Cooperative binding of acetaminophen and caffeine within the P450 3A4 active site.

Acetaminophen (N-acetyl-p-aminophenol, APAP) is a commonly used analgesic/antipyretic. When oxidized by P450, a toxic APAP metabolite is generated. Human P450 3A4 was expressed in Escherichia coli , purified, and reconstituted using artificial liposomes. Oxidation of APAP by P450 3A4, as detected by the formation of its glutathione adduct, was found to exhibit negative homotropic cooperativity with a Hill coefficient of 0.7. In the presence of caffeine, the observed kinetics were close to classical Michaelis-Menten kinetics with a Hill coefficient approaching 1. In order to probe for a potential repositioning of APAP within the P450 3A4 pocket in the presence of caffeine, NMR T1 paramagnetic relaxation techniques were used to calculate distances from the P450 3A4 heme iron to protons of APAP alone and in the presence of caffeine. Both APAP and caffeine were found to bind at the active site in proximity to the heme iron. When APAP was incubated with P450 3A4, the acetamido group of APAP was found to be closest to the heme iron consistent with the amide group of APAP weakly associating with the heme iron. The addition of caffeine disrupted the ability of APAP to coordinate with the heme iron of P450 3A4 and enhanced the rate of oxidation to its toxic metabolite.

NMR studies of ligand binding to P450(eryF) provides insight into the mechanism of cooperativity.

Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins. Although extensive studies have proven that some P450's demonstrate both homotropic and heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450(eryF). UV/vis absorbance spectra of P450(eryF) demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of K(S) = 4 and 200 microM, respectively. The broadening and shifting observed in the 2D-{1H,15N}-HSQC-monitored titrations of 15N-Phe-labeled P450(eryF) with 9-AP and TST indicated binding on intermediate and fast chemical exchange time scales, respectively, which was consistent with the Hill-equation-derived K(S) values for these two ligands. Regardless of the type of spectral perturbation observed (broadening for 9-AP and shifting for TST), the 15N-Phe NMR resonances most affected were the same in each titration, suggesting that the two ligands "contact" the same phenylalanines within the active site of P450(eryF). This finding is in agreement with X-ray crystal structures of bound P450(eryF) showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the low-affinity TST molecule or multiple TST-bound P450(eryF) conformational substates. A structural and energetic model is presented that combines the energetics and structural aspects of 9-AP and TST binding derived from these observations.

Sites of covalent attachment of CYP4 enzymes to heme: evidence for microheterogeneity of P450 heme orientation.

Typical cytochrome P450s secure the heme prosthetic group with a cysteine thiolate ligand bound to the iron, electrostatic interactions with the heme propionate carboxylates, and hydrophobic interactions with the heme periphery. In addition to these interactions, CYP4B1 covalently binds heme through a monoester link furnished, in part, by a conserved I-helix acid, Glu310. Chromatography, mass spectrometry, and NMR have now been utilized to identify the site of attachment on the heme. Native CYP4B1 covalently binds heme solely at the C-5 methyl position. Unexpectedly, recombinant CYP4B1 from insect cells and Escherichia coli also bound their heme covalently at the C-8 methyl position. Structural heterogeneity may be common among recombinant CYP4 proteins because CYP4A3 exhibited this duality. Attempts to evaluate functional heterogeneity were complicated by the complexity of the system. The phenomenon of covalent heme binding to P450 provides a novel method for assessing microheterogeneity in heme orientation and raises questions about the fidelity of heme incorporation in recombinant systems.

Cooperative binding of midazolam with testosterone and alpha-naphthoflavone within the CYP3A4 active site: a NMR T1 paramagnetic relaxation study.

Recent studies have indicated that CYP3A4 exhibits non-Michaelis-Menten kinetics for numerous substrates. Both homo- and heterotropic activation have been reported, and kinetic models have suggested multiple substrates within the active site. We provide some of the first physicochemical data supporting the hypothesis of allosteric substrate binding within the CYP3A4 active site. Midazolam (MDZ) is metabolized by CYP3A4 to two hydroxylated metabolites, 1'- and 4-hydroxymidazolam. Incubations using purified CYP3A4 and MDZ showed that both alpha-naphthoflavone (alpha-NF) and testosterone affect the ratio of formation rates of 1'- and 4-hydroxymidazolam. Similar to previous reports, alpha-NF was found to promote formation of 1'-hydroxymidazolam, while testosterone stimulated formation of 4-hydroxymidazolam. NMR was used to measure the closest approach of individual MDZ protons to the paramagnetic heme iron of CYP3A4 using paramagnetic T(1) relaxation measurements. Solutions of 0.2 microM CYP3A4 with 500 microM MDZ resulted in calculated distances between 7.4 and 8.3 A for all monitored MDZ protons. The distances were statistically equivalent for all protons except C3-H and were consistent with the rotation within the active site or sliding parallel to the heme plane. When 50 microM alpha-NF was added, proton-heme iron distances ranged from 7.3 to 10.0 A. Consistent with kinetics of activation, the 1' position was situated closest to the heme, while the fluorophenyl 5-H proton was the furthest. Proton-heme iron distances for MDZ with CYP3A4 and 50 microM testosterone ranged from 7.7 to 9.0 A, with the flourophenyl 5-H proton furthest from the heme iron and the C4-H closest to the heme, also consistent with kinetic observations. When titrated with CYP3A4 in the presence of MDZ, testosterone and alpha-NF resonances themselves exhibited significant broadening and enhanced relaxation rates, indicating that these effector molecules were also bound within the CYP3A4 active site near the paramagnetic heme iron. These results suggest that the effector exerts its cooperative effects on MDZ metabolism through simultaneous binding of MDZ and effector near the CYP3A4 heme.

The thermodynamic landscape of testosterone binding to cytochrome P450 3A4: ligand binding and spin state equilibria.

Human cytochrome P450 (CYP) 3A4 catalyzes the oxygen-dependent metabolism of greater than 60% of known drugs. CYP3A4 binds multiple ligands simultaneously, and this contributes to complex allosteric kinetic behavior. Substrates that bind to this enzyme change the ferric spin state equilibrium of the heme, which can be observed by optical absorbance and electron paramagnetic resonance (EPR) spectroscopy. The ligand-dependent spin state equilibrium has not been quantitatively understood for any ligands that exhibit multiple binding. The CYP3A4 substrate testosterone (TST) has been shown previously by absorbance spectroscopy to induce spin state changes that are characteristic of a low spin to high spin conversion. Here, EPR was used to examine the equilibrium binding of TST to CYP3A4 at [CYP3A4] > K(D), which allows for characterization of the singly occupied state (i.e., CYP3A4.TST). We also have used absorbance spectroscopy to examine equilibrium binding, where [CYP3A4] < K(D), which allows for determination of K(D)'s. The combination of absorbance and EPR spectroscopy at different CYP3A4 concentrations relative to K(D) and curve fitting of the resultant equilibrium binding titration curves to the Adair-Pauling equations, and modifications of it, reveals that the first equivalent of TST binds with higher affinity than the second equivalent of TST and its binding is positively cooperative with respect to ligand-dependent spin state conversion. Careful analysis of the EPR and absorbance spectral results suggests that the binding of the second TST induces a shift to the high spin state and thus that the second TST binding causes displacement of the bound water. A model involving six thermodynamic states is presented and this model is related to the turnover of the enzyme.

Structural and dynamic consequences of increasing repeats in a MUC1 peptide tumor antigen.

MUC1 mucin is a large transmembrane glycoprotein whose extracelluler domain is composed of repeating units of a 20 amino acid sequence. In the cancer associated state, this protein expression becomes upregulated and underglycosylated. Previous studies, which show an enhanced binding of a 5-repeat over a 1-repeat MUC1 peptide to a panel of anti-MUC1 antibodies, have led us to investigate the structural and dynamic consequences of increasing repeat number. Two MUC1 peptides were studied: a 16mer corresponding to slightly less than one full repeat of the MUC1 tandem repeat sequence (GVTSAPDTRPAPGSTA) and a 40mer corresponding to two full repeats of the MUC1 sequence (VTSAPDTRPAPGSTAPPAHG)2. Isotopically labeled versions of these MUC1 peptides were cloned, expressed, purified, and evaluated structurally and dynamically using 15N- and 13C-edited NMR approaches. The data show that MUC1 structure, dynamics, and antibody binding affinity are invariant with increasing repeat number. In light of these results, we conclude that the enhanced antibody affinity of the 5-repeat over the 1-repeat MUC1 peptide is due to multivalency effects, and not due to the development of higher order structure in the longer length peptides. The implications of these results are discussed within the context of a multiple repeat MUC1 breast cancer vaccine design.

"Allosterism" in the elementary steps of the cytochrome P450 reaction cycle.

Cytochrome P450-dependent drug metabolism in vitro frequently deviates from simple Michaelis-Menten kinetic models, and demonstrates both positive and negative homotropic and heterotropic effects. These complex "allsoteric" kinetics confound our ability to predict drug clearance, and they may provide a basis for drug-drug interactions. Although allosteric effects require that multiple substrates, or substrate and effector, are simultaneously bound to a cytochrome P450 (CYP), the mechanisms by which multiple ligand binding alters rates of individual steps in the CYP reaction cycle are incompletely characterized. In addition, it is unknown whether multiple ligands bind in discrete subsites within the large active site or whether they share a fluid dynamic site. These mechanistic aspects of multiple drug binding are addressed here via several spectroscopic probes including ultraviolet-vis difference spectroscopy, protein and ligand fluorescence, and 15N-edited HSQC nuclear magnetic resonance (NMR) with 15N-Phe-labeled CYPs. The results indicate a lack of correspondence between ligand binding per se and the ligand-dependent home spin state change when multiple ligands bind. Furthermore, the results provide proof of principle for NMR as a method for studying CYP allosterism, and demonstrate that the model ligand 9-aminophenanthrene binds in two discrete events to individual subsites within the active site of CYP(eryF).

Epitope mapping of antigenic MUC1 peptides to breast cancer antibody fragment B27.29: a heteronuclear NMR study.

MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant "reverse templates" of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], (1)H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including (15)N and (13)C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)(2). (15)N and (13)C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The (13)C(alpha) T(1) values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the (15)N- and (13)C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast cancer vaccine design.

Interaction of a peptide from the receptor-binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: changes in backbone dynamics induced by binding.

The C-terminal receptor-binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P. aeruginosa infections. We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein. The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond. The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142). The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135). These turns have been implicated in cross-reactive antibody recognition. (15)N NMR relaxation experiments of the (15)N-labeled recombinant PAK pilin peptide in complex with an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus, were performed in order to probe for changes in the mobilities and dynamics of the peptide backbone as a result of antibody binding. The major results of these studies are as follows: binding of Fab leads to the preferential ordering of the first turn over the second turn in each isomer, binding of Fab partially stabilizes peptide backbone regions undergoing slow (microsecond to millisecond) exchange-related motions, and binding of Fab leads to a greater loss in backbone conformational entropy at pH 7.2 versus pH 4.5. The biological implications of these results will be discussed in relation to the role that fast and slow backbone motions play in PAK pilin peptide immunogenicity and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains.

Effect of glycosylation on MUC1 humoral immune recognition: NMR studies of MUC1 glycopeptide-antibody interactions.

MUC1 mucin is a large transmembrane glycoprotein, of which the extracellular domain is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is underglycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above) as well as the normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc), and TF (Gal beta1-3 GalNAc) carbohydrates. In the present study, NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRP core epitope region to the recognition and binding of a monoclonal antibody, Mab B27.29, raised against the intact tumor-associated MUC1 mucin. Four peptides were studied: a MUC1 16mer peptide of the sequence Gly1-Val2-Thr3-Ser4-Ala5-Pro6-Asp7-Thr8-Arg9-Pro10-Ala11-Pro12-Gly13-Ser14-Thr15-Ala16, two singly Tn-glycosylated versions of this peptide at either Thr3 or Ser4, and a doubly Tn-glycosylated version at both Thr3 and Ser4. The results of these studies showed that the B27.29 MUC1 B-cell epitope maps to two separate parts of the glycopeptide, the core peptide epitope spanning the PDTRP sequence and a second (carbohydrate) epitope comprised of the Tn moieties attached at Thr3 and Ser4. The implications of these results are discussed within the framework of developing a glycosylated second-generation MUC1 glycopeptide vaccine.