Kinase conformations: a computational study of the effect of ligand binding.

Protein function is often controlled by ligand-induced conformational transitions. Yet, in spite of the increasing number of three-dimensional crystal structures of proteins in different conformations, not much is known about the driving forces of these transitions. As an initial step toward exploring the conformational and energetic landscape of protein kinases by computational methods, intramolecular energies and hydration free energies were calculated for different conformations of the catalytic domain of cAMP-dependent protein kinase (cAPK) with a continuum (Poisson) model for the electrostatics. Three protein kinase crystal structures for ternary complexes of cAPK with the peptide inhibitor PKI(5-24) and ATP or AMP-PNP were modeled into idealized intermediate and open conformations. Concordant with experimental observation, we find that the binding of PKI(5-24) is more effective in stabilizing the closed and intermediate forms of cAPK than ATP. PKI(5-24) seems to drive the final closure of the active site cleft from intermediate to closed state because ATP does not distinguish between these two states. Binding of PKI(5-24) and ATP is energetically additive.     

The dynamics of camphor in the cytochrome P450 CYP101D2

The recent crystal structures of CYP101D2, a cytochrome P450 protein from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 revealed that both the native (substrate‐free) and camphor‐soaked forms have open conformations. Furthermore, two other potential camphor‐binding sites were also identified from electron densities in the camphor‐soaked structure, one being located in the access channel and the other in a cavity on the surface near the F‐helix side of the F‐G loop termed the substrate recognition site. These latter sites may be key intermediate positions on the pathway for substrate access to or product egress from the active site. Here, we show via the use of unbiased atomistic molecular dynamics simulations that despite the open conformation of the native and camphor‐bound crystal structures, the underlying dynamics of CYP101D2 appear to be very similar to other CYP proteins. Simulations of the native structure demonstrated that the protein is capable of sampling many different conformational substates. At the same time, simulations with the camphor positioned at various locations within the access channel or recognition site show that movement towards the active site or towards bulk solvent can readily occur on a short timescale, thus confirming many previously reported in silico studies using steered molecular dynamics. The simulations also demonstrate how the fluctuations of an aromatic gate appear to control access to the active site. Finally, comparison of camphor‐bound simulations with the native simulations suggests that the fluctuations can be of similar level and thus are more representative of the conformational selection model rather than induced fit.     

Conformational dynamics of the F1-ATPase beta-subunit: a molecular dynamics study

According to the different nucleotide occupancies of the F(1)-ATPase beta-subunits and due to the asymmetry imposed through the central gamma-subunit, the beta-subunit adopts different conformations in the crystal structures. Recently, a spontaneous and nucleotide-independent closure of the open beta-subunit upon rotation of the gamma-subunit has been proposed. To address the question whether this closure is dictated by interactions to neighbored subunits or whether the open beta-subunit behaves like a prestressed "spring," we report multinanosecond molecular dynamics simulations of the isolated beta-subunit with different start conformations and different nucleotide occupancies. We have observed a fast, spontaneous closure motion of the open beta(E)-subunit, consistent with the available x-ray structures. The motions and kinetics are similar to those observed in simulations of the full (alpha beta)(3)gamma-complex, which support the view of a prestressed "spring," i.e., that forces internal to the beta(E)-subunit dominate possible interactions from adjacent alpha-subunits. Additionally, nucleotide removal is found to trigger conformational transitions of the closed beta(TP)-subunit; this provides evidence that the recently resolved half-closed beta-subunit conformation is an intermediate state before product release. The observed motions provide a plausible explanation why ADP and P(i) are required for the release of bound ATP and why gamma-depleted (alpha beta)(3) has a drastically reduced hydrolysis rate.     

Conformational states of cytochrome P450cam revealed by trapping of synthetic molecular wires

Members of the ubiquitous cytochrome P450 family catalyze a vast range of biologically significant reactions in mammals, plants, fungi, and bacteria. Some P450s display a remarkable promiscuity in substrate recognition, while others are very specific with respect to substrate binding or regio and stereo-selective catalysis. Recent results have suggested that conformational flexibility in the substrate access channel of many P450s may play an important role in controlling these effects. Here, we report the X-ray crystal structures at 1.8A and 1.5A of cytochrome P450cam complexed with two synthetic molecular wires, D-4-Ad and D-8-Ad, consisting of a dansyl fluorophore linked to an adamantyl substrate analog via an alpha,omega-diaminoalkane chain of varying length. Both wires bind with the adamantyl moiety in similar positions at the camphor-binding site. However, each wire induces a distinct conformational response in the protein that differs from the camphor-bound structure. The changes involve significant movements of the F, G, and I helices, allowing the substrate access channel to adapt to the variable length of the probe. Wire-induced opening of the substrate channel also alters the I helix bulge and Thr252 at the active site with binding of water that has been proposed to assist in peroxy bond cleavage. The structures suggest that the coupling of substrate-induced conformational changes to active-site residues may be different in P450cam and recently described mammalian P450 structures. The wire-induced changes may be representative of the conformational intermediates that must exist transiently during substrate entry and product egress, providing a view of how substrates enter the deeply buried active site. They also support observed examples of conformational plasticity that are believed be responsible for the promiscuity of drug metabolizing P450s. Observation of such large changes in P450cam suggests that substrate channel plasticity is a general property inherent to all P450 structures.     

Conformational equilibria and free energy profiles for the allosteric transition of the ribose-binding protein

The ribose-binding protein (RBP) is a sugar-binding bacterial periplasmic protein whose function is associated with a large allosteric conformational change from an open to a closed conformation upon binding to ribose. The crystal structures of RBP in open and closed conformations have been solved. It has been hypothesized that the open and closed conformations exist in a dynamic equilibrium in solution, and that sugar binding shifts the population from open conformations to closed conformations. Here, we study by computer simulations the thermodynamic changes that accompany this conformational change, and model the structural changes that accompany the allosteric transition, using umbrella sampling molecular dynamics and the weighted histogram analysis method. The open state is comprised of a diverse ensemble of conformations; the open ribose-free X-ray crystal conformations being representative of this ensemble. The unligated open form of RBP is stabilized by conformational entropy. The simulations predict detectable populations of closed ribose-free conformations in solution. Additional interdomain hydrogen bonds stabilize this state. The predicted shift in equilibrium from the open to the closed state on binding to ribose is in agreement with experiments. This is driven by the energetic stabilization of the closed conformation due to ribose-protein interactions. We also observe a significant population of a hitherto unobserved ribose-bound partially open state. We believe that this state is the one that has been suggested to play a role in the transfer of ribose to the membrane-bound permease complex.     

Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides

In addition to discriminating against base pair mismatches, DNA polymerases exhibit a high degree of selectivity for deoxyribonucleotides over ribo- or dideoxynucleotides. It has been proposed that a single active site residue (steric gate) blocks productive binding of nucleotides containing 2'-hydroxyls. Although this steric gate plays a role in sugar moiety discrimination, its interactions do not account fully for the observed behavior of mutants. Here we present 10 high resolution crystal structures and enzyme kinetic analyses of Bacillus DNA polymerase I large fragment variants complexed with deoxy-, ribo-, and dideoxynucleotides and a DNA substrate. Taken together, these data present a more nuanced and general mechanism for nucleotide discrimination in which ensembles of intermediate conformations in the active site trap non-cognate substrates. It is known that the active site O-helix transitions from an open state in the absence of nucleotide substrates to a ternary complex closed state in which the reactive groups are aligned for catalysis. Substrate misalignment in the closed state plays a fundamental part in preventing non-cognate nucleotide misincorpation. The structures presented here show that additional O-helix conformations intermediate between the open and closed state extremes create an ensemble of binding sites that trap and misalign non-cognate nucleotides. Water-mediated interactions, absent in the fully closed state, play an important role in formation of these binding sites and can be remodeled to accommodate different non-cognate substrates. This mechanism may extend also to base pair discrimination.     

Contributions of Ionic Interactions and Protein Dynamics to Cytochrome P450 2D6 (CYP2D6) Substrate and Inhibitor Binding

P450 2D6 contributes significantly to the metabolism of >15% of the 200 most marketed drugs. Open and closed crystal structures of P450 2D6 thioridazine complexes were obtained using different crystallization conditions. The protonated piperidine moiety of thioridazine forms a charge-stabilized hydrogen bond with Asp-301 in the active sites of both complexes. The more open conformation exhibits a second molecule of thioridazine bound in an expanded substrate access channel antechamber with its piperidine moiety forming a charge-stabilized hydrogen bond with Glu-222. Incubation of the crystalline open thioridazine complex with alternative ligands, prinomastat, quinidine, quinine, or ajmalicine, displaced both thioridazines. Quinine and ajmalicine formed charge-stabilized hydrogen bonds with Glu-216, whereas the protonated nitrogen of quinidine is equidistant from Asp-301 and Glu-216 with protonated nitrogen H-bonded to a water molecule in the access channel. Prinomastat is not ionized. Adaptations of active site side-chain rotamers and polypeptide conformations were evident between the complexes, with the binding of ajmalicine eliciting a closure of the open structure reflecting in part the inward movement of Glu-216 to form a hydrogen bond with ajmalicine as well as sparse lattice restraints that would hinder adaptations. These results indicate that P450 2D6 exhibits sufficient elasticity within the crystal lattice to allow the passage of compounds between the active site and bulk solvent and to adopt a more closed form that adapts for binding alternative ligands with different degrees of closure. These crystals provide a means to characterize substrate and inhibitor binding to the enzyme after replacement of thioridazine with alternative compounds.     

Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray structures of quinonoid intermediates

Amino acid transformations catalyzed by a number of pyridoxal 5'-phosphate (PLP)-dependent enzymes involve abstraction of the Calpha proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. Despite the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii tyrosine phenol-lyase with L-alanine and L-methionine in the crystalline state and determined their structures at 1.9- and 1.95-A resolution, respectively, by cryo-crystallography. The data reveal a network of protein-PLP-substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations, open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of phenol during the beta-elimination of L-tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys-257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Calpha proton.     

Probing the Periplasmic-Open State of Lactose Permease in Response to Sugar Binding and Proton Translocation

Based on the crystal structure of lactose permease (LacY) open to the cytoplasm, a hybrid molecular simulation approach with self-guided Langevin dynamics is used to describe conformational changes that lead to a periplasmic-open state. This hybrid approach consists of implicit (IM) and explicit (EX) membrane simulations and requires self-guided Langevin dynamics to enhance protein motions during the IM simulations. The pore radius of the lumen increases by 3.5 Å on the periplasmic side and decreases by 2.5 Å on the cytoplasmic side (relative to the crystal structure), suggesting a lumen that is fully open to the periplasm to allow for extracellular sugar transport and closed to the cytoplasm. Based on our simulations, the mechanism that triggers this conformational change to the periplasmic-open state is the protonation of Glu269 and binding of the disaccharide. Then, helix packing is destabilized by breaking of several side chains involved in hydrogen bonding (Asn245, Ser41, Glu374, Lys42, and Gln242). For the periplasmic-open conformations obtained from our simulations, helix-helix distances agree well with experimental measurements using double electron-electron resonance, fluorescence resonance energy transfer, and varying sized cross-linkers. The periplasmic-open conformations are also in compliance with various substrate accessibility/reactivity measurements that indicate an opening of the protein lumen on the periplasmic side on sugar binding. The comparison with these measurements suggests a possible incomplete closure of the cytoplasmic half in our simulations. However, the closure is sufficient to prevent the disaccharide from transporting to the cytoplasm, which is in accordance with the well-established alternating access model. Ser53, Gln60, and Phe354 are determined to be important in sugar transport during the periplasmic-open stage of the sugar transport cycle and the sugar is found to undergo an orientational change in order to escape the protein lumen.     

The crystal structure of rabbit phosphoglucose isomerase complexed with D-sorbitol-6-phosphate, an analog of the open chain form of D-glucose-6-phosphate

Phosphoglucose isomerase (PGI) catalyzes the isomerization of D-glucose-6-phosphate (G6P) and D-fructose-6-phosphate (F6P) in glycolysis and gluconeogenesis. Analysis of previously reported X-ray crystal structures of PGI without ligand, with the cyclic form of F6P, or with inhibitors that mimic the cis-enediol intermediate led to proposed mechanisms for the ring opening and isomerization steps in the multistep catalytic mechanism. To help complete our model of the overall mechanism, information is needed about the state of PGI between the ring opening and isomerization steps, in other words, a structure of the enzyme complexed with the open form of a substrate or an analog. Here, we report the crystal structure of rabbit PGI complexed with D-sorbitol-6-phosphate (S6P), an analog of the open chain form of G6P, at 2.0 A resolution. As was seen in the PGI/F6P structure, a helix containing amino acid residues 512-520 is found in the "out" position, which provides sufficient space in the active site for a substrate in its cyclic form and which is probably the location of that helix just after ring opening (or just before ring closure). However, the S6P ligand is in an extended conformation, as was seen previously with ligands that mimic the cis-enediol intermediate. The extended conformation enables the ligand to interact with Glu357, which transfers a proton during the isomerization step. The PGI/S6P structure represents the conformation of the enzyme and substrate between the ring opening (or ring closing) step and the isomerization step and helps to complete the model for PGI's catalytic mechanism.     

Crystal structure of human cytochrome P450 2D6 with prinomastat bound

Human cytochrome P450 2D6 contributes to the metabolism of >15% of drugs used in clinical practice. This study determined the structure of P450 2D6 complexed with a substrate and potent inhibitor, prinomastat, to 2.85 ? resolution by x-ray crystallography. Prinomastat binding is well defined by electron density maps with its pyridyl nitrogen bound to the heme iron. The structure of ligand-bound P450 2D6 differs significantly from the ligand-free structure reported for the P450 2D6 Met-374 variant (Protein Data Bank code 2F9Q). Superposition of the structures reveals significant differences for β sheet 1, helices A, F, F', G", G, and H as well as the helix B-C loop. The structure of the ligand complex exhibits a closed active site cavity that conforms closely to the shape of prinomastat. The closure of the open cavity seen for the 2F9Q structure reflects a change in the direction and pitch of helix F and introduction of a turn at Gly-218, which is followed by a well defined helix F' that was not observed in the 2F9Q structure. These differences reflect considerable structural flexibility that is likely to contribute to the catalytic versatility of P450 2D6, and this new structure provides an alternative model for in silico studies of substrate interactions with P450 2D6.     

Structural and spectroscopic characterization of P450 BM3 mutants with unprecedented P450 heme iron ligand sets. New heme ligation states influence conformational equilibria in P450 BM3

Two novel P450 heme iron ligand sets were generated by directed mutagenesis of the flavocytochrome P450 BM3 heme domain. The A264H and A264K variants produce Cys-Fe-His and Cys-Fe-Lys axial ligand sets, which were validated structurally and characterized by spectroscopic analysis. EPR and magnetic circular dichroism (MCD) provided fingerprints defining these P450 ligand sets. Near IR MCD spectra identified ferric low spin charge-transfer bands diagnostic of the novel ligands. For the A264K mutant, this is the first report of a Cys-Fe-Lys near-IR MCD band. Crystal structure determination showed that substrate-free A264H and A264K proteins crystallize in distinct conformations, as observed previously in substrate-free and fatty acid-bound wild-type P450 forms, respectively. This, in turn, likely reflects the positioning of the I alpha helix section of the protein that is required for optimal configuration of the ligands to the heme iron. One of the monomers in the asymmetric unit of the A264H crystals was in a novel conformation with a more open substrate access route to the active site. The same species was isolated for the wildtype heme domain and represents a novel conformational state of BM3 (termed SF2). The "locking" of these distinct conformations is evident from the fact that the endogenous ligands cannot be displaced by substrate or exogenous ligands. The consequent reduction of heme domain conformational heterogeneity will be important in attempts to determine atomic structure of the full-length, multidomain flavocytochrome, and thus to understand in atomic detail interactions between its heme and reductase domains.     

Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-A resolution: insight into the range of P450 conformations and the coordination of redox partner binding

A 1.9-A 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 beta(4) 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.     

Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4

The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form, P-420, involves a severe structural rearrangement in the heme binding pocket leading to drastic changes of the vinyl group conformations. The conformational differences of the active sites in cytochromes P-450 LM2 and LM4 observed in this work contribute to the understanding of the structural basis accounting for substrate and product specificity of cytochrome P-450 isozymes.     

Trapping conformational states along ligand-binding dynamics of peptide deformylase: the impact of induced fit on enzyme catalysis

For several decades, molecular recognition has been considered one of the most fundamental processes in biochemistry. For enzymes, substrate binding is often coupled to conformational changes that alter the local environment of the active site to align the reactive groups for efficient catalysis and to reach the transition state. Adaptive substrate recognition is a well-known concept; however, it has been poorly characterized at a structural level because of its dynamic nature. Here, we provide a detailed mechanism for an induced-fit process at atomic resolution. We take advantage of a slow, tight binding inhibitor-enzyme system, actinonin-peptide deformylase. Crystal structures of the initial open state and final closed state were solved, as well as those of several intermediate mimics captured during the process. Ligand-induced reshaping of a hydrophobic pocket drives closure of the active site, which is finally "zipped up" by additional binding interactions. Together with biochemical analyses, these data allow a coherent reconstruction of the sequence of events leading from the encounter complex to the key-lock binding state of the enzyme. A "movie" that reconstructs this entire process can be further extrapolated to catalysis.     

Plasticity of cytochrome P450 2B4 as investigated by hydrogen-deuterium exchange mass spectrometry and X-ray crystallography

Crystal structures of the xenobiotic metabolizing cytochrome P450 2B4 have demonstrated markedly different conformations in the presence of imidazole inhibitors or in the absence of ligand. However, knowledge of the plasticity of the enzyme in solution has remained scant. Thus, hydrogen-deuterium exchange mass spectrometry (DXMS) was utilized to probe the conformations of ligand-free P450 2B4 and the complex with 4-(4-chlorophenyl)imidazole (4-CPI) or 1-biphenyl-4-methyl-1H-imidazole (1-PBI). The results of DXMS indicate that the binding of 4-CPI slowed the hydrogen-deuterium exchange rate over the B'- and C-helices and portions of the F-G-helix cassette compared with P450 2B4 in the absence of ligands. In contrast, there was little difference between the ligand-free and 1-PBI-bound exchange sets. In addition, DXMS suggests that the ligand-free P450 2B4 is predominantly open in solution. Interestingly, a new high resolution structure of ligand-free P450 2B4 was obtained in a closed conformation very similar to the 4-CPI complex. Molecular dynamics simulations performed with the closed ligand-free structure as the starting point were used to probe the energetically accessible conformations of P450 2B4. The simulations were found to equilibrate to a conformation resembling the 1-PBI-bound P450 2B4 crystal structure. The results indicate that conformational changes observed in available crystal structures of the promiscuous xenobiotic metabolizing cytochrome P450 2B4 are consistent with its solution structural behavior.     

Ketoconazole-induced conformational changes in the active site of cytochrome P450eryF

The azole-based P450 inhibitor ketoconazole is used to treat fungal infections and functions by blocking ergosterol biosynthesis in yeast. Ketoconazole binds to mammalian P450 enzymes and this can result in drug-drug interactions and lead to liver damage. To identify protein-drug interactions that contribute to binding specificity and affinity, we determined the crystal structure of ketoconazole complexed with P450eryF. In the P450eryF/ketoconazole structure, the azole moiety and nearby rings of ketoconzole are positioned in the active site similar to the substrate, 6-deoxyerythronolide B, with the azole nitrogen atom coordinated to the heme iron atom. The remainder of the ketoconazole molecule extends into the active-site pocket, which is occupied by water in the substrate complex. Binding of ketoconazole led to unexpected conformational changes in the I-helix. The I-helix cleft near the active site has collapsed with a helical pitch of 5.4 A compared to 6.6 A in the substrate complex. P450eryF/ketoconazole crystals soaked in 6-deoxyerythronolide B to exchange ligands exhibit a structure identical with that of the original P450eryF/substrate complex, with the I-helix cleft restored to a pitch of 6.6 A. These findings indicate that the I-helix region of P450eryF is flexible and can adopt multiple conformations. An improved understanding of the flexibility of the active-site region of cytochrome P450 enzymes is important to gain insight into determinants of ligand binding/specificity as well as to evaluate models for catalytic mechanism based on static crystal structures.     

Allosteric transitions in cytochrome P450eryF explored with pressure-perturbation spectroscopy, lifetime FRET, and a novel fluorescent substrate, Fluorol-7GA

To establish a direct method for monitoring substrate binding in cytochrome P450eryF applicable at elevated hydrostatic pressures, we introduce a laser dye Fluorol-7GA (F7GA) as a novel fluorescent ligand. The high intensity of fluorescence and the reasonable resolution of the excitation band from the absorbance bands of P450 allowed us to establish highly sensitive binding assays compatible with pressure perturbation. The interactions of F7GA with P450eryF cause an ample spin shift revealing cooperative binding ( S50 = 8.2 +/- 1.3 microM; n = 2.3 +/- 0.1). Fluorescence resonance energy transfer (FRET) experiments suggest the presence of at least two substrate binding sites with apparent K D values in the ranges of 0.1-0.3 and 6-9 microM. Similar to that observed earlier with CYP3A4, increasing hydrostatic pressure does not cause either a complete dissociation of the substrate complexes or a displacement of the spin equilibrium toward the low-spin state. Rather, increased pressure enhances the cooperativity of the F7GA-induced spin shift, so that the Hill coefficient approaches 3 at 2 kbar. Lifetime FRET experiments revealed an important increase in the affinity of the enzyme for F7GA at elevated pressures, suggesting that the binding of the ligand induces a conformational transition associated with an important increase in the level of protein hydration. This transition largely attenuates the solvent accessibility of the heme pocket and causes an unusual stability of the high-spin, substrate-bound enzyme at elevated pressures.     

Human Cytochrome P450 17A1 Conformational Selection: MODULATION BY LIGAND AND CYTOCHROME b5*

Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1). This bifunctional enzyme is responsible for pregnenolone C17 hydroxylation, followed by a 17,20-lyase reaction to produce dehydroepiandrosterone, the key intermediate in human synthesis of androgen and estrogen sex steroids. The distribution of CYP17A1 conformational states was influenced by temperature, binding of these two substrates, and binding of the soluble domain of cytochrome b₅ (b₅). Notably, titration of b₅ to CYP17A1·pregnenolone induced a set of conformational states closely resembling those of CYP17A1·17α-hydroxypregnenolone without b_5, providing structural evidence consistent with the reported ability of b₅ to selectively enhance 17,20-lyase activity. Solution NMR thus revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating that this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states.     

A single mutation in cytochrome P450 BM3 induces the conformational rearrangement seen upon substrate binding in the wild-type enzyme

The multidomain fatty-acid hydroxylase flavocytochrome P450 BM3 has been studied as a paradigm model for eukaryotic microsomal P450 enzymes because of its homology to eukaryotic family 4 P450 enzymes and its use of a eukaryotic-like diflavin reductase redox partner. High-resolution crystal structures have led to the proposal that substrate-induced conformational changes lead to removal of water as the sixth ligand to the heme iron. Concomitant changes in the heme iron spin state and heme iron reduction potential help to trigger electron transfer from the reductase and to initiate catalysis. Surprisingly, the crystal structure of the substrate-free A264E heme domain mutant reveals the enzyme to be in the conformation observed for substrate-bound wild-type P450, but with the iron in the low-spin state. This provides strong evidence that the spin-state shift observed upon substrate binding in wild-type P450 BM3 not only is caused indirectly by structural changes in the protein, but is a direct consequence of the presence of the substrate itself, similar to what has been observed for P450cam. The crystal structure of the palmitoleate-bound A264E mutant reveals that substrate binding promotes heme ligation by Glu(264), with little other difference from the palmitoleate-bound wild-type structure observable. Despite having a protein-derived sixth heme ligand in the substrate-bound form, the A264E mutant is catalytically active, providing further indication for structural rearrangement of the active site upon reduction of the heme iron, including displacement of the glutamate ligand to allow binding of dioxygen.