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  DOI: 10.1126/science.1059344, 1160 (2001); 292 Science  Eva S. Istvan and Johann Deisenhofer Structural Mechanism for Statin Inhibition of HMG-CoA Reductase   This copy is for your personal, non-commercial use only.  clicking here.colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others  here.following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles    ): August 27, 2013 www.sciencemag.org (this information is current as of The following resources related to this article are available online at   http://www.sciencemag.org/content/292/5519/1160.full.htmlversion of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/content/292/5519/1160.full.html#ref-list-1, 4 of which can be accessed free: cites 18 articles This article 250 article(s) on the ISI Web of Science cited by This article has been http://www.sciencemag.org/content/292/5519/1160.full.html#related-urls43 articles hosted by HighWire Press; see: cited by This article has been http://www.sciencemag.org/cgi/collection/biochemBiochemistry subject collections: This article appears in the following registered trademark of AAAS. is a Science  2001 by the American Association for the Advancement of Science; all rights reserved. The title CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the Science   o  n   A  u  g  u  s   t   2   7 ,   2   0   1   3  w  w  w .  s  c   i  e  n  c  e  m  a  g .  o  r  g   D  o  w  n   l  o  a   d  e   d   f  r  o  m   tiousness from 3 days after infection until slaughter(for an average of eight infectious days).12. The effective neighborhood size,  n , in units of nearestneighbor farms, was estimated as  n   0   g  r   dr  /  0 R  g  r   dr  where  R  is given by the solution of   0 R  r   dr     1The connectedness of the contact network is given by    1  n 2   g ( r  )  g ( r   )  g (  r    r    )/  (  r    r    ) drdr   d   where  r    r     r  2  r   2  2 rr   cos(  )13. S. C. Howard, C. A. Donnelly,  Res. Vet. Sci.  69 , 189(2000).14. D. T. Haydon, M. E. J. Woolhouse, R. P. Kitching,  IMA J. Math. Appl. Med. Bio.  14 , 1 (1997).15. The population of farms was stratified into a suscep-tible class,  S ; sequential infection classes,  I i  ( i     1 .. M );and a slaughtered/vaccinated class,  D . Multiple in-fected classes were used to exactly reproduce thegamma distribution fits to the delay data shown inFig. 2 and to represent different stages of infectious-ness and diagnosis. The mixture model of the infec-tion-to-report distribution was represented by over-lapping sets of 30 classes (transit time    0.26 dayseach, weight 0.82) and 4 classes (transit times  3.73days, weight 0.18). Two classes (transit times    0.85to 0.21 days, time-dependent) represented farmsawaiting disease confirmation after report, and fourclasses (transit times    0.82 to 0.38 days, time-dependent)—overlapping the previous two—repre-sented farms awaiting culling after disease reporting.Infectiousness varies as a function of incubationstage, reaching significant levels after around 3.5days and then continuing at a constant level untildiagnosis, after which it remains constant untilslaughter at a level  r  I  times greater than beforereporting. The model is novel in tracking not only thenumbers of farms in each infection state throughtime, but also the numbers of pairs of farms connect-ed on the contact network used to represent spatiallylocalized disease transmission. For conciseness andclarity, we only present those for a simpler modelwith only two infected classes:  E   (uninfectious) and  I (infectious). Using [  X  ] to represent the mean numberin state  X  , [  XY  ] to represent the mean number of pairs of type  XY  , and [  XYZ  ] to represent the meannumber of triples, the dynamics can be representedby the following set of differential equations:  d  [  S ]/ dt     –(      )[  SI ] –  p  [  S ][ I ]/ N ,  d  [ E  ]/ dt     p  [  S ][ I ]/ N    [  SI ] –   [ E  ] –   [ EI ],  d  [ I ]/ dt     [ E  ] –  s [ I ] –  [II],  d  [  SS ]/ dt     –2(      )[  SSI ] – 2  p  [  SS ][ I ]/ N , d  [  SE  ]/ dt   ([  SSI ] – [ ISE  ]) –  ([  SEI ]  [ ISE  ]) –  [ ISE  ]   p  ([  SS ] – [  SE  ])[ I ]/ N ,  d  [  SI ]/ dt     [  SE  ] – (     )([ ISI ]    [  SI ]) –  p  [  SI ][ I ]/ N ,  d  [ EE  ]/ dt     [ ISE  ] –2  [ EEI ] – 2  [ EE  ]    2  p  [  SE  ][ I ]/ N ,  d  [ EI ]/ dt     [ EE  ] –  ([ EI ]  [ IEI ]) – (    )[ EI ]    p  [  SI ][ I ]/ N ,  d  [ II ]/ dt    2  [ EI ] – 2  [ II ] – 2  ([ II ]    [ III ]). The numbers of triplesare calculated with the closure approximation ( 16 )[  XYZ  ]    (  n  – 1)[  XY  ][ YZ  ](1 –      N [ YY  ]/  n [  X  ][  Z  ])/  n [ Y  ], where  n  is the mean contact neighborhood sizeof a farm,  is the proportion of triples in the network that are triangles, and  N  is the total number of farms[see ( 12 )].     (1 –  p )  /  n  is the transmission rateacross a contact, where    is the transmission coeffi-cient of the virus, and  p  is the proportion of contactsthat are long-range [see ( 9 )], both of which areestimated separately before and after the movementban.    is the rate of transit from the uninfectious tothe infectious class, and    is the rate of transit fromthe infectious to the removed class.    is the rate atwhich farms in the neighborhood of an infected farmare culled in ring culling, and    is the rate at whichfarms are vaccinated in ring vaccination. It is assumedthat vaccination has no effect on previously infectedfarms.16. M. J. Keeling,  Proc. R. Soc. London B  266 , 859 (1999).17. Removal by culling of an infected herd and theremoval of contiguous holdings of animals have dif-ferent impacts on  R 0  and the scale of the epidemic.The former acts directly to reduce  R 0 , whereas thelatter serves to significantly reduce the overall scaleof the epidemic by stopping second-generationtransmission events [hence reducing the effectivereproductive number ( 10 )].18.  Northumberland Report: The Report of the Commit-tee of Inquiry on Food and Mouth Disease  (Her Maj-esty’s Stationery Office, London, 1968).19. June 2000 Agricultural and Horticultural Census, Min-istry of Agriculture, Fisheries and Food, National As-sembly for Wales Agriculture Department and Scot-tish Executive Rural Affairs Department; Crown copy-right, 2001.20. The rapid decline in case incidence seen after com-pletion of the analysis presented in this paper hasgiven new estimates of   r  I  significantly above 1,though more precise estimation awaits availability of detailed data on all slaughter schemes in operationsince 30 March 2001.21. We are extremely grateful for help in the provisionof data and for invaluable advice from J. Wilesmith(Veterinary Laboratory Agency), D. Reynolds (FoodStandards Agency and Ministry of Agriculture, Fish-eries and Food), and D. Thompson (Ministry of Agriculture, Fisheries and Food) and to the manygovernment epidemiologists and veterinary staff who collected the unique contact tracing dataon FMD spread in the current epidemic. In addition,we thank D. King (Office of Science and Technol-ogy), B. Grenfell, M. Keeling, M. Woolhouse, andother members of the FMD Official Science Groupfor stimulating discussions; Sir Robert May andSir David Cox for valuable advice and discussions;three anonymous referees for comments; and S.Dunstan, S. Riley, and H. Carabin for valuableassistance. N.M.F. thanks the Royal Society and theHoward Hughes Medical Institute for fellowshipand research funding support. C.A.D. and R.M.A.thank the Wellcome Trust for research funding.23 March 2001; accepted 10 April 2001Published online 12 April 2001;10.1126/science.1061020Include this information when citing this paper. Structural Mechanism for StatinInhibition of HMG-CoAReductase Eva S. Istvan 1 and Johann Deisenhofer 1,2 * HMG-CoA (3-hydroxy-3-methylglutaryl–coenzyme A) reductase (HMGR) cat-alyzes the committed step in cholesterol biosynthesis. Statins are HMGR in-hibitors with inhibition constant values in the nanomolar range that effectivelylower serum cholesterol levels and are widely prescribed in the treatment of hypercholesterolemia. We have determined structures of the catalytic portionof human HMGR complexed with six different statins. The statins occupy aportion of the binding site of HMG-CoA, thus blocking access of this substrateto the active site. Near the carboxyl terminus of HMGR, several catalyticallyrelevant residues are disordered in the enzyme-statin complexes. If these res-idues were not flexible, they would sterically hinder statin binding. Elevated cholesterol levels are a primary risk factor for coronary artery disease. This dis-ease is a major problem in developed coun-tries and currently affects 13 to 14 millionadults in the United States alone. Dietarychanges and drug therapy reduce serum cho-lesterol levels and dramatically decrease therisk of stroke and overall mortality ( 1 ). Inhib-itors of HMGR, commonly referred to asstatins, are effective and safe drugs that arewidely prescribed in cholesterol-loweringtherapy. In addition to lowering cholesterol,statins appear to have a number of additionaleffects, such as the nitric oxide–mediated promotion of new blood vessel growth ( 2 ),stimulation of bone formation ( 3 ), protectionagainst oxidative modification of low-densitylipoprotein, as well as anti-inflammatory ef-fects and a reduction in C-reactive proteinlevels ( 4 ). All statins curtail cholesterol bio-synthesis by inhibiting the committed step inthe biosynthesis of isoprenoids and sterols( 5 ). This step is the four-electron reductivedeacylation of HMG-CoA to CoA and meva-lonate. It is catalyzed by HMGR in a reactionthat proceeds as follows(S)-HMG-CoA    2NADPH    2H  3   (R)-mevalonate    2NADP    CoASHwhere NADP  is the oxidized form of nico-tinamide adenine dinucelotide, NADPH isthe reduced form of NADP  , and CoASH isthe reduced form of CoA.Several statins are available or in late-stageclinical development (Fig. 1). All share anHMG-like moiety, which may be present inan inactive lactone form. In vivo, these pro-drugs are enzymatically hydrolyzed to their active hydroxy-acid forms ( 5 ). The statins 1 Department of Biochemistry,  2 Howard Hughes Med-ical Institute, University of Texas Southwestern Med-ical Center at Dallas, TX 75390–9050, USA.*To whom correspondence should be addressed. E-mail: Johann.Deisenhofer@UTSouthwestern.edu R  E P O R T S 11 MAY 2001 VOL 292 SCIENCE www.sciencemag.org 1160    o  n   A  u  g  u  s   t   2   7 ,   2   0   1   3  w  w  w .  s  c   i  e  n  c  e  m  a  g .  o  r  g   D  o  w  n   l  o  a   d  e   d   f  r  o  m   share rigid, hydrophobic groups that arecovalently linked to the HMG-like moiety.Lovastatin, pravastatin, and simvastatin re-semble the substituted decalin-ring structureof compactin (also known as mevastatin). Weclassify this group of inhibitors as type 1statins. Fluvastatin, cerivastatin, atorvastatin,and rosuvastatin (in development by Astra-Zeneca) are fully synthetic HMGR inhibitorswith larger groups linked to the HMG-likemoiety. We refer to these inhibitors as type 2statins. The additional groups range in char-acter from very hydrophobic (e.g., cerivasta-tin) to partly hydrophobic (e.g., rosuvastatin).All statins are competitive inhibitors of HMGR with respect to binding of the sub-strate HMG-CoA, but not with respect to binding of NADPH ( 6  ). The  K  i  (inhibitionconstant) values for the statin-enzyme com- plexes range between 0.1 to 2.3 nM ( 5 ),whereas the Michaelis constant,  K  m , for HMG-CoA is 4   M ( 7  ).Although the structure of the catalytic portion of human HMGR in complex withsubstrates and with products has recently been elucidated ( 8 ,  9 ), it yields little informa-tion concerning statin binding. The proteinforms a tightly associated tetramer with bi- partite active sites, in which neighboringmonomers contribute residues to the activesites. The HMG-binding pocket is character-ized by a loop (residues 682–694, referred toas “cis loop”) (Fig. 2A). Because statins arecompetitive with respect to HMG-CoA, itappeared likely that their HMG-like moietiesmight bind to the HMG-binding portion of the enzyme active site. However, in this bind-ing mode their bulky hydrophobic groupswould clash with residues that compose thenarrow pocket which accommodates the pan-tothenic acid moiety of CoA; thus, the mech-anism of inhibition has remained unresolved.To determine how statins prevent the binding of HMG-CoA, we solved six crystalstructures of the catalytic portion of humanHMGR bound to six different statin inhibitorsat resolution limits of 2.3 Å or higher (Table1) ( 10 ). For each structure, the bound inhib-itors are well defined in the electron-densitymaps (Fig. 3). They extend into a narrow pocket where HMG is normally bound andare kinked at the O5-hydroxyl group of theHMG-like moiety, which replaces the thio-ester oxygen atom found in the HMG-CoAsubstrate. The hydrophobic-ring structures of the statins contact residues within helicesL  1 and L  10 of the enzyme’s large domain(Fig. 2B). No portion of the elongated NADP(H) binding site is occupied by statins.The structures presented here illustrate thatstatins inhibit HMGR by binding to the activesite of the enzyme, thus sterically preventingsubstrate from binding. This agrees well withkinetic studies that indicate that statins com- petitively inhibit HMG-CoA but do not affect NADPH binding ( 6  ).A comparison between substrate-boundand inhibitor-bound HMGR structures clearlyillustrates rearrangement of the substrate-bind-ing pocket to accommodate statin molecules(Fig. 2). The structures differ in the COOH-terminal 28 amino acids of the protein. In theelectron-density maps of the statin-complexstructures, residues COOH-terminal to Gly 860 are missing. In the substrate-complex structure,these residues encompass part of helix L  10and all of helix L  11, fold over the substrate,and participate in the formation of the narrow pantothenic acid–binding pocket (Fig. 2A). Inthe statin-bound structures, these residues aredisordered, revealing a shallow hydrophobicgroove that accommodates the hydrophobicmoieties of the statins. Fig. 1.  Structural formulas of statin inhibitors and the enzyme substrateHMG-CoA. ( A ) Structure of several statin inhibitors. Compactin and simva-statin are examples of type 1 statins; not shown are the other type 1 statins,lovastatin and pravastatin. Fluvastatin, cerivastatin, atorvastatin, androsuvastatin are type 2 statins. The HMG-like moiety that is conserved in allstatins is colored in red. The IC 50  (median inhibitory concentration) values of the statins are indicated ( 21 ). ( B ) Structure of HMG-CoA. The HMG-moietyis colored in red, and the  K  m  value of HMG-CoA is indicated ( 7 ). Fig. 2.  Statins exploit the conformational flexibility of HMGR to create a hydrophobic bindingpocket near the active site. ( A ) Active site of human HMGR in complex with HMG, CoA, and NADP.The active site is located at a monomer-monomer interface. One monomer is colored yellow, theother monomer is in blue. Selected side chains of residues that contact the substrates or the statinare shown in a ball-and-stick representation ( 20 ). Secondary structure elements are marked byblack labels. HMG and CoA are colored in magenta; NADP is colored in green. To illustrate themolecular volume occupied by the substrates, transparent spheres with a radius of 1.6 Å are laidover the ball-and-stick representation of the substrates or the statin. ( B ) Binding of rosuvastatin toHMGR. Rosuvastatin is colored in purple; other colors and labels are as in (A). This figure and Figs.3 and 4 were prepared with Bobscript ( 22 ), GLR ( 23 ), and POV-Ray ( 24 ). R  E P O R T S www.sciencemag.org SCIENCE VOL 292 11 MAY 2001  1161    o  n   A  u  g  u  s   t   2   7 ,   2   0   1   3  w  w  w .  s  c   i  e  n  c  e  m  a  g .  o  r  g   D  o  w  n   l  o  a   d  e   d   f  r  o  m   Although the structural changes in thecomplexes with statin had not been predicted,the COOH-terminal residues of HMGR areknown to be a mobile element in this protein.In structures of the human enzyme in com- plex with HMG-CoA alone, helix L  11 was partially disordered ( 8 ). Similarly, in struc-tures of a bacterial homolog of HMGR from  Pseudomonas mevalonii , a larger COOH-ter-minal domain that is not present in the human protein is disordered when no substrates are present ( 11 ) but ordered in the ternary com- plex ( 12 ). It appears that the innate flexibilityof the COOH-terminal region of HMGR isfortuitously exploited by statins to create a binding site for the inhibitor molecules.How is the specificity and tight binding of statin inhibitors achieved? The HMG-moi-eties of the statins occupy the enzyme activesite of HMGR. The orientation and bondinginteractions of the HMG moieties of the in-hibitors clearly resemble those of the sub- Fig. 3.  Stereoview of the electron-density map of atorvastatin bound to the HMGR active site. This2.2 Å simulated-annealing omit map, contoured at 1   , was calculated by omitting all atoms of theatorvastatin molecule shown, as well as protein atoms within 4.5 Å of the inhibitor. The electrondensity is overlaid on the final, refined model. The electron density covering atorvastatin is in green,whereas the electron density covering the protein is in blue. Carbon atoms of one of the twoprotein monomers are colored yellow, those of the neighboring monomer are in blue, and those of atorvastatin are in gray. In all molecules oxygen atoms are red, nitrogen atoms are blue, sulfuratoms are yellow, and the fluorine atoms are green. Fig. 4.  Mode of binding of compactin ( A ), simvastatin ( B ), fluvastatin ( C ),cerivastatin ( D ), atorvastatin ( E ), and rosuvastatin ( F ) to human HMGR.Interactions between the HMG moieties of the statins and the proteinare mostly ionic or polar. They are similar for all inhibitors and areindicated by the dotted lines. Numbers next to the lines indicate dis-tances in Å ( 13 ). The rigid hydrophobic groups of the statins aresituated in a shallow groove between helices L  1 and L  10.Additional interactions between Arg 590 and the fluorophenyl groupare present in the type 2 statins (C, D, E, F). Atorvastatin androsuvastatin form a hydrogen bond between Ser 565 and a carbonyloxygen atom (atorvastatin) (E) or a sulfone oxygen atom (rosuv-astatin) (F). R  E P O R T S 11 MAY 2001 VOL 292 SCIENCE www.sciencemag.org 1162    o  n   A  u  g  u  s   t   2   7 ,   2   0   1   3  w  w  w .  s  c   i  e  n  c  e  m  a  g .  o  r  g   D  o  w  n   l  o  a   d  e   d   f  r  o  m 
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