Class Note for BIOC 462A at UA
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Date Created: 02/06/15
STRUCTURAL BIOLOGY Breaching the Barrier Kaspar P Locher Randal B Bass Douglas C Rees brane proteins that selectively medi ate the passage of molecules across the otherwise impermeable barrier im posed by the phospholipid bilayer that sur rounds all cells and organelles The identi fication of more than 360 families of trans porters through bio Enhanced online at Chemical and ge39 www5ciencemagorgcgi nomic analyses 1 contentfulV30lS6336OZ highlights the impor tance of transport processes to cells Among the most fasci nating transporters are those that act as mo lecular pumps translocating their sub strates across membranes against a concen tration gradient this thermodynamically unfavorable process is powered by coupling to a second energetically favorable process such as ATP hydrolysis or the movement of a second solute down a transmembrane concentration gradient The latter mecha nism is used by members of the maj or fa cilitator superfamily MFS of transporters which includes the bacterial lactose perme ase LacY and the glycerol3phosphate transporter GlpT LacY mediates the cou pled cotransport of lactose and HT whereas GlpT catalyzes the exchange of glycerol3 phosphate for phosphate see the figure On pages 610 2 and 616 3 of this issue two groups present their structural determi nations of the E coli LacY and GlpT trans porters The structures of LacY and GlpT together with those of CaZTATPase 4 MsbA 5 Ach 6 and BtuCD 7 mark a new era in defining transport mechanisms in molecular detail The LacY and GlpT structures reveal that the expected 12 transmembrane helices in each protein are organized into two dis tinct domains composed of six helices in the amino terminus and six helices in the carboxyl terminus see the figure These domains have equivalent helixpacking arrangements and are approximately relat ed by an intramolecular twofold rotation This general arrangement had been antici pated by electron microscopy studies on the OxlT transporter 8 At the interface be Transporter proteins are integral mem K P Locher is at the Institut fiJr Molekularbiologie und Biophysik Eidgenossische Technische Hochschule Zurich Ziirich CH 8093 Switzerland R B Bass and D C Rees are at the Division of Chemistry and Chemical Engineering California Institute of Technology Pasadena CA 91125 USA E mail dcreescaltechedu www5ciencemagorg SCIENCE VOL301 tween the amino and carboxylterminal domains a crevice containing residues im plicated in substrate binding is formed along the twofold axis In both LacY and GlpT this crevice opens into the cytoplasm whereas access to the periplasm is blocked The LacY and GlpT structures support the alternating access mechanism of trans port 9 10 in which the protein undergoes a series of conformational transitions such that the ligandbinding site is alternatively acces sible to one side of the membrane or the oth er but not to both sides simultaneously The key to successful operation of transporters is PERSPECTIVES to eliminate shortcircuiting by preventing the uncoupled processes that is substrate translocation or ATP hydrolysis from occur ring individually in their energetically favor able directions The challenge to biochemists has been to define the molecular details of a mechanism that goes beyond the cartoon level of resolution depicted in such general schemes as part A of the figure Although still far from clear this mechanistic analysis is most advanced for LacY in large part ow ing to the experimental efforts of Kaback s group 11 The lactose and protonbinding sites contain distinct sets of amino acids that are linked by critical residues In the pres ence of the appropriate set of bound ligands these residues apparently adopt different conformations that switch the transporter be tween inward and outwardfacing orienta A Extracellular outside i Hquot39 Lactose Inwardlooking membrane proteins A The alternatingaccess model of transport for the bac terial transporters LacY left and GlpT right LacY catalyzes the coupled transport of lactose and H from one side of the membrane to the other whereas GlpT mediates the exchange of glycerol 3phosphate GBP for inorganic phosphate P The pair of structures for each transporter show the outwardfacing conformation exposed to the extracellular side on the left and the inward facing conformation exposed to the cytoplasm on the rightThe bottom surface of the membrane bilayer is depicted in contact with the cytoplasm there is no standard convention in the trans porter field for this topbottom orientation and different conventions are used in the LacY and GlpT papers 2 3 B Ribbon representation of the polypeptide folds of LacY left and GlpT right Shown are the aminoterminal and carboxylterminal domains blue and green respectively the substratebinding site is located at their interface Left Shown for LacY are a lactose analog yel low amino acids in the sugarbinding site ballandsticks and residues implicated in proton translocation spacefilling models Right Shown for GlpT are arginine residues 45 and 269 ball andsticks at the proposed substratebinding site Bends and other irregularities in the or helices are indicated by deviations from ideally straight and continuous helical ribbons The crystallo graphically determined structures for LacY and GlpT are both inward facing and would open into the cytoplasm Part B of the figure was prepared with the program MOLSCRIPT 18 I AUGUST 2003 603 604 PERSPECTIVES tions At a minimum this rearrangement must reposition the amino and carboxyl terminal domains through reconfiguration of helixhelix interactions along the interface surrounding the substratebinding site Both groups describe plausible models for this transition 2 3 Beyond mechanistic insights the new structures provide an opportrmity to evalu ate extensive biochemical studies on LacY that were designed to define functionally important residues and helixpacking inter actions II In general the biochemical studies correctly identified mechanistically critical residues in the LacY ligandbinding site However an additional important ob servation from the structures of LacY GlpT and other membrane proteins is that transmembrane helices can be bent or ex hibit other types of irregular features see the figure Together with the conforma tional exibility inherent in these trans porters which is necessary for their activi ty deviations from ideal 0c helices have confounded attempts to unambiguously es tablish helixhelix interactions through bio chemical studies such as crosslinking 12 The mechanistic importance of heli cal distortions and irregularities for trans port processes is underscored by the role these features have been proposed to play during substrate translocation by the Ca2 ATPase 13 as well as gating transitions in K r I4 and acetylcholine receptor chan nels 13 Why did the LacY and GlpT structure determinations succeed Although the terse method sections may imply otherwise magic was not involved rather both structures are the result of extended and committed efforts to surmount the irmumer able experimental barriers encountered in these projects Perhaps even more daunting was the necessity to survive promotion and mding hurdles long enough to see the structures through to completion The LacY and GlpT structure determinations illustrate different emphases in tackling the mem brane protein structure problem In the case of LacY decades of experimental studies on E coli LacY dictated that this protein would be the target of structural inquiry Ultimately suitable crystals were prepared of a mutant form of LacY trapped in the iri wardfacing conformation to minimize con formational dynamics In contrast the GlpT structure was the result of a more general desire to crystallographically characterize a member of the MFS family The E coli GlpT was identified through the screening of a number of homologs to find the most suitable candidate for structural work 16 In both cases the structures could only have been solved through ready access to syn chrotron xray sources and their staffs to surmount the limitations of modest diffrac tion quality and substantial crystaltocrys tal variability Because LacY and GlpT were both crystallized in the inwardfacing con formation a high priority for future studies will be to develop approaches to trap each protein in the outwardfacing state to define the endpoints of the transporter mechanism The LacY and GlpT structures provide significant advances in our understanding not only of the structure and mechanism of an im portant class of transporters but also of mem brane proteins in general The development of new approaches encourages the hope that 18 years after the determination of the photosyn thetic reaction center structure I 7 the crys tallography of membrane proteins is coming of age With sufficient effort the major barri ers to the structural determinations of mem brane proteins are finally being breached References and Notes 1 W Busch M H J Saier Crit Rev Eiochem Mal Eiol 27 287 2002 httptcdbursdedutcdb 2 JAbramson etal Science 301 610 2003 3 Y Huang etal Science 301 616 2003 4 CToyoshima etal Nature405 647 2000 5 0 Chang c 13 Roth Science 293 1793 2001 6 S Murakami etal Nature419 587 2002 7 K P Locher etal Science 296 1091 2002 8 T Hirai et al Nature Strum Eiol 9 597 2002 9 w FWiddas j Physiology118 23 1952 0 o Jardetsky Nature 211 969 1966 1 H R Kabark et al Nature Rev Mol Cell Eiol Z 610 2001 12 P L Sorgen et al Proc Natl Acad Sci USA 99 14037 2002 13 CToyoshima H Nomura Nature418 605 2002 14 Y Jiang etal Nature417 523 2002 15 A Miyazawa etal Nature423 949 2003 15 0 Chang etal Science 282 2220 1998 17 J Deisenhofer etal Nature 318 618 1985 18 P J Kraulisj Appl Crystallogr 24 946 1991 19 D C Rees is an Investigator with the Howard Hughes Medical Institute PHYSICS Getting Entangled in Free Space J G Rarity theory that allows two particles such as photons to be much more strong ly correlated than is possible in classical physics Conventional quantum theory sets no limit on the range or duration of these correlations Over the past 20 years many optical experiments have demon strated these apparently nonlocal effects in the laboratory 14 and more recently over ranges of up to 10 km in optical fibers 577 On page 621 of this issue Aspelrneyer et al 8 report the latest of these demon strations In contrast to earlier studies they study entanglement not in optical fibers but in free space The authors show that strong correlations can be maintained in E ntanglement is a property of quantum The author is in the Department of Electrical Engineering Bristol University Bristol 558 1UB UK Emailjohnraritybristolacuk 1 AUGUST 2003 VOL 301 free space over a separation of 600 m The optical losses were comparable to those in a putative spacebased experiment in which a satellite would send correlated photon pairs to two separate observers on Earth about 600 km away A simplified version of the experimen tal setup used by Aspelrneyer et al is shown in the figure Key to the experiment is a source that creates entangled pairs of photons with anticorrelated polarizations If the source emits a horizontal photon in direction 1 a vertical photon is emitted in direction 2 and vice versa This anticorre lation holds for any polarization direction The photons are sent in opposite directions to two wellseparated polarizers and sin glephoton detectors The polarizers which can be rotated to any angle allow those photons to pass that are polarized parallel to this angle while blocking all photons polarized at 900 to this angle The detectors fire when photons are de tected producing a series of clicks that measure the arrival times of photons Separate measurements in arm 1 or arm 2 show no change in the click rate when the polarizer angle is changed Hence there is no favored polarization emitted by the source But when both detectors fire simul taneously indicating the detection of pho ton pairs the click rate increases when the polarizers are set at 900 to each other For instance a detector click in channel 1 behind a vertical V polarizer immedi ately implies a click in channel 2 when using a horizontal H polarizer In a classical or local realistic view we would make the naive assumption that photons have a real locally labeled polar ization when they leave the crystal For iri stance an H polarized photon emitted in direction 1 would imply a Vpolarized pho ton emitted in direction 2 After emission we could set the angle for the polarizer to 45 in charmel 1 745 in charmel 2 such that only half the intensity of horizontally or vertically polarized light will pass through the polarizer Each individual H or Vphoton then has a 50 chance ofpassing SCIENCE www5ciencemagorg
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