Structure of Mammalian Cytochrome P450 2B4 Complexed with 4-(4-Chlorophenyl)imidazole at 1.9-Å Resolution 论文

摘要

A 1.9-Å molecular structure of the microsomal cytochrome P450 2B4 with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI) in the active site was determined by x-ray crystallography. In contrast to the previous experimentally determined 2B4 structure, this complex adopted a closed conformation similar to that observed for the mammalian 2C enzymes. The differences between the open and closed structures of 2B4 were primarily limited to the lid domain of helices F through G, helices B′ and C, the N terminus of helix I, and the β4 region. These large-scale conformational changes were generally due to the relocation of conserved structural elements toward each other with remarkably little remodeling at the secondary structure level. For example, the F′ and G′ helices were maintained with a sharp turn between them but are placed to form the exterior ceiling of the active site in the CPI complex. CPI was closely surrounded by residues from substrate recognition sites 1, 4, 5, and 6 to form a small, isolated hydrophobic cavity. The switch from open to closed conformation dramatically relocated helix C to a more proximal position. As a result, heme binding interactions were altered, and the putative NADPH-cytochrome P450 reductase binding site was reformed. This suggests a structural mechanism whereby ligand-induced conformational changes may coordinate catalytic activity. Comparison of the 2B4/CPI complex with the open 2B4 structure yields insights into the dynamics involved in substrate access, tight inhibitor binding, and coordination of substrate and redox partner binding. A 1.9-Å molecular structure of the microsomal cytochrome P450 2B4 with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI) in the active site was determined by x-ray crystallography. In contrast to the previous experimentally determined 2B4 structure, this complex adopted a closed conformation similar to that observed for the mammalian 2C enzymes. The differences between the open and closed structures of 2B4 were primarily limited to the lid domain of helices F through G, helices B′ and C, the N terminus of helix I, and the β4 region. These large-scale conformational changes were generally due to the relocation of conserved structural elements toward each other with remarkably little remodeling at the secondary structure level. For example, the F′ and G′ helices were maintained with a sharp turn between them but are placed to form the exterior ceiling of the active site in the CPI complex. CPI was closely surrounded by residues from substrate recognition sites 1, 4, 5, and 6 to form a small, isolated hydrophobic cavity. The switch from open to closed conformation dramatically relocated helix C to a more proximal position. As a result, heme binding interactions were altered, and the putative NADPH-cytochrome P450 reductase binding site was reformed. This suggests a structural mechanism whereby ligand-induced conformational changes may coordinate catalytic activity. Comparison of the 2B4/CPI complex with the open 2B4 structure yields insights into the dynamics involved in substrate access, tight inhibitor binding, and coordination of substrate and redox partner binding. Cytochromes P450 (P450) 1The abbreviations used are: P450, cytochrome P450; CPI, 4-(4-chlorophenyl)imidazole; FOM, figure of merit; PDB, Protein Data Bank; r.m.s., root mean square; SRS, substrate recognition site. are involved in steroidogenesis, fatty acid metabolism, synthesis of bile and retinoid acids, and production of plant toxins, but it is their function in the elimination of xenobiotics that has received the most attention. In mammals, xenobiotic metabolizing P450s play the central role in detoxification of hydrophobic drugs, carcinogens, and toxins by decreasing the lipid solubility of these chemicals and, thus, promoting excretion. In contrast to the strict substrate selectivity of classical enzymes, xenobiotic-metabolizing P450s can each bind and oxidize a set of substrates with distinct sizes, shapes, and stereochemical features. Although the variety of substrates binding to a given P450 is often broad, the oxidation of each is usually remarkably regiospecific and stereospecific. Identification of the structural basis for the specific monooxygenation and binding of a diverse but select set of substrates has been a particularly challenging goal, which is an important prerequisite for understanding selective substrate oxidation. Although the diversity of substrates might suggest an easily accessible active site, initial structures of both soluble bacterial (1Poulos T.L. Finzel B.C. Howard A.J. J. Mol. Biol. 1987; 195: 687-700Crossref PubMed Scopus (1296) Google Scholar) and microsomal mammalian (2Williams P.A. Cosme J. Sridhar V. Johnson E.F. McRee D.E. Mol. Cell. 2000; 5: 121-131Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar) P450s revealed active sites buried within the globular structure of the protein. A few recent bacterial structures, however, suggest a “lid” domain composed of helices F and G, the motion of which controls substrate entry (3Haines D.C. Tomchick D.R. Machius M. Peterson J.A. Biochemistry. 2001; 40: 13456-13465Crossref PubMed Scopus (302) Google Scholar, 4Serbe K. Pylypenko O. Vitali F. Zhang W. Rouset S. Heck M. Vrijbloed J.W. Bischoff D. Bister B. Sussmuth R.D. Pelzer S. Wohllenben W. Robinson J.A. Schlichting I. J. Biol. Chem. 2002; 277: 47476-47485Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 5Podust L.M. Kim Y. Arase M. Neely B.A. Beck B.J. Bach H. Sherman D.H. Lamb D.C. Kelly S.L. Waterman M.R. J. Biol. Chem. 2003; 278: 12214-12221Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Protein flexibility has also been implicated in dictating the regiospecificity of oxidation once a substrate is present in the active site. Structures of mammalian P450 2C5 with different substrates have shown that specific regions of the protein alter their conformation in response to the ligand present (6Wester M.R. Johnson E.F. Marques-Soares C. Dansette P. Mansuy D. Stout C.D. Biochemistry. 2003; 42: 6370-6379Crossref PubMed Scopus (207) Google Scholar, 7Wester M.R. Johnson E.F. Marques-Soares C. Dijols S. Dansette P.M. Mansuy D. Stout C.D. Biochemistry. 2003; 42: 9335-9345Crossref PubMed Scopus (190) Google Scholar). Structures of P450 119 with different bound ligands have revealed flexibility in regions composing both the upper active site and a putative substrate access channel (8Yano J.K. Koo L.S. Schuller D.J. Li H. Ortiz de Montellano P.R. Poulos T.L. J. Biol. Chem. 2000; 275: 31086-31092Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 9Park S.Y. Yamane K. Adachi S. Shiro Y. Maves S.A. Weiss K.E. Sligar S.G. J. Inorg. Biochem. 2002; 91: 491-501Crossref PubMed Scopus (107) Google Scholar). The most dramatic differences in P450 conformation were recently observed for P450 2B4. This structure revealed that the protein can adopt a wide open conformation, allowing direct transfer of substrates from the protein exterior to the active site. This open cleft was grossly similar to openings observed in a few recent bacterial P450 structures (4Serbe K. Pylypenko O. Vitali F. Zhang W. Rouset S. Heck M. Vrijbloed J.W. Bischoff D. Bister B. Sussmuth R.D. Pelzer S. Wohllenben W. Robinson J.A. Schlichting I. J. Biol. Chem. 2002; 277: 47476-47485Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 5Podust L.M. Kim Y. Arase M. Neely B.A. Beck B.J. Bach H. Sherman D.H. Lamb D.C. Kelly S.L. Waterman M.R. J. Biol. Chem. 2003; 278: 12214-12221Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and was stabilized by dimerization of the protein, which occurs both in the crystals and in solution. In the symmetric homodimer, a portion of one molecule (helices F′–G′) partially fills the cleft of the second molecule, with His-226 forming an intermolecular coordinate bond to the sixth position of the heme iron. Although reversible at low protein concentrations, formation of this symmetric dimer is spontaneous at the high protein concentrations required for crystallization. This dimerization precluded investigation of the structural consequences of ligand binding in the P450 2B4 active site. The present report describes modifications of both the protein and crystallization conditions to yield an x-ray structure of the P450 2B4 complexed with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI). This experimentally determined structure was significantly different from the previous open structure of 2B4 and revealed a closed conformation more similar to that observed for the mammalian 2C enzymes. The structural differences between the open and closed structures of 2B4 yielded insights into the control of substrate access and the structural changes involved in tight inhibitor binding as well as the coordination of substrate and redox partner binding. Site-directed Mutagenesis, Expression, and Purification—2B4dH differs from the wild type protein by truncation and modification of the N-terminal transmembrane domain and the addition of a C-terminal His4 tag as described (10Scott E.E. He Y.A. M.R. Johnson E.F. Stout C.D. S. 2003; PubMed Scopus Google Scholar). For the present an was the and and the P450 was and as described for (10Scott E.E. He Y.A. M.R. Johnson E.F. Stout C.D. S. 2003; PubMed Scopus Google Scholar). protein was in with the by the addition of to The were by and in the of high and the the was and The and oxidation was as described E.E. He Chem. 2002; PubMed Scopus Google Scholar) with of P450, of NADPH-cytochrome P450 of cytochrome and and were from the in was from and as a in CPI was to the protein in and the complex was by a The 2B4/CPI complex was from CPI, and The were and at were in the with as a by in In a was by for in the solution. were a to a x-ray and a and Data were the to a x-ray and a and and S. of the Data and and and Scholar). were to a of the a and of Scholar) and Biol. PubMed Scopus Google Scholar). are shown in and for the are in of of for the are in in a The complex in a different the 2B4 The 2B4 structure was used as a for molecular Biol. PubMed Scopus Google Scholar). of the helices B′ through C and F through G, the their from the initial The was used to The one which was at the position. The the of sites in the The site and the sites were used as a for a J. Biol. PubMed Scopus Google Scholar). sites for a of The by was to most of the protein and the Biol. 2000; PubMed Scopus Google Scholar) was used to and The experimentally by was to the acid and the CPI was CPI was and stereochemical were the M. J. Mol. PubMed Scopus Google Scholar). The was P. J. L.M. Biol. Scopus Google Scholar). was by of and Biol. PubMed Scopus Google Scholar) the which was by the of structure is from the M. W. The structure were for and a Scopus Google Scholar) into the The of D.E. J. Biol. PubMed Scopus Google Scholar) was used for into the and, the 1.9-Å with J. Google Scholar) that the has with residues in regions and and one in a of the and the N terminus and are is well and also occurs as an in the open 2B4 structure, which is to role in heme binding and interactions the proximal heme The have been in the of 2B4/CPI the conformation that 2B4 a ligand is were used to the symmetric spontaneous that occurs the crystallization conditions that to the previous 2B4 structure (10Scott E.E. He Y.A. M.R. Johnson E.F. Stout C.D. S. 2003; PubMed Scopus Google Scholar). to the between the of His-226 and the heme His-226 was to a The protein for the substrate that were similar to observed for a complex with the specific inhibitor CPI was to protein for crystallization. CPI and to 2B4 with an of M. He M.R. Johnson E.F. Mol. 2001; PubMed Scopus Google Scholar). of CPI to 2B4 wild in an of the from of the low to of to the heme The complex of CPI with in the with a molecule in the Although crystals similar conditions the were significantly more and, to a initial were for one and one The structure was by molecular the set and the previous structure of P450 The initial were easily for most of the protein, but structural changes were more by the from the and iron. The of the was this The was from a to 1.9-Å and The is by an of and an of CPI in a to the in P450 CPI the structure 2B4 a conformation in which the inhibitor is within the active site of the protein with entry channel In the regions the open cleft observed in the 2B4 structure have toward each other to the bound inhibitor from the exterior of the protein. In the CPI helices F through the active site and with the N-terminal structures, the the N terminus of helix I, and the β4 The is a P450 conformation more similar to the closed form observed for the mammalian P450s 2C5 (2Williams P.A. Cosme J. Sridhar V. Johnson E.F. McRee D.E. Mol. Cell. 2000; 5: 121-131Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, M.R. Johnson E.F. Marques-Soares C. Dansette P. Mansuy D. Stout C.D. Biochemistry. 2003; 42: 6370-6379Crossref PubMed Scopus (207) Google Scholar, 7Wester M.R. Johnson E.F. Marques-Soares C. Dijols S. Dansette P.M. Mansuy D. Stout C.D. Biochemistry. 2003; 42: 9335-9345Crossref PubMed Scopus (190) Google J.K. M.R. Stout C.D. Johnson E.F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and P.A. Cosme J. D. H. 2003; PubMed Scopus Google Scholar) the recent 2B4 open structure of the 2B4/CPI complex with other P450 of the 2B4/CPI structure the Structures were by the conserved structure residues and of the open 2B4 structure the 2B4/CPI structure The structures were by the conserved structure and were PubMed Scopus Google Scholar). The heme and CPI are shown in and helices between 2B4 and 2B4/CPI are This conformational between the open and structures of 2B4 occurs with strict of the of the protein an of the 2B4 structures yields an of regions in an of that the of the protein is remarkably These regions with the differences between the 2B4 structures helices B′ and C, helices F through G, the N terminus of helix and the and the β4 little is required at the secondary structure to the different of 2B4 The of the in active site occurs of the relocation of residues in the helix F through the helix region. In contrast to the open 2B4 conformation, the CPI complex of 2B4 has an turn at the C terminus of the F helix and the helix to at the position as the F helices of the closed 2C structures The position of the F helix is that the turn the helix the heme Although the CPI complex the F′ and G′ helices observed in the 2B4 open structure the active site In residues in helix G′ of the CPI complex by B′ residues in the open As occurs in the open structure, is a sharp between helices F′ and G′ at The occurs at the C terminus of helix G′ and is to with interactions that by the Although the secondary structure of helix is also it a more helix in the CPI complex This of helix C terminus in the of N terminus the active site to partially the cleft observed in the open conformation of 2B4. The of the active site is also significantly by the and of the helix B′ through the The B′ helix is by In the open 2B4 structure, helix B′ is from helix by but in the CPI residues of helix B′ and the with residues in helices F′ and The of the residues between these to exterior interactions between the of the the B′ and the of the protein primarily In the structure of the CPI complex that the C helix is C terminus toward the proximal of the protein and that the is residues that are similar to the of these elements in other closed P450 The a that the in the C helix the of helix D. The the B′ helix to and the in the to the of 2B4 to significantly between these but of the structures them The cleft of the open 2B4 structure is also partially closed by a of the N-terminal of helix I. In the open conformation, helix a that at as the helix the helix of the CPI complex is As a result, the helix has by at N helices the turn to and to involved in The conformation of the N terminus of helix is by in helices and C, as structural elements to the The between helix and the N terminus of helix to a as helix is placed in both 2B4 In the CPI this is by primarily at the of a turn of helix present in the open conformation the of the from the N-terminal helix is the β4 In the CPI form the of a sharp turn that toward helix F This with the open conformation, the is and the of the is from helix F to open a portion of the putative substrate access revealed for CPI in the active site of 2B4 The molecule in a with an to the heme at a bond of The of CPI is at an of to the of the toward the B′ The CPI are with an of between The active site residues the inhibitor that of the inhibitor a conformational the of the protein. within a of from CPI 1, 4, 5, and and most closely with the These with and form a hydrophobic the upper The is by and with the of and forming a with the of the and a The complex is stabilized by this bond between and CPI, which is of of the at In contrast to the 2C5 structures with and with the Comparison of the 2B4 and 2B4/CPI structures that residues helix B′ and and in the β4 and have the from to which are primarily a of the of the protein differences observed in the of residues in helix are from in conformation the protein in and are in their little between the 2B4 of the proximal of the protein, which is implicated in cytochrome P450 reductase and cytochrome binding J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. K. P. J. Biochem. 2000; PubMed Scopus Google is significantly different between the 2B4 structures, primarily as a of differences in the of the C helix and the The of the C helix in the closed 2B4/CPI conformation most closely the of observed in the structure of the P450 heme and the reductase domain as well as in the closed structures of P450s from the 2C In the open 2B4 conformation, a helices C and but in the CPI complex the from the similar to the position in the 2C structures of the with residues in the a in the between the 2B4 between the and may a mechanism for ligand-induced conformational changes the of the protein to the proximal the 2B4/CPI residues that with the heme to this function in the closed of the 2C enzymes. the A is to and the also with as well as with and This to the open conformation of and switch that with both with the the of is to this but both the and the of the are A more in the interactions with the occurs as the with in the open form is by interactions with and in the closed CPI This in heme binding is due to the relocation of the C helix that for example, a of from a position in the open form to a buried position the heme in the closed CPI is the ligand to in a mammalian P450 active site. The active site is that of the structures of 2C enzymes, which in addition to the bound Although the structures of 2C5 with and within of the bound residues are within this from CPI in the 2B4/CPI complex. In contrast to the 2C5 structures, residues from the regions with In the 2B4/CPI helix residues in with the inhibitor are limited to the β4 residues are limited to at the of the The CPI residues at and have been implicated in the of and T.L. 2001; PubMed Scopus Google Scholar) and to of the active site for of these Site-directed of has revealed that the set of residues regiospecificity and is that by residues forming the CPI active site, an active site that to substrates of different and In of with substrates have that ligand residues might also at and in T.L. 2001; PubMed Scopus Google Scholar) E.E. He Chem. 2002; PubMed Scopus Google Scholar, Zhang H. J. 2003; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J.A. He Y. L.S. Mol. Google Scholar, T.L. He E.E. Biochem. 2001; PubMed Scopus Google Scholar, He Y.A. Biochemistry. PubMed Scopus Google Scholar). These residues in the the CPI active site to CPI of as of the active site and with a of The of important residues to CPI residues suggests a of the active site, the role of residues suggests an of the of substrates in this important residues that are implicated in ligand the between protein and alter the of the protein to adopt a substrate binding Although 2B4 and are the in for CPI between the is M. He M.R. Johnson E.F. Mol. 2001; PubMed Scopus Google Scholar). Although most of the acid differences are from the active site, the and are within of these the of hydrophobic residues CPI maintained in of by at position in the of the active site and CPI binding by the of the active site, residues and their and in 2B4. the active site may between 2B4 and the inhibitor protein with significantly binding to the iron. The entry of a ligand and the binding in the 2B4 active site to the of the protein the As a result, the structure of P450 2B4 complexed with the specific inhibitor CPI is of the closed structures of P450 from the 2C that have been recently (2Williams P.A. Cosme J. Sridhar V. Johnson E.F. McRee D.E. Mol. Cell. 2000; 5: 121-131Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, M.R. Johnson E.F. Marques-Soares C. Dansette P. Mansuy D. Stout C.D. Biochemistry. 2003; 42: 6370-6379Crossref PubMed Scopus (207) Google Scholar, 7Wester M.R. Johnson E.F. Marques-Soares C. Dijols S. Dansette P.M. Mansuy D. Stout C.D. Biochemistry. 2003; 42: 9335-9345Crossref PubMed Scopus (190) Google Scholar, J.K. M.R. Stout C.D. Johnson E.F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P.A. Cosme J. D. H. 2003; PubMed Scopus Google Scholar). interactions with the bound ligand in the closed form of 2B4 conformational changes in helix form the C-terminal turn of helix and helices and but structural changes have been observed in structures of other mammalian and bacterial Structures of 2C5 with and from each other in the of the residues in helix B′ and the of helices F and as a of interactions with ligands of different and (6Wester M.R. Johnson E.F. Marques-Soares C. Dansette P. Mansuy D. Stout C.D. Biochemistry. 2003; 42: 6370-6379Crossref PubMed Scopus (207) Google Scholar, 7Wester M.R. Johnson E.F. Marques-Soares C. Dijols S. Dansette P.M. Mansuy D. Stout C.D. Biochemistry. 2003; 42: 9335-9345Crossref PubMed Scopus (190) Google Scholar). In the that helices F through has the in most P450 structures, Although bacterial P450 structures have the F′ and G′ of the from the protein, of the C-terminal turn of helix and of helices F and have also been observed in structures of the bacterial P450 119 (8Yano J.K. Koo L.S. Schuller D.J. Li H. Ortiz de Montellano P.R. Poulos T.L. J. Biol. Chem. 2000; 275: 31086-31092Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 9Park S.Y. Yamane K. Adachi S. Shiro Y. Maves S.A. Weiss K.E. Sligar S.G. J. Inorg. Biochem. 2002; 91: 491-501Crossref PubMed Scopus (107) Google Scholar). The most between structures of the closely bacterial P450s with a and in an also of helices F and L.M. Bach H. Kim Y. Lamb D.C. Arase M. Sherman D.H. Kelly S.L. Waterman M.R. Protein 2003; Scopus Google Scholar). these the of a P450 structure conformation is to the of a Comparison of the open and closed structures of 2B4 also suggests a structural mechanism for coordination of substrate binding and redox partner binding. In the 2B4 structures, the and the interactions with the significantly alter the of helix C. The residues helix C play a role in P450 with redox J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). ligand binding the of the protein may partially with transfer the proximal This is with that substrate binding both reductase binding J. Inorg. Biochem. 2003; PubMed Scopus Google Scholar) and 2B4 D.R. PubMed Scopus Google Scholar) that of the The flexibility of helix is also to important in allowing diverse substrates to bind as well as in redox partner binding L.M. Poulos T.L. Waterman M.R. S. 2001; PubMed Scopus Google Scholar) in at The heme redox is to by the proximal heme ligand and and and interactions with the protein the structure H. Poulos T.L. Biol. PubMed Scopus Google Scholar) and of Biochemistry. PubMed Scopus Google Scholar) that substrate binding a conformational in one of the a bond with the protein. of 2B4 that the addition of a substrate the in a Biochem. PubMed Scopus Google Scholar). The 2B4 structures a of a bond to the of the 2B4 open conformation to the closed These in heme coordination may the heme redox a structural between substrate binding and the binding of redox catalytic Although the open 2B4 structure is an of the conformation required for entry of it accessible in solution. A with the present closed structure yields insights into the structural elements of P450s that both substrate binding and redox partner binding and, thus, may at partially coordinate CPI is a these 2B4 structures the conformational changes to These structures that the lid active site, and redox partner binding are with recent structures of 2C5 with different bound suggests that the residues in the 2B4/CPI complex a set that for with CPI a closed conformation structural the B′ helix and the to the C with redox binding in heme binding are with the relocation of helix C and may also redox and activity. These of dynamics may a for protein The and the of the conformational changes observed for 2B4 are investigation but are with structural observed for the P450 119 (8Yano J.K. Koo L.S. Schuller D.J. Li H. Ortiz de Montellano P.R. Poulos T.L. J. Biol. Chem. 2000; 275: 31086-31092Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 9Park S.Y. Yamane K. Adachi S. Shiro Y. Maves S.A. Weiss K.E. Sligar S.G. J. Inorg. Biochem. 2002; 91: 491-501Crossref PubMed Scopus (107) Google and the bacterial P450 (3Haines D.C. Tomchick D.R. Machius M. Peterson J.A. Biochemistry. 2001; 40: 13456-13465Crossref PubMed Scopus (302) Google Scholar). He for the of the with and and the of the for at that