on May 12, 2015
` on May 12, 2015
` on May 12, 2015
` on May 12, 2015
` on May 12, 2015
`
`www.sciencemag.org
`www.sciencemag.org
`www.sciencemag.org
`www.sciencemag.org
`www.sciencemag.org
`
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`Downloaded from
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`
`R E P O R T S
`
`farms are vaccinated in ring vaccination. It is assumed
`that vaccination has no effect on previously infected
`farms.
`16. M. J. Keeling, Proc. R. Soc. London B 266, 859 (1999).
`17. Removal by culling of an infected herd and the
`removal of contiguous holdings of animals have dif-
`ferent impacts on R0 and the scale of the epidemic.
`The former acts directly to reduce R0, whereas the
`latter serves to signi(cid:222)cantly reduce the overall scale
`of
`the epidemic by stopping second-generation
`transmission events [hence reducing the effective
`reproductive number (10)].
`18. Northumberland Report: The Report of the Commit-
`tee of Inquiry on Food and Mouth Disease (Her Maj-
`esty(cid:213)s Stationery Of(cid:222)ce, 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 has
`given new estimates of rI signi(cid:222)cantly above 1,
`though more precise estimation awaits availability of
`detailed data on all slaughter schemes in operation
`since 30 March 2001.
`
`21. We are extremely grateful for help in the provision
`of data and for invaluable advice from J. Wilesmith
`(Veterinary Laboratory Agency), D. Reynolds (Food
`Standards Agency and Ministry of Agriculture, Fish-
`eries and Food), and D. Thompson (Ministry of
`Agriculture, Fisheries and Food) and to the many
`government epidemiologists and veterinary staff
`who collected the unique contact tracing data
`on FMD spread in the current epidemic. In addition,
`we thank D. King (Of(cid:222)ce of Science and Technol-
`ogy), B. Grenfell, M. Keeling, M. Woolhouse, and
`other members of the FMD Of(cid:222)cial Science Group
`for stimulating discussions; Sir Robert May and
`Sir David Cox for valuable advice and discussions;
`three anonymous referees for comments; and S.
`Dunstan, S. Riley, and H. Carabin for valuable
`assistance. N.M.F. thanks the Royal Society and the
`Howard Hughes Medical
`Institute for fellowship
`and research funding support. C.A.D. and R.M.A.
`thank the Wellcome Trust for research funding.
`
`23 March 2001; accepted 10 April 2001
`Published online 12 April 2001;
`10.1126/science.1061020
`Include this information when citing this paper.
`
`Structural Mechanism for Statin
`Inhibition of HMG-CoA
`Reductase
`Eva S. Istvan1 and Johann Deisenhofer1,2*
`
`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 effectively
`lower serum cholesterol levels and are widely prescribed in the treatment of
`hypercholesterolemia. We have determined structures of the catalytic portion
`of human HMGR complexed with six different statins. The statins occupy a
`portion of the binding site of HMG-CoA, thus blocking access of this substrate
`to the active site. Near the carboxyl terminus of HMGR, several catalytically
`relevant residues are disordered in the enzyme-statin complexes. If these res-
`idues were not (cid:223)exible, 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 million
`adults in the United States alone. Dietary
`changes and drug therapy reduce serum cho-
`lesterol levels and dramatically decrease the
`risk of stroke and overall mortality (1). Inhib-
`itors of HMGR, commonly referred to as
`statins, are effective and safe drugs that are
`widely prescribed in cholesterol-lowering
`therapy. In addition to lowering cholesterol,
`statins appear to have a number of additional
`effects, such as the nitric oxide–mediated
`promotion of new blood vessel growth (2),
`stimulation of bone formation (3), protection
`against oxidative modification of low-density
`
`1Department of Biochemistry, 2Howard 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
`
`lipoprotein, as well as anti-inflammatory ef-
`fects and a reduction in C-reactive protein
`levels (4). All statins curtail cholesterol bio-
`synthesis by inhibiting the committed step in
`the biosynthesis of isoprenoids and sterols
`(5). This step is the four-electron reductive
`deacylation of HMG-CoA to CoA and meva-
`lonate. It is catalyzed by HMGR in a reaction
`that proceeds as follows
`(S)-HMG-CoA 1 2NADPH 1 2H1 3
`mevalonate 1 2NADP1 1 CoASH
`
`(R)-
`
`where NADP1 is the oxidized form of nico-
`tinamide adenine dinucelotide, NADPH is
`the reduced form of NADP1, and CoASH is
`the reduced form of CoA.
`Several statins are available or in late-stage
`clinical development (Fig. 1). All share an
`HMG-like moiety, which may be present in
`an inactive lactone form. In vivo, these pro-
`drugs are enzymatically hydrolyzed to their
`active hydroxy-acid forms (5). The statins
`
`tiousness from 3 days after infection until slaughter
`(for an average of eight infectious days).
`12. The effective neighborhood size, n, in units of nearest
`neighbor farms, was estimated as
`‘
`R
`
`n 5E
`
`g~r!dr/E
`
`g~r!dr
`
`0
`0
`where R is given by the solution of
`R
`
`k~r!dr 5 1
`
`E 0
`
`f 5
`
`The connectedness of the contact network is given by
`1
`
`n2EEE g(r)g(r9)g((cid:239) r 2 r9(cid:239) )/k((cid:239) r 2 r9(cid:239) )drdr9du
`
`where
`
`(cid:239) r 2 r*(cid:239) 5 r2 1 r92 2 2rr9cos(u)
`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 strati(cid:222)ed into a suscep-
`tible class, S; sequential infection classes, Ii (i 5 1..M);
`and a slaughtered/vaccinated class, D. Multiple in-
`fected classes were used to exactly reproduce the
`gamma distribution (cid:222)ts to the delay data shown in
`Fig. 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 5 0.26 days
`each, weight 0.82) and 4 classes (transit times 5 3.73
`days, weight 0.18). Two classes (transit times 5 0.85
`to 0.21 days, time-dependent) represented farms
`awaiting disease con(cid:222)rmation after report, and four
`classes (transit times 5 0.82 to 0.38 days, time-
`dependent)(cid:209)overlapping the previous two(cid:209)repre-
`sented farms awaiting culling after disease reporting.
`Infectiousness varies as a function of incubation
`stage, reaching signi(cid:222)cant levels after around 3.5
`days and then continuing at a constant level until
`diagnosis, after which it remains constant until
`slaughter at a level rI times greater than before
`reporting. The model is novel in tracking not only the
`numbers of farms in each infection state through
`time, but also the numbers of pairs of farms connect-
`ed on the contact network used to represent spatially
`localized disease transmission. For conciseness and
`clarity, we only present those for a simpler model
`with only two infected classes: E (uninfectious) and I
`(infectious). Using [X] to represent the mean number
`in state X, [XY] to represent the mean number of
`pairs of type XY, and [XYZ] to represent the mean
`number of triples, the dynamics can be represented
`by the following set of differential equations: d[S]/
`dt 5 —(t 1 m 1 v)[SI] — pb[S][I]/N, d[E]/dt 5
`pb[S][I]/N 1 t[SI] — n[E] — m[EI], d[I]/dt 5 n[E] — s[I] —
`m[II], d[SS]/dt 5 —2(t 1 m 1 v)[SSI] — 2pb[SS][I]/N,
`d[SE]/dt 5 t([SSI] — [ISE]) — m([SEI] 1 [ISE]) — v[ISE] 1
`pb([SS] — [SE])[I]/N, d[SI]/dt 5 n[SE] — (t 1 m 1
`v)([ISI] 1 [SI]) — pb[SI][I]/N, d[EE]/dt 5 t[ISE] —
`2m[EEI] — 2n[EE] 1 2pb[SE][I]/N, d[EI]/dt 5 n[EE] —
`m([EI]1[IEI]) — (n 1 s)[EI] 1 pb[SI][I]/N, d[II]/dt 5
`2n[EI] — 2s[II] — 2m([II] 1 [III]). The numbers of triples
`are calculated with the closure approximation (16)
`[XYZ] ’ (n — 1)[XY][YZ](1 — f 1 f N[YY]/n[X][Z])/
`n[Y], where n is the mean contact neighborhood size
`of a farm, f is the proportion of triples in the network
`that are triangles, and N is the total number of farms
`[see (12)]. t 5 (1 — p)b/n is the transmission rate
`across a contact, where b is the transmission coef(cid:222)-
`cient of the virus, and p is the proportion of contacts
`that are long-range [see (9)], both of which are
`estimated separately before and after the movement
`ban. n is the rate of transit from the uninfectious to
`the infectious class, and s is the rate of transit from
`the infectious to the removed class. m is the rate at
`which farms in the neighborhood of an infected farm
`are culled in ring culling, and v is the rate at which
`
`1160
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`
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`

`
`share rigid, hydrophobic groups that are
`covalently linked to the HMG-like moiety.
`Lovastatin, pravastatin, and simvastatin re-
`semble the substituted decalin-ring structure
`of compactin (also known as mevastatin). We
`classify this group of inhibitors as type 1
`statins. Fluvastatin, cerivastatin, atorvastatin,
`and rosuvastatin (in development by Astra-
`Zeneca) are fully synthetic HMGR inhibitors
`with larger groups linked to the HMG-like
`moiety. We refer to these inhibitors as type 2
`statins. 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 Ki (inhibition
`constant) values for the statin-enzyme com-
`plexes range between 0.1 to 2.3 nM (5),
`for
`whereas the Michaelis constant, Km,
`HMG-CoA is 4 mM (7 ).
`Although the structure of the catalytic
`portion of human HMGR in complex with
`substrates and with products has recently
`been elucidated (8, 9), it yields little informa-
`tion concerning statin binding. The protein
`forms a tightly associated tetramer with bi-
`partite active sites,
`in which neighboring
`monomers contribute residues to the active
`sites. The HMG-binding pocket is character-
`ized by a loop (residues 682– 694, referred to
`as “cis loop”) (Fig. 2A). Because statins are
`competitive with respect to HMG-CoA, it
`appeared likely that their HMG-like moieties
`might bind to the HMG-binding portion of
`the enzyme active site. However, in this bind-
`ing mode their bulky hydrophobic groups
`would clash with residues that compose the
`narrow 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 crystal
`structures of the catalytic portion of human
`
`R E P O R T S
`
`HMGR bound to six different statin inhibitors
`at resolution limits of 2.3 Å or higher (Table
`1) (10). For each structure, the bound inhib-
`itors are well defined in the electron-density
`maps (Fig. 3). They extend into a narrow
`pocket where HMG is normally bound and
`are kinked at the O5-hydroxyl group of the
`HMG-like moiety, which replaces the thio-
`ester oxygen atom found in the HMG-CoA
`substrate. The hydrophobic-ring structures of
`the statins contact residues within helices
`La1 and La10 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 that
`statins inhibit HMGR by binding to the active
`site of the enzyme, thus sterically preventing
`substrate from binding. This agrees well with
`kinetic studies that indicate that statins com-
`
`petitively inhibit HMG-CoA but do not affect
`NADPH binding (6 ).
`A comparison between substrate-bound
`and inhibitor-bound HMGR structures clearly
`illustrates 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 the
`electron-density maps of the statin-complex
`structures, residues COOH-terminal to Gly860
`are missing. In the substrate-complex structure,
`these residues encompass part of helix La10
`and all of helix La11, fold over the substrate,
`and participate in the formation of the narrow
`pantothenic acid– binding pocket (Fig. 2A). In
`the statin-bound structures, these residues are
`disordered, revealing a shallow hydrophobic
`groove that accommodates the hydrophobic
`moieties of the statins.
`
`Fig. 2. Statins exploit the conformational (cid:223)exibility of HMGR to create a hydrophobic binding
`pocket 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, the
`other monomer is in blue. Selected side chains of residues that contact the substrates or the statin
`are shown in a ball-and-stick representation (20). Secondary structure elements are marked by
`black labels. HMG and CoA are colored in magenta; NADP is colored in green. To illustrate the
`molecular volume occupied by the substrates, transparent spheres with a radius of 1.6 (cid:129) are laid
`over the ball-and-stick representation of the substrates or the statin. (B) Binding of rosuvastatin to
`HMGR. Rosuvastatin is colored in purple; other colors and labels are as in (A). This (cid:222)gure and Figs.
`3 and 4 were prepared with Bobscript (22), GLR (23), and POV-Ray (24).
`
`Fig. 1. Structural formulas of statin inhibitors and the enzyme substrate
`HMG-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, and
`
`rosuvastatin are type 2 statins. The HMG-like moiety that is conserved in all
`statins is colored in red. The IC50 (median inhibitory concentration) values of
`the statins are indicated (21). (B) Structure of HMG-CoA. The HMG-moiety
`is colored in red, and the Km value of HMG-CoA is indicated (7).
`
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`
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`
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`

`
`R E P O R T S
`
`Although the structural changes in the
`complexes with statin had not been predicted,
`the COOH-terminal residues of HMGR are
`known to be a mobile element in this protein.
`In structures of the human enzyme in com-
`plex with HMG-CoA alone, helix La11 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 flexibility
`of the COOH-terminal region of HMGR is
`fortuitously 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 active
`site of HMGR. The orientation and bonding
`interactions 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. This
`2.2 (cid:129) simulated-annealing omit map, contoured at 1 s, was calculated by omitting all atoms of the
`atorvastatin molecule shown, as well as protein atoms within 4.5 (cid:129) of the inhibitor. The electron
`density is overlaid on the (cid:222)nal, re(cid:222)ned 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 two
`protein 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, sulfur
`atoms are yellow, and the (cid:223)uorine atoms are green.
`
`Fig. 4. Mode of binding of compactin (A), simvastatin (B), (cid:223)uvastatin (C),
`cerivastatin (D), atorvastatin (E), and rosuvastatin (F) to human HMGR.
`Interactions between the HMG moieties of the statins and the protein
`are mostly ionic or polar. They are similar for all inhibitors and are
`indicated by the dotted lines. Numbers next to the lines indicate dis-
`tances in (cid:129) (13). The rigid hydrophobic groups of the statins are
`
`situated in a shallow groove between helices La1 and La10.
`Additional interactions between Arg590 and the (cid:223)uorophenyl group
`are present in the type 2 statins (C, D, E, F). Atorvastatin and
`rosuvastatin form a hydrogen bond between Ser565 and a carbonyl
`oxygen atom (atorvastatin) (E) or a sulfone oxygen atom (rosuv-
`astatin) (F).
`
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`
`R E P O R T S
`Table 1. Data collection and re(cid:222)nement statistics. Constants a, b, and c are in (cid:129); b is in degrees. n, number; Rmsd, root mean square deviation.
`
`Crystal
`
`Compactin
`
`Simvastatin
`
`Fluvastatin
`
`Cerivastatin
`
`Atorvastatin
`
`Rosuvastatin
`
`Cell constants
`
`a 5 74.4
`a 5 74.6
`a 5 74.6
`a 5 74.8
`a 5 74.6
`a 5 73.8
`b 5 172.5
`b 5 172.7
`b 5 173.0
`b 5 175.1
`b 5 172.8
`b 5 173.0
`c 5 80.0
`c 5 80.0
`c 5 80.2
`c 5 74.8
`c 5 80.0
`c 5 75.2
`b 5 117.4
`b 5 117.7
`b 5 117.4
`b5118.3
`b 5 117.6
`b 5 118.4
`2
`1
`1
`1
`1
`1
`Crystals (n)
`43.3 to 2.10
`43.4 to 2.22
`43.5 to 2.26
`43.8 to 2.30
`43.4 to 2.33
`43.1 to 2.10
`Resolution ((cid:129))
`101,733
`86,963
`80,409
`73,193
`73,699
`89,377
`Unique re(cid:223)ections (n)
`5.0
`3.7
`4.2
`3.6
`3.9
`2.4
`Redundancy
`97.6
`98.6
`96.0
`97.6
`96.4
`92.7
`Completeness (%)
`Rsym (%)*
`7.2
`3.8
`4.7
`10.0
`6.4
`5.4
`^I/sI&
`21.1
`30.8
`28.7
`11.8
`20.7
`14.8
`11,764
`11,772
`11,938
`11,398
`11,750
`11,565
`Protein atoms (n)
`182
`225
`186
`199
`176
`287
`Water molecules (n)
`213
`299
`294
`201
`259
`170
`Heterogen atoms (n)
`0.087
`0.011
`0.010
`0.009
`0.009
`0.011
`Rmsd bond lengths ((cid:129))
`1.7
`1.4
`1.4
`1.4
`1.3
`1.5
`Rmsd bond angles (¡)
`Average B factor ((cid:129)2)
`55.4
`52.7
`55.1
`28.3
`60.4
`36.8
`Rworking (%)†
`21.9
`21.2
`22.1
`18.6
`22.2
`19.1
`Rfree (%)‡
`23.9
`23.5
`23.7
`21.4
`24.8
`22.3
`1HWL
`1HWK
`1HWJ
`1HWI
`1HW9
`1HW8
`PDB accession no.
`?) / (SFobs), where Fobs and Fcalc are observed and calculated structure
`†R 5 (S?Fobs — Fcalc
`5 S?(Ihkl) — ^I&? / S (Ihkl), where Ihkl is the integrated intensity of a given re(cid:223)ection.
`*Rmerge
`factors, respectively; no I/sI cutoff was used in the re(cid:222)nement.
`‡For each crystal, about 2000 re(cid:223)ections were excluded from the re(cid:222)nement to calculate Rfree.
`
`strate complex (Fig. 2). Several polar inter-
`actions are formed between the HMG-moi-
`eties and residues that are located in the cis
`loop (Ser684, Asp690, Lys691, Lys692). Lys691
`also participates in a hydrogen-bonding net-
`work with Glu559, Asp767 and the O5-hy-
`droxyl of the statins. The terminal carboxyl-
`ate of the HMG moiety forms a salt bridge to
`Lys735. The large number of hydrogen bonds
`and ion pairs results in charge and shape
`complementarity between the protein and the
`HMG-like moiety of the statins. Identical
`bonding interactions are observed between
`the protein and HMG and presumably also
`with the reaction product mevalonate (Fig.
`2A). Because mevalonate is released from the
`active site,
`it
`is likely that not all of its
`interactions with the protein are stabilizing.
`These observations suggest that the hydro-
`phobic groups of the inhibitors are predomi-
`nately responsible for the nanomolar Ki val-
`ues; they may also change the context of the
`HMG-like polar interactions such that the ion
`pairs contribute favorably to the binding of
`statins.
`Hydrophobic side chains of the enzyme
`involving residues Leu562, Val683, Leu853,
`Ala856, and Leu857 participate in van der
`Waals contacts with the statins. The surface
`complementarity between HMGR and the hy-
`drophobic ring structures of the statins is
`present in all enzyme-inhibitor complexes,
`despite the structural diversity of these com-
`pounds. This is possible because the type 1
`and type 2 statins adopt different conforma-
`tions that allow their hydrophobic groups to
`maximize contacts with the hydrophobic
`pocket on the protein (Fig. 4). Functionally,
`the methylethyl group attached to the central
`ring of the type 2 statins replaces the decalin
`of the type 1 statins. The butyryl group of the
`
`type 1 statins occupies a region similar to the
`fluorophenyl group present
`in the type 2
`inhibitors.
`A comparison between the six complex
`structures illustrates subtle differences in
`their modes of binding. Rosuvastatin has the
`greatest number of bonding interactions with
`HMGR (Fig. 4F). In addition to numerous
`contacts present in other statin-HMGR com-
`plex structures, a polar interaction between
`the Arg568 side chain and the electronegative
`sulfone group is unique to rosuvastatin.
`Present only in atorvastatin and rosuvastatin
`are hydrogen bonds between Ser565 and ei-
`ther a carbonyl oxygen atom (atorvastatin) or
`a sulfone oxygen atom (rosuvastatin) (Fig. 4,
`E and F). The fluorophenyl groups of type 2
`statins are one of the main features distin-
`guishing type 2 from the type 1 statins. Here,
`the guanidinium group of Arg590 stacks on
`the fluorophenyl group, and polar interac-
`tions between the arginine e nitrogen atoms
`and the fluorine atoms are observed. No dif-
`ferences between the type 1 statins compactin
`and simvastatin are apparent (Fig. 4, A and
`B). With the exception of the larger atorva-
`statin,
`the solvent-accessible areas of un-
`bound or bound statins and the buried areas
`upon statin binding to HMGR are similar for
`all inhibitors (13).
`In summary, these studies reveal how st-
`atins bind to and inhibit their target, human
`HMGR. The bulky, hydrophobic compounds
`of statins occupy the HMG-binding pocket
`and part of the binding surface for CoA.
`Thus, access of the substrate HMG-CoA to
`HMGR is blocked when statins are bound.
`The tight binding of statins is probably due to
`the large number of van der Waals interac-
`tions between inhibitors and with HMGR.
`The structurally diverse, rigid hydrophobic
`
`groups of the statins are accommodated in a
`shallow non-polar groove that is present only
`when COOH-terminal residues of HMGR are
`disordered. Although the statins that are cur-
`rently available or in late-stage development
`excel in curtailing the biosynthesis of meva-
`lonate, the precursor of cholesterol, it is pos-
`sible that the visualization of statin bound to
`HMGR will assist in the development of even
`better inhibitors. In particular, it should be
`noted that the nicotinamide-binding site of
`HMGR is not occupied by statin inhibitors
`and that the covalent attachment of a nicoti-
`namide-like moiety to statins might improve
`their potency.
`
`References and Notes
`1. D. A. Eisenberg, Am. J. Med. 104, 2S (1998).
`2. Y. Kureishi et al., Nature Med. 6, 1004 (2000).
`3. G. Mundy et al., Science 286, 1946 (1999).
`4. J. Davignon, R. Laaksonen, Curr. Opin. Lipidol. 10, 543
`(1999).
`5. A. Corsini, F. M. Maggi, A. L. Catapano, Pharmacol.
`Res. 31, 9 (1995).
`6. A. Endo, M. Kuroda, K. Tanzawa, FEBS Lett. 72, 323
`(1976).
`7. K. M. Bischoff, V. W. Rodwell, Biochem. Med. Metab.
`Biol. 48, 149 (1992).
`8. E. S. Istvan, M. Palnitkar, S. K. Buchanan, J. Deisen-
`hofer, EMBO J. 19, 819 (2000).
`9. E. S. Istvan, J. Deisenhofer, Biochim. Biophys. Acta
`1529, 9 (2000).
`10. The catalytic portion of human HMGR was puri(cid:222)ed as
`described (8). Concentrated stock solutions of the
`inhibitors were prepared in methanol and added to
`the protein in three- or fourfold molar excess. Sim-
`vastatin, (cid:223)uvastatin, cerivastatin, atorvastatin, and
`rosuvastatin were received from AstraZeneca and
`were in their active hydroxy-acid form. Compactin
`was purchased from Sigma and activated by convert-
`ing the lactone form to the sodium salt with NaOH
`as described (14). After a 6 to 24 hour incubation of
`protein with inhibitor at 4¡C, batch crystallization
`trials at 21¡C were set up. Crystals were grown at a
`protein concentration of 3 to 5 mg/ml and in solu-
`tions containing 12 to 15 % [weight/volume (w/v)]
`polyethylene glycol (PEG) 4000, 0.15 to 0.2 M am-
`monium acetate, 25 mM Na-Hepes ( pH 7.5), 50 mM
`
`www.sciencemag.org SCIENCE VOL 292 11 MAY 2001
`
`1163
`
`NCI Exhibit 2028
`Page 4 of 6
`
`

`
`dithiothreitol (DT T), 10 mM adenosine diphosphate
`(ADP), and 10% glycerol. Crystallization was initiated
`by the addition of microseeds, prepared from sub-
`strate crystals, after 14 to 20 hours. Plate-like crys-
`tals grew in about 10 days. The crystals were har-
`vested in solutions containing 20% (w/v) PEG 4000,
`0.3 M ammonium acetate, 25 mM Na-Hepes ( pH
`7.5), 50 mM DT T, 10 mM ADP and 10% glycerol. For
`cryoprotection, the crystals were transferred to so-
`lutions containing increasing glycerol (15, 20, and
`25%) for about 1 min each and (cid:223)ash-cooled in liquid
`propane. Initial data for a rosuvastatin complex struc-
`ture to a resolution of 2.4 (cid:129) were collected at beam-
`line 5.0.2 of the Advanced Light Source (ALS) syn-
`chrotron, which is supported by the Director, Of(cid:222)ce
`of Science, Of(cid:222)ce of Basic Energy Sciences, Materials
`Sciences Division of the U.S. Department of Energy
`under Contract No. DE-AC03-76SF00098 at Law-
`rence Berkeley National Laboratory. Data for the
`other inhibitor complexes and higher resolution data
`for the rosuvastatin complex were collected at beam-
`line F1 at the Cornell High Energy Synchrotron
`Source (CHESS), which is supported by the National
`Science Foundation under award DMR-9311772, us-
`ing the Macromolecular Diffraction at CHESS (Mac-
`CHESS) facility, which is supported by award RR-
`01646 from the National Institutes of Health. Data
`reduction and processing were carried out with the
`HKL package (15). Because the low-resolution data
`for the rosuvastatin complex crystal was incomplete
`for the data collected at CHESS, the reduced data
`were merged with the reduced data collected at ALS
`during scaling. All crystals have the symmetry of
`space group P21 and contain four HMGR monomers
`in each asymmetric unit, although two different crys-
`tal forms were observed ( Table 1). The protein por-
`tion of the structure of human HMGR in complex
`with HMG, CoA, and NADP1 [Protein Data Bank
`(PDB) code 1dqa] was used as the starting model for
`the re(cid:222)nement. Initially, the inhibitor molecules were
`placed into Fo-Fc electron-density maps. Subsequent-
`ly, their positions were modi(cid:222)ed by consulting s
`A
`weighted 2Fo-Fc maps (16) and simulated-annealing
`omit maps (17). The models were built using the
`program O (18) and re(cid:222)ned with CNS (19). Bulk
`solvent, overall aniosotropic B-factor scaling, and
`noncrystallographic symmetry restraints were ap-
`plied throughout the re(cid:222)nement process. For each of
`the six HMGR-statin complexes, the electron-density
`maps were excellent for all four statin molecules
`bound to the four crystallographically independent
`monomers. Additionally, poor electron density was
`located close to residues Y479 and F629 (20) and was
`interpreted as ADP. The positions of the ADP mole-
`cules resemble the positions of the adenosine moi-
`eties of the substrates CoA or NADPH. ADP was
`bound only to some of the CoA or NADPH binding
`sites and the number of ADP molecules is different
`for the six structures.
`11. C. M. Lawrence, V. W. Rodwell, C. V. Stauffacher,
`Science 268, 1758 (1995).
`12. L. Tabernero, D. A. Bochar, V. W. Rodwell, C. V.
`Stauffacher, Proc. Natl. Acad. Sci. U.S.A. 96, 7167
`(1999).
`13. All calculations on accessible or buried surface areas
`for the statins or the protein, as well as distance
`information between speci(cid:222)c groups, represent aver-
`ages for the four crystallographically independent
`statin molecules observed in each complex structure.
`The surface accessible areas for the unbound statins,
`the bound statins, and the buried surface areas upon
`statin binding to HMGR, respectively, are as follows:
`compactin 670 (cid:129)2, 100 (cid:129)2, 880 (cid:129)2; simvastatin 670
`(cid:129)2, 110 (cid:129)2, 880 (cid:129)2; (cid:223)uvastatin 660 (cid:129)2, 80 (cid:129)2, 870 (cid:129)2;
`cerivastatin 720 (cid:129)2, 100 (cid:129)2, 880 (cid:129)2; atorvastatin 840
`(cid:129)2, 150 (cid:129)2, 1060 (cid:129)2; and rosuvastatin 710 (cid:129)2, 130 (cid:129)2,
`880 (cid:129)2.
`14. M. S. Brown, J. R. Faust, J. L. Goldstein, J. Biol. Chem.
`253, 1121 (1978).
`15. Z. Otwinowski, W. Minor, Methods Enzymol. 276,
`306 (1997).
`16. A. Hodel, S.-H. Kim, A. T. Bru‹nger, Acta Crystallogr. A
`48, 851 (1992).
`17. R. J. Read, Acta Crystallogr. A 1986, 140 (1986).
`
`R E P O R T S
`
`18. T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, Acta
`Crystallogr. A 47, 110 (1991).
`19. A. T. Bru‹nger et al., Acta Crystallogr. D 54, 905
`(1998).
`20. Single-letter abbreviations for the amino acid resi-
`dues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F,
`Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn;
`P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and
`Y, Tyr.
`21. G. A. Holdgate et al., in preparation.
`22. R. M. Esnouf, Acta Crystallogr. D 55, 938 (1999).
`23. L. Esser, personal communication.
`
`24. Persistence of Vision Ray Tracer v.3.02, Copyright
`1997, POV-Team. www.povray.org
`25. We thank AstraZeneca for providing simvastatin, (cid:223)u-
`vastatin, cerivastatin, atorvastatin, and rosuvastatin
`and for stimulating discussions; S. Jeong for convert-
`ing compactin to the active sodium salt form; the
`personnel at ALS beamline 5-1 and CHESS beamline
`F1 for their assistance in data collection; and C. A.
`Brautigam for critical reading of the manuscript. The
`coordinates are available from the PDB (accession
`numbers are indicated in Table 1).
`
`26 January 2001; accepted 3 April 2001
`
`Control of a Genetic Regulatory
`Network by a Selector Gene
`Kirsten A. Guss,* Craig E. Nelson,* Angela Hudson,
`Mary Ellen Kraus, Sean B. Carroll†
`
`The formation of many complex structures is controlled by a special class of
`transcription factors encoded by selector genes. It is shown that SCALLOPED,
`the DNA binding component of the selector protein complex for the Drosophila
`wing (cid:222)eld, binds to and directly regulates the cis-regulatory elements of many
`individual target genes within the genetic regulatory network controlling wing
`development. Furthermore, combinations of binding sites for SCALLOPED and
`transcriptional effectors of signaling pathways are necessary and suf(cid:222)cient to
`specify wing-speci(cid:222)c responses to different signaling pathways. The obligate
`integration of selector and signaling protein inputs on cis-regulatory DNA may
`be a general mechanism by which selector proteins control extensive genetic
`regulatory networks during development.
`
`The concept of the morphogenetic field, a dis-
`crete set of cells in the embryo that gives rise to
`a particular structure, has held great importance
`in experimental embryology (1). The discovery
`of genes whose products control the formation
`and identity of various fields, dubbed “selector
`genes” (2), has enabled the recognition and
`redefinition of fields as discrete territories of
`selector gene activity (3). Although the term has
`been used somewhat liberally, two kinds of
`selector genes have been of central interest to
`understanding the development of embryonic
`fields. These include the Hox genes, whose
`products differentiate the identity of homolo-
`gous fields, and field-specific selector genes
`such as eyeless (4), Distal-less (5), and vesti-
`gial-scalloped (vg-sd) (6–8), whose products
`have the unique property of directing the for-
`mation of entire complex structures. The mech-
`anisms by which field-specific selector proteins
`direct the development of these structures are
`not well understood. In principle, selector pro-
`teins could directly regulate the expression of
`only a few genes, thus exerting much of their
`effect indirectly, or they may regulate the tran-
`
`Howard Hughes Medical Institute and Laboratory of
`Molecular Biology, University of Wisconsin, Madison,
`WI 53706, USA.
`
`*These authors contributed equally to this work.
`†To whom correspondence should be addressed. E-
`mail: sbcarrol@facstaff.wisc.edu
`
`scription of many genes distributed throughout
`genetic regulatory networks.
`In the Drosophila wing imaginal disc, the
`VG-SD selector protein complex regulates
`wing formation and identity (7, 8). SD is a
`TEA-domain protein (9) that binds to DNA in
`a sequence-specific manner (7), whereas VG,
`a novel nuclear protein (10), functions as a
`trans-activator (11). To determine whether
`direct regulation by SD is widely required for
`gene expression in the wi