Introduction

Top Abstract Introduction Homology modeling of mammalian... Application of homology models... Conclusions References

There is considerable interest in the function of cytochromes P450 (P450)¹ because of their involvement in drug metabolism as well as carcinogen bioactivation. Based on their structure, P450 enzymes are classified into gene families and subfamilies, with members of the same family exhibiting at least 40% amino acid sequence identity, and the members of the same subfamily >55% identity (Nelson et al., 1996). The main xenobiotic metabolizing enzymes belong to families 1 through 4 (Wrighton and Stevens, 1992). Many forms of P450 display broad substrate specificities, but individual isoforms often exhibit strict regio- and stereospecificity toward a particular compound. The elucidation of the structural basis for such specificity is of great importance in understanding enzyme function and mechanism. It may also help to predict the possible metabolic fate of drugs and carcinogens, as well as provide a foundation for the rational design of drugs and inhibitors.

In recent years, site-directed mutagenesis has become an important tool to study the structure-function relationships of mammalian cytochromes P450. The concept of substrate recognition sites (SRSs) was introduced by Gotoh (Gotoh, 1992) for the P450 2 family, based on the alignment of mammalian P450s with a bacterial enzyme, P450cam. This was the only enzyme at the time for which the crystal structure was solved. The SRS concept has provided an excellent guide in exploring the basis for P450 specificity, and a number of key amino acid residues responsible for substrate specificity in various mammalian P450s have been determined (von Wachenfeldt and Johnson, 1995). However, further advances in understanding enzyme function require information about the three-dimensional (3D) structure. To date, four bacterial P450 structures solved by X-ray crystallography have been published: namely, P450cam (Poulos et al., 1985 and 1987), P450 BM-3 (Ravichandran et al., 1993), P450terp (Hasemann et al., 1994) and P450eryF (Cupp-Vickery and Poulos, 1995). Therefore, molecular models of various mammalian enzymes have been constructed in order to explain substrate specificity and to relate enzyme function to structure. However, because of inaccuracies inherent in homology modeling, all modeling predictions must be verified experimentally. A recent review summarizes the state of the art in the field of homology modeling with an emphasis on the utilization of models in conjunction with site-directed mutagenesis studies to investigate mammalian P450s (Szklarz and Halpert, 1997). This article will describe our recent studies in which models of cytochromes P450 2B1 and 3A4 have been utilized to study enzyme function. The models have been used to identify key residues, to explain changes in regio- and stereospecificity of substrate oxidation upon site-directed mutagenesis, and to aid in the analysis of P450 inhibition and activation.

	Homology Modeling of Mammalian Cytochromes P450

Top Abstract Introduction Homology modeling of mammalian... Application of homology models... Conclusions References

Homology Modeling Methods. In homology modeling, a 3D model of the protein is constructed based on its amino acid sequence and the crystal structure of one or more reference proteins. The model can be built using molecular replacement or consensus methods. In both methods, the first step involves a sequence alignment between the protein to be modeled and the template(s). In regions of low homology, secondary structure predictions, as well as additional information, such as site-directed mutagenesis data (Szklarz et al., 1995), can be invaluable. In the next step, the structurally conserved regions (SCRs) of the modeled protein are determined. When the model is built using molecular replacement methods, the coordinates for SCRs are copied directly from those of the reference protein(s). The coordinates for the variable regions, such as loops, are calculated or obtained from those for the similar loops in known protein structures. The initial model is then refined using minimization and molecular dynamics methods.

Limitations of Homology Modeling. The basic assumption in homology modeling is that a modeled protein resembles the structure(s) used as templates; therefore, the choice of the template(s) is of crucial importance. The final 3D homology structure is highly dependent upon the modeling procedure and is influenced by a number factors. The most important of them is sequence alignment, which may lead to differences in the location of some residues and thus result in different models. For example, in P450 2B1 models, the identity of active site residues depended upon the alignment (Szklarz et al., 1994). Thus the accuracy of the alignment is of crucial importance for the final structure. Another important factor is the choice of the modeling method. In molecular replacement methods, the coordinates of the SCRs are identical to those of a given reference protein, while in consensus models, they are averages of those of several templates. In the case of cytochromes P450, a model based on structures of several known enzymes should be more accurate than one based on the crystal structure of only a single protein, especially in view of the low sequence identity between mammalian and bacterial P450s. Moreover, the choice of coordinates for loops can alter the location of key residues, as shown in the case of P450 2B1 models (Szklarz et al., 1994).

Docking of Substrates/Inhibitors Into the Active Site of P450 Models. Docking of enzyme substrates or inhibitors into the active site of the homology model can help to explain enzyme-substrate interactions as well as the role of particular residues in catalysis. An important issue is the orientation of the substrate bound in the active site. Several choices are possible: (1) docking a compound based solely on steric considerations, (2) docking a compound based on steric considerations but orienting the site of metabolism toward heme and ferryl oxygen, and (3) docking a compound in a reactive (productive) binding orientation. The first choice can be appropriate for the docking of competitive inhibitors that are not metabolized by the enzyme. It may also reflect the preferred binding orientation of the substrate in the initial enzyme-substrate complex. Approach 2, based on the orientation of camphor in the crystallized P450cam, has frequently been used in docking P450 substrates. That orientation may also reflect the enzyme-substrate complex. In contrast, approach 3 represents an orientation of the substrate that is necessary for the first oxidative event in the P450 catalytic cycle. Thus the substrate is oriented in the active site to allow for the initial hydrogen or electron abstraction. The latter approach has been successfully utilized to interpret changes in regiospecificity of substrate oxidation and susceptibility to inactivation upon residue replacement by site-directed mutagenesis (e.g. Szklarz et al., 1995; Kent et al., 1997; Kobayashi et al., 1998). Additional methods, such as molecular dynamics and evaluation of enzyme-substrate interaction energies, may further increase our understanding of P450 catalysis and the motion of the substrate in the active site.

	Application of Homology Models to Study P450 Function

Top Abstract Introduction Homology modeling of mammalian... Application of homology models... Conclusions References

Identification of Key Amino Acid Residues. With a 3D P450 model, the location of amino acid residues of interest can readily be visualized. Key residues should be present in the active site and be able to interact with a substrate. Docking of the substrate in the active site of the model should also make it possible to determine the prevalent enzyme-substrate interactions. Moreover, the model should be in agreement with experimental results.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   The active site of P450 3A4 with key residues determined to date. Substitution of residues shown in magenta primarily affects steroid metabolite profiles, while replacement of residues shown in yellow affects cooperativity.

Interpretation of Alterations in Substrate Specificity Upon Site-Directed Mutagenesis. Site-directed mutagenesis can pinpoint key amino acid residues, but the interpretation of changes in regio- and stereospecificity of substrate oxidation upon residue replacement is uncertain. However, these difficulties can be overcome with the help of 3D molecular models, which may also give some insights into structural basis of enzyme function. The usual procedure involves the replacement in the model of a given amino acid to mimic the mutant, and docking of the substrate in an orientation leading to the expected product. The binding of the substrate in the active site of the mutant is then compared with that of the wild-type enzyme. In general, the loss of activity upon mutation of a key residue can be a result of (1) van der Waals overlaps that hinder substrate binding, or (2) increased substrate mobility when enzyme-substrate interactions become too weak. In contrast, an increase in activity can be related to the stabilization of a given binding orientation through an increase in van der Waals contacts and decreased substrate mobility. Changes in substrate mobility may alter the coupling efficiency of the mutant.³

View larger version (30K): [in this window] [in a new window]   Fig. 2.   7-Ethoxycoumarin docked into the active site of P450 2B1 model in an orientation leading to O-dealkylation. The substrate is shown in grey, with all hydrogens displayed. The labeled residues are located within 5 Å from the substrate.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   7-Butoxycoumarin docked into the active site of P450 2B1 V363A mutant in an orientation leading to O-dealkylation. The substrate is shown in grey, with all hydrogens displayed. Ala at position 363 allows 7-butoxycoumarin to assume the orientation allowing for O-dealkylation, while the larger Val present in the wild-type enzyme would create van der Waals overlaps with the substrate.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   7-Butoxycoumarin docked into the active site of P450 2B1 V363L mutant in an orientation leading to (-1) hydroxylation. The substrate is shown in grey, with all hydrogens displayed. Leu-363 stabilizes the binding of the substrate in this orientation.

Utilization of Models in Inhibition Studies. Homology models can be of great help in the analysis of P450 inhibition or inactivation. The models can be used to explain changes in enzyme inhibition upon residue replacement, provide additional information on the mechanisms of P450 inhibition or inactivation, and aid in the design of better inhibitors.

View larger version (51K): [in this window] [in a new window]   Fig. 5.   BBT docked in the active site of the P450 2B1 model in an orientation allowing for oxidation at the 1-amino nitrogen of the substrate. The substrate is shown in orange and heme in red. Ala at position 478 (green) hinders binding of BBT in this orientation because of van der Waals overlaps with the substrate.

Stimulation of P450 3A4 by -Naphthoflavone. P450 3A4 is known to catalyze the oxidation of a number of substrates in a cooperative manner. Metabolic activities of the enzyme are also modulated by naturally occurring phenolic compounds known as flavonoids, resulting in either stimulation or inhibition of activity. An allosteric mechanism is usually invoked to explain cooperativity. Recent studies in our laboratory provided the first evidence for the location of residues that influence flavonoid stimulation of P450 3A4 (Harlow and Halpert, 1997; He et al., 1997). These residues are proposed to constitute part of the active site of the enzyme (Szklarz and Halpert, 1997; He et al., 1997). In addition to bound progesterone, up to two molecules of -naphthoflavone (-NF) can be fitted in the model (fig. 6). Instead of -NF, the active site of P450 3A4 is able to accommodate a second molecule of progesterone, which may explain the homotropic enzyme stimulation observed with steroids (Harlow and Halpert, 1997).

View larger version (53K): [in this window] [in a new window]   Fig. 6.   Progesterone and two molecules of -NF docked into the P450 3A4 model. Side chains of residues 211 and 214 are displayed. Progesterone is shown in orange; one molecule of -NF is yellow and the other green. Heme is shown in red. -NF can bind in the vicinity of the substrate close to residues 211 and 214.

	Conclusions

Top Abstract Introduction Homology modeling of mammalian... Application of homology models... Conclusions References

The combination of homology modeling and site-directed mutagenesis has provided an important insight into P450 structure-function relationships. The simplest application of the homology models involves the determination of the "hot spots," or key residues. The location of key residues in the active site and their interactions with docked enzyme substrates can be readily ascertained. Moreover, additional residues that may be important for enzymatic activity can be pinpointed. However, we should keep in mind that the identity of residues able to contact the substrate depends upon the structure of the compound and the specific binding orientations it assumes in the active site. P450 models have been successfully utilized to explain changes in regio- and stereospecificity of substrate oxidation, as well as alterations in susceptibility toward inactivation upon site-directed mutagenesis. These changes can be related to the removal or appearance of van der Waals overlaps in the mutant proteins and changes in substrate/inhibitor mobility. With P450 models, we can postulate plausible mechanisms for enzyme catalysis, inhibition, and activation, based on the 3D structure. In the last case, the model of P450 3A4 suggested a plausible hypothesis concerning the location of the effector site, which has been supported experimentally. In summary, homology modeling allows for a mechanistic interpretation of various aspects of P450 function and, in conjunction with experimental methods, is likely to continue as an important tool in studies of mammalian P450s. We can expect further development of homology modeling methods, as well as methods for structural verification of the models. The improvement of forcefields for protein modeling and generation of better parameters for heme will allow for the introduction of molecular dynamics methods to analyze substrate or inhibitor motion in the active site and to calculate binding free energies, as has been done for P450cam (Paulsen and Ornstein, 1992 and 1996, Paulsen et al., 1993). When the structure of a eukaryotic enzyme is solved, the methodology established and verified in studies utilizing homology models can be easily adapted and refined for use with the "real" structures.