The Structure of Human Cytochrome P450 2C9 Complexed with Flurbiprofen at 2.0-Å Resolution

Abstract

The structure of human P450 2C9 complexed with flurbiprofen was determined to 2.0 Å by x-ray crystallography. In contrast to other structurally characterized P450 2C enzymes, 2C5, 2C8, and a 2C9 chimera, the native catalytic domain of P450 2C9 differs significantly in the conformation of the helix F to helix G region and exhibits an extra turn at the N terminus of helix A. In addition, a distinct conformation of the helix B to helix C region allows Arg-108 to hydrogen bond with Asp-293 and Asn-289 on helix I and to interact directly with the carboxylate of flurbiprofen. These interactions position the substrate in a and to the catalytic by P450 2C9 the of The structure a of a of the substrate of P450 2C9 and the of on The structure of human P450 2C9 complexed with flurbiprofen was determined to 2.0 Å by x-ray crystallography. In contrast to other structurally characterized P450 2C enzymes, 2C5, 2C8, and a 2C9 chimera, the native catalytic domain of P450 2C9 differs significantly in the conformation of the helix F to helix G region and exhibits an extra turn at the N terminus of helix A. In addition, a distinct conformation of the helix B to helix C region allows Arg-108 to hydrogen bond with Asp-293 and Asn-289 on helix I and to interact directly with the carboxylate of flurbiprofen. These interactions position the substrate in a and to the catalytic by P450 2C9 the of The structure a of a of the substrate of P450 2C9 and the of on P450 2C9 of human a a with a on structure structure substrate a a with a on structure structure substrate in 2C to the of The P450 2C9 to a to in P450 2C9 of of interactions to at P450 2C9 in the of in other P450 the 2C the 2C and to of in and substrate of the and substrate 2C9 and with distinct substrate to the on the of other A. the conformation to in the substrate of 2C9 and and the of by of 2C9 and to the substrate of distinct catalytic 2C9 exhibits a the of the and the a in the of P450 2C9 the of A. A. the structure of a of P450 2C9 a The of the by the structure the of P450 2C9 to in the structure of P450 2C9 was to the native of the to the and to the with a substrate in the to the substrate of the with the a helix at the N terminus in of the domain structure of the catalytic of in a to to In the of P450 and the P450 the C terminus of the to a to of the P450 2C9 was in the and the P450 In contrast to the P450 2C9 to the structure A. the catalytic domain of the the structure of the flurbiprofen of differs significantly the structure of the 2C9 by A. in and The 2C9 in the of A. and to in and the to to of in region the structure In addition, was to the in the of the with the of in an position Å the of the of an the in the substrate A. In the structure flurbiprofen with the of by the of by the The structure in an Arg-108 in the of flurbiprofen and a conformation of the helix B to helix C Arg-108 The structure significantly to the of Arg-108 Asn-289 and Asp-293 in substrate and a the by 2C9 was by of the a the by with the and the of with was the of the in The the C terminus was the a to a at the C terminus of to an of the of to a the The was with the and and the to the the of 2C9 at with the of the N terminus to P450 and the with an a to and was and with of with of an of the the of of and of the was at with at by and in of the of and was and the with at of was and the with an The by at the was and the in and at The was in and with on the by at at was to a of and the was with at the was at at The was with of was with of and the was by The was with of and with of with of and was in a The was with and and with of and The was at with with of was with the The the was in a and with of the The P450 in and the the was to a with a 2C9 and with P450 P450 in and a the was by the of of and on The and substrate the and by was by at and the with by of in the of P450 a to a in the of flurbiprofen was by was to in and in a of in to the and the in the and The was and in and The with a to the and by by the and of and and at x-ray a of The by in a and The was to the and at the at a and a and The and of and on to the and the in and of the in the in the in of of in in in and in and of the was to and in the of of of of of the in of in in in and in and of the was to and in the in a The structure of 2C9 was with the structure a of and of the and the and The the and the the of and flurbiprofen was the at in the of the in the at of the of with flurbiprofen in the of the of the of the flurbiprofen was a a was to and of the with a of Å at Å the was in the to the the by and the to flurbiprofen and the The of and the the of the of the a was to of the catalytic domain the The the with the was the of the N terminus the and of P450 The differs by A. to the and of with and to and in the the and in the and the the to of the P450 2C9 to in on and with to in P450 with P450 the of with a of of P450 with of P450 the to P450 with an of determined by the of the to the with a of the These the to the N and C significantly flurbiprofen P450 2C9 of a to 2.0 Å in with a in the and on the structure of was and a the and in a structure with an of and an of The These the of the the and helix F and and the of the of the an of a of the the and The at the position and of flurbiprofen in the was on The determined structure of with of the in of the structure of the flurbiprofen of to the structure in with a of in the N terminus in helix B and helix F the of helix and the turn in the These in of the structure and These of the structure in other The structure exhibits an extra turn at the of helix in other P450 The of the helix with the the position in the of the structure with the structure the of the the of helix A. and in the exhibits a conformation of the region F and G and a helix A. in the The region an structure with in the conformation of to and In to the of the differs the native in the helix region The the helix region of the to the of the in the and in to a position in and 2C5, a turn and The of the the helix region of P450 2C9 in a conformation to in and In addition, the and the of region to The distinct conformation of the helix region by the native in the structure with of 2C9 and of the and the to the distinct of the and to the to to the in the conformation of region the and to the substrate of 2C9 in the of an of flurbiprofen. The of flurbiprofen in the by the by the the substrate of the the of the and the of the with the a with by the other to by 2C9 The carboxylate of flurbiprofen a with Arg-108 and hydrogen to Arg-108 in the structure of 2C9 in the of and the In the flurbiprofen of in the helix B to helix C region a conformation Arg-108 to interact with the substrate and Asn-289 and Asp-293 on helix I region exhibits in the of and and the structure and of the of and These the region the conformation significantly other of the at and to The was with the substrate the the was other of the The and the of the and substrate and of in the and The of the the position of the of In addition, flurbiprofen to the in the and on the other of of and to in of Arg-108 with on helix Asp-293 and the conformation of Arg-108 in the Arg-108 to the of flurbiprofen to of other Arg-108 to flurbiprofen and to the of and of a substrate of hydrogen with was by the of Asp-293 the of by the of the Asp-293 exhibits hydrogen interactions to the structurally turn by the at The Asn-289 of 2C9 hydrogen interactions the conformation of Arg-108 in the with the the of the to of the 2C9 the structure a the in substrate the of Å the with the in other P450 substrate flurbiprofen and the The Å the and Å flurbiprofen. The by interactions with the and hydrogen interactions with the of a in to the with by of the of the in the in the of helix I the in other P450 Å of flurbiprofen. a and flurbiprofen with in and and the a and a of helix I a with the position of and to helix I in a by and in the and in and in and in and in The flurbiprofen on flurbiprofen to in the and a the substrate and the turn in the The helix helix and the turn in the The of and the of and the with the of flurbiprofen. by and with to the of flurbiprofen by P450 and with a and flurbiprofen the The with the of flurbiprofen to with flurbiprofen in the to the of in the structure of the flurbiprofen a with of The the in The of the helix F flurbiprofen and the the to the was by at The of in position to the of flurbiprofen in the and the hydrogen the These to the of catalytic the of flurbiprofen in the of of of the substrate of The by a and was a with the of the the a and the a and and the and and the structure of the P450 substrate to the The structure with to on the The structure Arg-108 a in the of flurbiprofen to the and to to of other P450 2C9 and The conformation of Arg-108 in the structure by interactions with on helix Asp-293 and Arg-108 and Asp-293 in P450 Asn-289 and of at position in P450 to the The conformation of the helix B to helix C region in the structure differs significantly the of in of and and The of the and and of P450 the helix B to helix C and helix F to helix G and to in A. the of the P450 2C9 the the and to the of the of the of in the of by the of and of P450 the of substrate P450 2C9 of human a a with a on structure structure substrate a a with a on structure structure substrate in 2C to the of The P450 2C9 to a to in P450 2C9 of of interactions to at P450 2C9 in the of in other P450 the 2C the 2C and to of in and substrate of the and substrate 2C9 and with distinct substrate to the on the of other A. the conformation to in the substrate of 2C9 and and the of by of 2C9 and to the substrate of distinct catalytic P450 2C9 exhibits a the of the and the a in the of P450 2C9 the of A. A. the structure of a of P450 2C9 a The of the by the structure the of P450 2C9 to in the structure of P450 2C9 was to the native of the to the and to the with a substrate in the to the substrate of the with the a helix at the N terminus in of the domain structure of the catalytic of in a to to In the of P450 and the P450 the C terminus of the to a to of the P450 2C9 was in the and the P450 In contrast to the P450 2C9 to the structure A. the catalytic domain of the the The structure of the flurbiprofen of differs significantly the structure of the 2C9 by A. in and The 2C9 in the of A. and to in and the to to of in region the structure In addition, was to the in the of the with the of in an position Å the of the of an the in the substrate A. In the structure flurbiprofen with the of by the of by the The structure in an Arg-108 in the of flurbiprofen and a conformation of the helix B to helix C Arg-108 The structure significantly to the of Arg-108 Asn-289 and Asp-293 in substrate and a the by 2C9 was by of the a the by with the and the of with was the of the in The the C terminus was the a to a at the C terminus of to an of the of to a the The was with the and and the to the the of 2C9 at with the of the N terminus to P450 and the with an a to and was and with of with of an of the the of of and of the was at with at by and in of the of and was and the with at of was and the with an The by at the was and the in and at The was in and with on the by at at was to a of and the was with at the was at at The was with of was with of and the was by The was with of and with of with of and was in a The was with and and with of and The was at with with of was with the The the was in a and with of the The P450 in and the the was to a with a 2C9 and with P450 P450 in and a the was by the of of and on The and substrate the and by was by at and the with by of in the of P450 a to a in the of flurbiprofen was by was to in and in a of in to the and the in the and The was and in and The with a to the and by by the and of and and at x-ray a of The by in a and The was to the and at the at a and a and The and of and on to the and the in and of the in the in the in of of in in in and in and of the was to and in the of of of of of the in of in in in and in and of the was to and in the in a The structure of 2C9 was with the structure a of and of the and the and The the and the the of and flurbiprofen was the at in the of the in the at of the of with flurbiprofen in the of the of the of the flurbiprofen was a a was to and of the with a of Å at Å the was in the to the the by and the to flurbiprofen and the The of and the the of the of the a 2C9 was by of the a the by with the and the of with was the of the in The the C terminus was the a to a at the C terminus of to an of the of to a the The was with the and and the to the the of 2C9 at with the of the N terminus to P450 and the with an a to and was and with of with of an of the the of of and of the was at with at by and in of the of and was and the with at of was and the with an The by at the was and the in and at The was in and with on the by at at was to a of and the was with at the was at at The was with of was with of and the was by The was with of and with of with of and was in a The was with and and with of and The was at with with of was with the The the was in a and with of the The P450 in and the the was to a with a 2C9 and with P450 P450 in and a the was by the of of and on The and substrate the and by was by at and the with by of in the of P450 a to a in the of flurbiprofen was by was to in and in a of in to the and the in the and The was and in and The with a to the and by by the and of and and at x-ray a of The by in a and The was to the and at the at a and a and The and of and on to the and the in The structure of 2C9 was with the structure a of and of the and the and The the and the the of and flurbiprofen was the at in the of the in the at of the of with flurbiprofen in the of the of the of the flurbiprofen was a a was to and of the with a of Å at Å the was in the to the the by and the to flurbiprofen and the The of and the the of the of the a was to of the catalytic domain the The the with the was the of the N terminus the and of P450 The differs by A. to the and of with and to and in the the and in the and the the to of the P450 2C9 to in on and with to in P450 with P450 the of with a of of P450 with of P450 the to P450 with an of determined by the of the to the with a of the These the to the N and C significantly flurbiprofen P450 2C9 of a to 2.0 Å in with a in the and on the structure of was and a the and in a structure with an of and an of The These the of the the and helix F and and the of the of the an of a of the the and The at the position and of flurbiprofen in the was on The determined structure of with of the in of the structure of the flurbiprofen of to the structure in with a of in the N terminus in helix B and helix F the of helix and the turn in the These in of the structure and These of the structure in other The structure exhibits an extra turn at the of helix in other P450 The of the helix with the the position in the in the The region an structure with in the conformation of to and In to the of the differs the native in the helix region The the helix region of the to the of the in the and in to a position in and 2C5, a turn and The of the the helix region of P450 2C9 in a conformation to in and In addition, the and the of region to The distinct conformation of the helix region by the native in the structure with of 2C9 and of the and the to the distinct of the and to the to to the in the conformation of region the and to the substrate of 2C9 in the of an of flurbiprofen. The of flurbiprofen in the by the by the the substrate of the the of the and the of the with the a with by the other to by 2C9 The carboxylate of flurbiprofen a with Arg-108 and hydrogen to Arg-108 in the structure of 2C9 in the of and the In the flurbiprofen of in the helix B to helix C region a conformation Arg-108 to interact with the substrate and Asn-289 and Asp-293 on helix I region exhibits in the of and and the structure and of the of and These the region the conformation significantly other of the at and to The was with the substrate the the was other of the The and the of the and substrate and of in the and The of the the position of the of In addition, flurbiprofen to the in the and on the other of of and to in of Arg-108 with on helix Asp-293 and the conformation of Arg-108 in the Arg-108 to the of flurbiprofen to of other Arg-108 to flurbiprofen and to the of and of a substrate of hydrogen with was by the of Asp-293 the of by the of the Asp-293 exhibits hydrogen interactions to the structurally turn by the at The Asn-289 of 2C9 hydrogen interactions the conformation of Arg-108 in the with the the of the to of the 2C9 the structure a the in substrate the of Å the with the in other P450 substrate flurbiprofen and the The Å the and Å flurbiprofen. The by interactions with the and hydrogen interactions with the of a in to the with by of the of the in the in the of helix I the in other P450 Å of flurbiprofen. a and flurbiprofen with in and and the a and a of helix I a with the position of and to helix I in a by and in the and in and in and in and in The flurbiprofen on flurbiprofen to in the and a the substrate and the turn in the The helix helix and the turn in the The of and the of and the with the of flurbiprofen. by and with to the of flurbiprofen by P450 and with a and flurbiprofen the The with the of flurbiprofen to with flurbiprofen in the to the of in the structure of the flurbiprofen a with of The the in The of the helix F flurbiprofen and the the to the was by at The of in position to the of flurbiprofen in the and the hydrogen the These to the of catalytic the of flurbiprofen in the of of of the substrate of The by a and was a with the of the the a and the a and and the and and the structure of the P450 substrate to the The structure with to on the The structure Arg-108 a in the of flurbiprofen to the and to to of other P450 2C9 and The conformation of Arg-108 in the structure by interactions with on helix Asp-293 and Arg-108 and Asp-293 in P450 Asn-289 and of at position in P450 to the The conformation of the helix B to helix C region in the structure differs significantly the of in of and and The of the and and of P450 the helix B to helix C and helix F to helix G and to in A. the of the P450 2C9 the the and to the of the of the of in the of by the of and of P450 the of substrate The was to of the catalytic domain the The the with the was the of the N terminus the and of P450 The differs by A. to the and of with and to and in the the and in the and the the to of the P450 2C9 The to in on and with to in P450 with P450 the of with a of of P450 with of P450 the to P450 with an of determined by the of the to the with a of the These the to the N and C significantly flurbiprofen P450 2C9 of a to 2.0 Å in with a in the and on the structure of was and a the and in a structure with an of and an of The These the of the the and helix F and and the of the of the an of a of the the and The at the position and of flurbiprofen in the was on The determined structure of with of the in of the The structure of the flurbiprofen of to the structure in with a of in the N terminus in helix B and helix F the of helix and the turn in the These in of the structure and These of the structure in other The structure exhibits an extra turn at the of helix in other P450 The of the helix with the the position in the in the The region an structure with in the conformation of to and In to the of the differs the native in the helix region The the helix region of the to the of the in the and in to a position in and 2C5, a turn and The of the the helix region of P450 2C9 in a conformation to in and In addition, the and the of region to The distinct conformation of the helix region by the native in the structure with of 2C9 and of the and the to the distinct of the and to the to to the in the conformation of region the and to the substrate of 2C9 The in the of an of flurbiprofen. The of flurbiprofen in the by the by the the substrate of the the of the and the of the with the a with by the other to by 2C9 The carboxylate of flurbiprofen a with Arg-108 and hydrogen to Arg-108 in the structure of 2C9 in the of and the In the flurbiprofen of in the helix B to helix C region a conformation Arg-108 to interact with the substrate and Asn-289 and Asp-293 on helix I region exhibits in the of and and the structure and of the of and These the region the conformation significantly other of the of Arg-108 with on helix Asp-293 and the conformation of Arg-108 in the Arg-108 to the of flurbiprofen to of other Arg-108 to flurbiprofen and to the of and of a substrate of hydrogen with was by the of Asp-293 the of by the of the Asp-293 exhibits hydrogen interactions to the structurally turn by the at The Asn-289 of 2C9 hydrogen interactions the conformation of Arg-108 in the with the the of the to of the 2C9 the structure a the in The substrate the of Å the with the in other P450 substrate flurbiprofen and the The Å the and Å flurbiprofen. The by interactions with the and hydrogen interactions with the of a in to the with by of the of the in the in the of helix I the in other P450 to helix I in a by and in the and in and in and in and in The flurbiprofen on flurbiprofen to in the and a the substrate and the turn in the The helix helix and the turn in the The of and the of and the with the of flurbiprofen. by and with to the of flurbiprofen by P450 and with a and flurbiprofen the The with the of flurbiprofen to with flurbiprofen in the to the of in the structure of the flurbiprofen a with of The the in The of the helix F flurbiprofen and the the to the was by at The of in position to the of flurbiprofen in the and the hydrogen the These to the of catalytic the of flurbiprofen in the of of In the structure of the P450 substrate to the The structure with to on the The structure Arg-108 a in the of flurbiprofen to the and to to of other P450 2C9 and The conformation of Arg-108 in the structure by interactions with on helix Asp-293 and Arg-108 and Asp-293 in P450 Asn-289 and of at position in P450 to the The conformation of the helix B to helix C region in the structure differs significantly the of in of and and The of the and and of P450 the helix B to helix C and helix F to helix G and to in A. the of the P450 2C9 the the and to the of the of the of in the of by the of and of P450 the of substrate