the lysineretinal chromophore (dark purple) and the arginine counterions R108 and R82, respectively. Only functionally important residues are s

48、hown with their hydrogen bonds (orange lines) along theCa traces of helices C, F and G, which line the cytoplasmic and extracellular pathways. The locations of the halide transport and cytoplasmic release sites are indicated by red and blue arrows, respectively. CP, cytoplasmic side; EC, extracellul

49、ar side.the NO transition 34,41, but a careful kinetic analysis of the photocycle of N. pharaonis halorhodopsin 25 placed the N state (lmax 520 nm) after O state decay. In this analysis, Cl release and thermal reisomerisation were suggested to occur during an L2O1 transition (Figure 4). After closur

50、e of the cytosolic pathway (O1O2), reuptake of Cl into the transport site would proceed during the O2N transition 25, instead of during the OHR transition, which was postulated to precede a spectroscop- ically silent restoration of the unphotolysed HR state 41. Obviously, more structural data for la

51、te photocycle inter- mediates are needed to get reliable correlations between spectroscopic states and transport states.only be related to the transport site. For example, in the D85T mutant, Cl translocation induces a transient deprotonation of E204 45, a residue that is part of an extra- cellular

52、proton-release group. Such a compensatory proton release is not required in halorhodopsins, as the corresponding residue is replaced by a neutral threonine (T230).Second-site mutations in the extracellular half of the D85T mutant showed a similar important role for T178 in Cl translocation towards t

53、he cytoplasmic side as T203 in halorhodopsin 44. Apart from this residue, which is predicted to line the cytoplasmic release site for Cl, no other residues that are crucial to halide transport were identified in the cytoplasmic half of either halorhodopsin31 or the bacteriorhodopsin D85T mutant 44.

54、Consequently, the walling of the cytosolic exit pathway for Cl can be rather unspecific and may be compatible with the passive diffusion of Cl into the cytosol 32.A common mechanism for ion transport by archaeal rhodopsins?What is the essence of halorhodopsin being a Cl pump? A single-site mutation

55、in bacteriorhodopsin, D85S or D85T, converted it from a proton pump to a Cl pump 42. The photocycles of halorhodopsin and this bacteriorhodopsin D85T mutant resembled each other, but bacterio- rhodopsin D85T exhibited a 10-fold lower transportInterestingly, halorhodopsin can be converted to an outwa

56、rdly directed proton pump by replacing Cl in the transport site by the pseudohalide azide 46,4749. The azide anion acts as a proton acceptor in much the same way as D85 in bacteriorhodopsin and shuttles protons along the cytoplasmic pathway between the protein surface and the Schiff base 48.efficien

57、cy and a 20-fold lower affifor Cl 43,44 thanhalorhodopsin. Furthermore, the photocycle of the D85T mutant comprised a rate-limiting Cl uptake step, presumablybecause access from the extracellular side to the transport site is sterically hindered, whereas Cl release was faster than in halorhodopsin 43,44. The structural reasons why halorhodopsin is still a better catalyst than D85T bacterio- rhodopsin for Cl pumping are unclear, but they