Structural Mechanism for Statin Inhibition of HMG-CoA Reductase

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Science  11 May 2001:
Vol. 292, Issue 5519, pp. 1160-1164
DOI: 10.1126/science.1059344


HMG-CoA (3-hydroxy-3-methylglutaryl–coenzyme A) reductase (HMGR) catalyzes the committed step in cholesterol biosynthesis. Statins are HMGR inhibitors 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 residues were not flexible, they would sterically hinder statin binding.

Elevated cholesterol levels are a primary risk factor for coronary artery disease. This disease is a major problem in developed countries and currently affects 13 to 14 million adults in the United States alone. Dietary changes and drug therapy reduce serum cholesterol levels and dramatically decrease the risk of stroke and overall mortality (1). Inhibitors 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 lipoprotein, as well as anti-inflammatory effects and a reduction in C-reactive protein levels (4). All statins curtail cholesterol biosynthesis 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 mevalonate. It is catalyzed by HMGR in a reaction that proceeds as followsEmbedded Image Embedded Imagewhere NADP+ is the oxidized form of nicotinamide adenine dinucelotide, NADPH is the reduced form of NADP+, 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 prodrugs are enzymatically hydrolyzed to their active hydroxy-acid forms (5). The statins share rigid, hydrophobic groups that are covalently linked to the HMG-like moiety. Lovastatin, pravastatin, and simvastatin resemble 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 AstraZeneca) 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 character from very hydrophobic (e.g., cerivastatin) to partly hydrophobic (e.g., rosuvastatin). All statins are competitive inhibitors of HMGR with respect to binding of the substrate HMG-CoA, but not with respect to binding of NADPH (6). The K i(inhibition constant) values for the statin-enzyme complexes range between 0.1 to 2.3 nM (5), whereas the Michaelis constant,K m, for HMG-CoA is 4 μM (7).

Figure 1

Structural formulas of statin inhibitors and the enzyme substrate HMG-CoA. (A) Structure of several statin inhibitors. Compactin and simvastatin 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 theK m value of HMG-CoA is indicated (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 information concerning statin binding. The protein forms a tightly associated tetramer with bipartite active sites, in which neighboring monomers contribute residues to the active sites. The HMG-binding pocket is characterized 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 binding mode their bulky hydrophobic groups would clash with residues that compose the narrow pocket which accommodates the pantothenic acid moiety of CoA; thus, the mechanism of inhibition has remained unresolved.

Figure 2

Statins exploit the conformational flexibility 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 Å 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 figure and Figs. 3 and 4 were prepared with Bobscript (22), GLR (23), and POV-Ray (24).

To determine how statins prevent the binding of HMG-CoA, we solved six crystal structures of the catalytic portion of human HMGR bound to six different statin inhibitors at resolution limits of 2.3 Å or higher (Table 1) (10). For each structure, the bound inhibitors 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 thioester oxygen atom found in the HMG-CoA substrate. The hydrophobic-ring structures of the statins contact residues within helices Lα1 and Lα10 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 competitively inhibit HMG-CoA but do not affect NADPH binding (6).

Figure 3

Stereoview of the electron-density map of atorvastatin bound to the HMGR active site. This 2.2 Å simulated-annealing omit map, contoured at 1 σ, was calculated by omitting all atoms of the atorvastatin molecule shown, as well as protein atoms within 4.5 Å of the inhibitor. The electron density 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 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 fluorine atoms are green.

Table 1

Data collection and refinement statistics. Constants a, b, and c are in Å; β is in degrees. n, number; Rmsd, root mean square deviation.

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A comparison between substrate-bound and inhibitor-bound HMGR structures clearly illustrates rearrangement of the substrate-binding 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 Lα10 and all of helix Lα11, 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.

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 complex with HMG-CoA alone, helix Lα11 was partially disordered (8). Similarly, in structures of a bacterial homolog of HMGR from Pseudomonas mevalonii, a larger COOH-terminal domain that is not present in the human protein is disordered when no substrates are present (11) but ordered in the ternary complex (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-moieties of the statins occupy the enzyme active site of HMGR. The orientation and bonding interactions of the HMG moieties of the inhibitors clearly resemble those of the substrate complex (Fig. 2). Several polar interactions are formed between the HMG-moieties and residues that are located in the cis loop (Ser684, Asp690, Lys691, Lys692). Lys691 also participates in a hydrogen-bonding network with Glu559, Asp767 and the O5-hydroxyl of the statins. The terminal carboxylate 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 hydrophobic groups of the inhibitors are predominately responsible for the nanomolar K i values; 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 hydrophobic ring structures of the statins is present in all enzyme-inhibitor complexes, despite the structural diversity of these compounds. This is possible because the type 1 and type 2 statins adopt different conformations 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.

Figure 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 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 distances in Å (13). The rigid hydrophobic groups of the statins are situated in a shallow groove between helices Lα1 and Lα10. Additional interactions between Arg590 and the fluorophenyl 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 (rosuvastatin) (F).

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 complex 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 either 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 distinguishing type 2 from the type 1 statins. Here, the guanidinium group of Arg590 stacks on the fluorophenyl group, and polar interactions between the arginine ɛ nitrogen atoms and the fluorine atoms are observed. No differences between the type 1 statins compactin and simvastatin are apparent (Fig. 4, A and B). With the exception of the larger atorvastatin, the solvent-accessible areas of unbound or bound statins and the buried areas upon statin binding to HMGR are similar for all inhibitors (13).

In summary, these studies reveal how statins 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 interactions 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 currently available or in late-stage development excel in curtailing the biosynthesis of mevalonate, the precursor of cholesterol, it is possible 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 nicotinamide-like moiety to statins might improve their potency.

  • * To whom correspondence should be addressed. E-mail: Johann.Deisenhofer{at}


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