Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry.

Neuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The binding of calmodulin (CaM) to a linker region between the FAD/FMN-containing reductase domain, and the heme-containing oxygenase domain is needed for electron transfer reactions, reduction of the heme, and NO synthesis. Due to the dynamic nature of the reductase domain and low resolution of available full-length structures, the exact conformation of the CaM-bound active complex during heme reduction is still unresolved. Interestingly, hydrogen-deuterium exchange and mass spectrometry studies revealed interactions of the FMN domain and CaM with the oxygenase domain for iNOS, but not nNOS. This finding prompted us to utilize covalent crosslinking and mass spectrometry to clarify interactions of CaM with nNOS. Specifically, MS-cleavable bifunctional crosslinker disuccinimidyl dibutyric urea was used to identify thirteen unique crosslinks between CaM and nNOS as well as 61 crosslinks within the nNOS. The crosslinks provided evidence for CaM interaction with the oxygenase and reductase domain residues as well as interactions of the FMN domain with the oxygenase dimer. Cryo-EM studies, which gave a high-resolution model of the oxygenase domain, along with crosslink-guided docking provided a model of nNOS that brings the FMN within 15 Å of the heme in support for a more compact conformation than previously observed. These studies also point to the utility of covalent crosslinking and mass spectrometry in capturing transient dynamic conformations that may not be captured by hydrogen-deuterium exchange and mass spectrometry experiments.

Dynamic cycling with a unique Hsp90/Hsp70-dependent chaperone machinery and GAPDH is needed for heme insertion and activation of neuronal NO synthase.

Heat shock protein 90 (Hsp90) is known to mediate heme insertion and activation of heme-deficient neuronal nitric oxide (NO) synthase (apo-nNOS) in cells by a highly dynamic interaction that has been extremely difficult to study mechanistically with the use of subcellular systems. In that the heme content of many critical hemeproteins is regulated by Hsp90 and the heme chaperone GAPDH, the development of an in vitro system for the study of this chaperone-mediated heme regulation would be extremely useful. Here, we show that use of an antibody-immobilized apo-nNOS led not only to successful assembly of chaperone complexes but the ability to show a clear dependence on Hsp90 and GAPDH for heme-mediated activation of apo-nNOS. The kinetics of binding for Hsp70 and Hsp90, the ATP and K+ dependence, and the absolute requirement for Hsp70 in assembly of Hsp90•apo-nNOS heterocomplexes all point to a similar chaperone machinery to the well-established canonical machine regulating steroid hormone receptors. However, unlike steroid receptors, the use of a purified protein system containing Hsp90, Hsp70, Hsp40, Hop, and p23 is unable to activate apo-nNOS. Thus, heme insertion requires a unique Hsp90-chaperone complex. With this newly developed in vitro system, which recapitulates the cellular process requiring GAPDH as well as Hsp90, further mechanistic studies are now possible to better understand the components of the Hsp90-based chaperone system as well as how this heterocomplex works with GAPDH to regulate nNOS and possibly other hemeproteins.

Computational redesign of cytochrome P450 CYP102A1 for highly stereoselective omeprazole hydroxylation by UniDesign.

Cytochrome P450 CYP102A1 is a prototypic biocatalyst that has great potential in chemical synthesis, drug discovery, and biotechnology. CYP102A1 variants engineered by directed evolution and/or rational design are capable of catalyzing the oxidation of a wide range of organic compounds. However, it is difficult to foresee the outcome of engineering CYP102A1 for a compound of interest. Here, we introduce UniDesign as a computational framework for enzyme design and engineering. We tested UniDesign by redesigning CYP102A1 for stereoselective metabolism of omeprazole (OMP), a proton pump inhibitor, starting from an active but nonstereoselective triple mutant (TM: A82F/F87V/L188Q). To shift stereoselectivity toward (R)-OMP, we computationally scanned three active site positions (75, 264, and 328) for mutations that would stabilize the binding of the transition state of (R)-OMP while destabilizing that of (S)-OMP and picked three variants, namely UD1 (TM/L75I), UD2 (TM/A264G), and UD3 (TM/A328V), for experimentation, based on computed energy scores and models. UD1, UD2, and UD3 exhibit high turnover rates of 55 ± 4.7, 84 ± 4.8, and 79 ± 5.7 min-1, respectively, for (R)-OMP hydroxylation, whereas the corresponding rates for (S)-OMP are only 2.2 ± 0.19, 6.0 ± 0.68, and 14 ± 2.8 min-1, yielding an enantiomeric excess value of 92, 87, and 70%, respectively. These results suggest the critical roles of L75I, A264G, and A328V in steering OMP in the optimal orientation for stereoselective oxidation and demonstrate the utility of UniDesign for engineering CYP102A1 to produce drug metabolites of interest. The results are discussed in the context of protein structures.

The Versatile Biocatalyst of Cytochrome P450 CYP102A1: Structure, Function, and Engineering.

Wild-type cytochrome P450 CYP102A1 from Bacillus megaterium is a highly efficient monooxygenase for the oxidation of long-chain fatty acids. The unique features of CYP102A1, such as high catalytic activity, expression yield, regio- and stereoselectivity, and self-sufficiency in electron transfer as a fusion protein, afford the requirements for an ideal biocatalyst. In the past three decades, remarkable progress has been made in engineering CYP102A1 for applications in drug discovery, biosynthesis, and biotechnology. The repertoire of engineered CYP102A1 variants has grown tremendously, whereas the substrate repertoire is avalanched to encompass alkanes, alkenes, aromatics, organic solvents, pharmaceuticals, drugs, and many more. In this article, we highlight the major advances in the past five years in our understanding of the structure and function of CYP102A1 and the methodologies used to engineer CYP102A1 for novel applications. The objective is to provide a succinct review of the latest developments with reference to the body of CYP102A1-related literature.

Amphipol-facilitated elucidation of the functional tetrameric complex of full-length cytochrome P450 CYP2B4 and NADPH-cytochrome P450 oxidoreductase.

Interactions of membrane-bound mammalian cytochromes P450 (CYPs) with NADPH-cytochrome P450 oxidoreductase (POR), which are required for metabolism of xenobiotics, are facilitated by membrane lipids. A variety of membrane mimetics, such as phospholipid liposomes and nanodiscs, have been used to simulate the membrane to form catalytically active CYP:POR complexes. However, the exact mechanism(s) of these interactions are unclear because of the absence of structural information of full-length mammalian CYP:POR complexes in membranes. Herein, we report the use of amphipols (APols) to form a fully functional, soluble, homogeneous preparation of full-length CYP:POR complexes amenable to biochemical and structural study. Incorporation of CYP2B4 and POR into APols resulted in a CYP2B4:POR complex with a stoichiometry of 1:1, which was fully functional in demethylating benzphetamine at a turnover rate of 37.7 ± 2.2 min-1, with a coupling efficiency of 40%. Interestingly, the stable complex had a molecular weight (Mw) of 338 ± 22 kDa determined by multiangle light scattering, suggestive of a tetrameric complex of 2CYP2B4:2POR embedded in one APol nanoparticle. Moreover, negative stain electron microscopy (EM) validated the homogeneity of the complex and allowed us to generate a three-dimensional EM map and model consistent with the tetramer observed in solution. This first report of the full-length mammalian CYP:POR complex by transmission EM not only reveals the architecture that facilitates electron transfer but also highlights a potential use of APols in biochemical and structural studies of functional CYP complexes with redox partners.

Cryo-EM reveals the architecture of the dimeric cytochrome P450 CYP102A1 enzyme and conformational changes required for redox partner recognition.

Cytochrome P450 family 102 subfamily A member 1 (CYP102A1) is a self-sufficient flavohemeprotein and a highly active bacterial enzyme capable of fatty acid hydroxylation at a >3,000 min-1 turnover rate. The CYP102A1 architecture has been postulated to be responsible for its extraordinary catalytic prowess. However, the structure of a functional full-length CYP102A1 enzyme remains to be determined. Herein, we used a cryo-EM single-particle approach, revealing that full-length CYP102A1 forms a homodimer in which both the heme and FAD domains contact each other. The FMN domain of one monomer was located close to the heme domain of the other monomer, exhibiting a trans configuration. Moreover, full-length CYP102A1 is highly dynamic, existing in multiple conformational states, including open and closed states. In the closed state, the FMN domain closely contacts the FAD domain, whereas in the open state, one of the FMN domains rotates away from its FAD domain and traverses to the heme domain of the other monomer. This structural arrangement and conformational dynamics may facilitate rapid intraflavin and trans FMN-to-heme electron transfers (ETs). Results with a variant having a 12-amino-acid deletion in the CYP102A1 linker region, connecting the catalytic heme and the diflavin reductase domains, further highlighted the importance of conformational dynamics in the ET process. Cryo-EM revealed that the Δ12 variant homodimer is conformationally more stable and incapable of FMN-to-heme ET. We conclude that closed-to-open alternation is crucial for redox partner recognition and formation of an active ET complex for CYP102A1 catalysis.

Targeting Hsp70 facilitated protein quality control for treatment of polyglutamine diseases.

The polyglutamine (polyQ) diseases are a group of nine fatal, adult-onset neurodegenerative disorders characterized by the misfolding and aggregation of mutant proteins containing toxic expansions of CAG/polyQ tracts. The heat shock protein 90 and 70 (Hsp90/Hsp70) chaperone machinery is a key component of cellular protein quality control, playing a role in the regulation of folding, aggregation, and degradation of polyQ proteins. The ability of Hsp70 to facilitate disaggregation and degradation of misfolded proteins makes it an attractive therapeutic target in polyQ diseases. Genetic studies have demonstrated that manipulation of Hsp70 and related co-chaperones can enhance the disaggregation and/or degradation of misfolded proteins in models of polyQ disease. Therefore, the development of small molecules that enhance Hsp70 activity is of great interest. However, it is still unclear if currently available Hsp70 modulators can selectively enhance disaggregation or degradation of misfolded proteins without perturbing other Hsp70 functions essential for cellular homeostasis. This review discusses the multifaceted role of Hsp70 in protein quality control and the opportunities and challenges Hsp70 poses as a potential therapeutic target in polyQ disease.

Hsp70:CHIP Ubiquitinates Dysfunctional but Not Native Neuronal NO Synthase.

Heat shock protein (Hsp) 70 modulators are being developed to enhance the removal of toxic proteins in a variety of protein misfolding diseases. In the course of our studies on neuronal nitric oxide synthase (nNOS), a client of the Hsp90 and Hsp70 chaperone system, we have established that inactivation of nNOS by heme or tetrahydrobiopterin (BH4) alteration and loss triggers ubiquitination by the Hsp70-associated E3 ligase c-terminus of Hsp70-interacting protein (CHIP) and subsequent degradation in cells. Although in cells Hsp90 and Hsp70 work together to maintain protein quality control, in this study, we specifically developed an assay to assess the selectivity of the Hsp70:CHIP complex for inactivated nNOS. We developed a highly sensitive ELISA to measure Hsp70:CHIP-dependent nNOS ubiquitination without interference from direct ubiquitination by CHIP, as evidenced by Bcl-2 associated athanogene 1-M completely abolishing ubiquitination. To further validate the assay we demonstrated, JG-98, a rhodocyanin compound that acts on Hsp70 but not its inactive structural analog JG-258, enhances the ubiquitination of nNOS 3-fold. Utilizing this assay, we have shown that the Hsp70:CHIP complex preferentially ubiquitinates heme-deficient nNOS (apo-nNOS) over heme-containing nNOS (holo-nNOS). Moreover, depletion of nNOS-bound BH4 triggers ubiquitination of holo-nNOS by the Hsp70:CHIP complex. Most importantly, JG-98 was shown to enhance the ubiquitination of only dysfunctional nNOS while leaving the native functional nNOS untouched. Thus, the finding that enhancing Hsp70:CHIP-mediated ubiquitination does not affect native proteins has important pharmacological implications. Moreover, development of a facile in vitro method for Hsp70:CHIP-mediated ubiquitination will be beneficial for testing other Hsp70 modulators. SIGNIFICANCE STATEMENT: The heat shock protein 70 (Hsp70):c-terminus of Hsp70-interacting protein (CHIP) complex facilitates the ubiquitination and subsequent degradation of several hundred-client proteins, and activation of Hsp70 has been suggested as a therapeutic strategy to enhance the degradation of disease-causing proteins. The current study shows that the pharmacological activation of Hsp70 enhances the ubiquitination of dysfunctional but not native nNOS, and it suggests that this therapeutic strategy will likely be highly selective.

The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis.

The cytochrome P450 enzyme CYP102A1 from Bacillus megaterium is a highly efficient hydroxylase of fatty acids, and there is a significant interest in using CYP102A1 for biotechnological applications. Here, we used size-exclusion chromatography-multiangle light scattering (SEC-MALS) analysis and negative-stain EM to investigate the molecular architecture of CYP102A1. The SEC-MALS analysis yielded a homogeneous peak with an average molecular mass of 235 ± 5 kDa, consistent with homodimeric CYP102A1. The negative-stain EM of dimeric CYP102A1 revealed four distinct lobes, representing the two heme and two reductase domains. Two of the lobes were in close contact, whereas the other two were often observed apart and at the ends of a U-shaped configuration. The overall dimension of the dimer was ∼130 Å. To determine the identity of the lobes, we FLAG-tagged the N or C terminus of CYP102A1 to visualize additional densities in EM and found that anti-FLAG Fab could bind only the N-tagged P450. Single-particle analysis of this anti-Flag Fab-CYP102A1 complex revealed additional density in the N-terminally tagged heme domains, indicating that the heme domains appear flexible, whereas the reductase domains remain tightly associated. The effects of truncation on CYP102A1 dimerization, identification of cross-linked sites by peptide mapping, and molecular modeling results all were consistent with the dimerization of the reductase domain. We conclude that functional CYP102A1 is a compact globular protein dimerized at its reductase domains, with its heme domains exhibiting multiple conformations that likely contribute to the highly efficient catalysis of CYP102A1.

Chaperone Activity and Dimerization Properties of Hsp90α and Hsp90ß in Glucocorticoid Receptor Activation by the Multiprotein Hsp90/Hsp70-Dependent Chaperone Machinery.

Several hundred proteins cycle into heterocomplexes with a dimer of the chaperone heat shock protein 90 (Hsp90), regulating their activity and turnover. There are two isoforms of Hsp90, Hsp90α and Hsp90ß, and their relative chaperone activities and composition in these client protein•Hsp90 heterocomplexes has not been determined. Here, we examined the activity of human Hsp90α and Hsp90ß in a purified five-protein chaperone machinery that assembles glucocorticoid receptor (GR)•Hsp90 heterocomplexes to generate high-affinity steroid-binding activity. We found that human Hsp90α and Hsp90ß have equivalent chaperone activities, and when mixed together in this assay, they formed only GR•Hsp90αα and GR•Hsp90ßß homodimers and no GR•Hsp90αß heterodimers. In contrast, GR•Hsp90 heterocomplexes formed in human embryonic kidney (HEK) cells also contain GR•Hsp90αß heterodimers. The formation of GR•Hsp90αß heterodimers in HEK cells probably reflects the longer time permitted for exchange to form Hsp90αß heterodimers in the cell versus in the cell-free assembly conditions. This purified GR-activating chaperone machinery can be used to determine how modifications of Hsp90 affect its chaperone activity. To that effect, we have tested whether the unique phosphorylation of Hsp90α at threonines 5 and 7 that occurs during DNA damage repair affects its chaperone activity. We showed that the phosphomimetic mutant Hsp90α T5/7D has the same intrinsic chaperone activity as wild-type human Hsp90α in activation of GR steroid-binding activity by the five-protein machinery, supporting the conclusion that T5/7 phosphorylation does not affect Hsp90α chaperone activity.

Targeting Hsp90/Hsp70-based protein quality control for treatment of adult onset neurodegenerative diseases.

Currently available therapies for adult onset neurodegenerative diseases provide symptomatic relief but do not modify disease progression. Here we explore a new neuroprotective approach based on drugs targeting chaperone-directed protein quality control. Critical target proteins that unfold and aggregate in these diseases, such as the polyglutamine androgen receptor in spinal and bulbar muscular atrophy, huntingtin in Huntington's disease, α-synuclein in Parkinson's disease, and tau in Alzheimer's disease, are client proteins of heat shock protein 90 (Hsp90), and their turnover is regulated by the protein quality control function of the Hsp90/Hsp70-based chaperone machinery. Hsp90 and Hsp70 have opposing effects on client protein stability in protein quality control; Hsp90 stabilizes the clients and inhibits their ubiquitination, whereas Hsp70 promotes ubiquitination dependent on CHIP (C terminus of Hsc70-interacting protein) and proteasomal degradation. We discuss how drugs that modulate proteostasis by inhibiting Hsp90 function or promoting Hsp70 function enhance the degradation of the critical aggregating proteins and ameliorate toxic symptoms in cell and animal disease models.

Significant Improvement of Antithrombotic Responses to Clopidogrel by Use of a Novel Conjugate as Revealed in an Arterial Model of Thrombosis.

Clopidogrel is a prodrug that requires bioactivation by cytochrome P450 (P450) enzymes to a pharmacologically active metabolite for antiplatelet action. The clinical limitations of clopidogrel are in large part due to its poor pharmacokinetics resulting from inefficient bioactivation by P450s. In this study, we determined the pharmacokinetics and pharmacodynamics of a novel conjugate of clopidogrel, referred to as ClopNPT, in animal models and we evaluated its potential to overcome the limitations of clopidogrel. Results from pharmacokinetic (PK) studies showed that ClopNPT released the active metabolite with a time to maximal plasma concentration of <5 minutes in C57BL/6 mice after either oral or intravenous administration, and plasma concentrations of the active metabolite reached Cmax values of 1242 and 1100 ng/ml after a 10-mg/kg oral dose and a 5-mg/kg intravenous dose, respectively. Furthermore, ClopNPT was highly effective in preventing arterial thrombosis in rabbits and mice after vascular injuries. Formation of occlusive thrombi was prevented by ClopNPT at the 1-mg/kg dose with no significant increase in tongue bleeding time, whereas clopidogrel was ineffective at the same dose. These results suggest that ClopNPT has favorable PK/pharmacodynamic properties that can potentially overcome the attenuated PK properties of clopidogrel and thus significantly improve the efficacy of antiplatelet therapy.

Improved method for assembly of hemeprotein neuronal NO-synthase heterodimers.

The assembly of mutated and wild type monomers into functional heterodimeric hemeproteins has provided important mechanistic insights. As in the case of NO synthase (NOS), the existing methods to make such heterodimeric NOSs are inefficient and labor intensive with typical yields of about 5%. We have found that expression of neuronal NOS heterodimers in insect cells, where we take advantage of an exogenous heme-triggered chaperone-assisted assembly process, provides an approximately 43% yield in heterodimeric NOS. In contrast, in Escherichia coli little heterodimerization occurred. Thus, insect cells are preferred and may represent a valuable method for assembly of other dimeric hemeproteins.

Architecture of the nitric-oxide synthase holoenzyme reveals large conformational changes and a calmodulin-driven release of the FMN domain.

Nitric-oxide synthase (NOS) is required in mammals to generate NO for regulating blood pressure, synaptic response, and immune defense. NOS is a large homodimer with well characterized reductase and oxygenase domains that coordinate a multistep, interdomain electron transfer mechanism to oxidize l-arginine and generate NO. Ca(2+)-calmodulin (CaM) binds between the reductase and oxygenase domains to activate NO synthesis. Although NOS has long been proposed to adopt distinct conformations that alternate between interflavin and FMN-heme electron transfer steps, structures of the holoenzyme have remained elusive and the CaM-bound arrangement is unknown. Here we have applied single particle electron microscopy (EM) methods to characterize the full-length of the neuronal isoform (nNOS) complex and determine the structural mechanism of CaM activation. We have identified that nNOS adopts an ensemble of open and closed conformational states and that CaM binding induces a dramatic rearrangement of the reductase domain. Our three-dimensional reconstruction of the intact nNOS-CaM complex reveals a closed conformation and a cross-monomer arrangement with the FMN domain rotated away from the NADPH-FAD center, toward the oxygenase dimer. This work captures, for the first time, the reductase-oxygenase structural arrangement and the CaM-dependent release of the FMN domain that coordinates to drive electron transfer across the domains during catalysis.

Activation of Hsp70 reduces neurotoxicity by promoting polyglutamine protein degradation.

We sought new strategies to reduce amounts of the polyglutamine androgen receptor (polyQ AR) and achieve benefits in models of spinobulbar muscular atrophy, a protein aggregation neurodegenerative disorder. Proteostasis of the polyQ AR is controlled by the heat shock protein 90 (Hsp90)- and Hsp70-based chaperone machinery, but mechanisms regulating the protein's turnover are incompletely understood. We demonstrate that overexpression of Hsp70 interacting protein (Hip), a co-chaperone that enhances binding of Hsp70 to its substrates, promotes client protein ubiquitination and polyQ AR clearance. Furthermore, we identify a small molecule that acts similarly to Hip by allosterically promoting Hsp70 binding to unfolded substrates. Like Hip, this synthetic co-chaperone enhances client protein ubiquitination and polyQ AR degradation. Both genetic and pharmacologic approaches targeting Hsp70 alleviate toxicity in a Drosophila model of spinobulbar muscular atrophy. These findings highlight the therapeutic potential of allosteric regulators of Hsp70 and provide new insights into the role of the chaperone machinery in protein quality control.

Destabilization of the epidermal growth factor receptor (EGFR) by a peptide that inhibits EGFR binding to heat shock protein 90 and receptor dimerization.

An eight-amino acid segment is known to be responsible for the marked difference in the rates of degradation of the EGF receptor (ErbB1) and ErbB2 upon treatment of cells with the Hsp90 inhibitor geldanamycin. We have scrambled the first six amino acids of this segment of the EGF receptor (EGFR), which lies in close association with the ATP binding cleft and the dimerization face. Scrambling these six amino acids markedly reduces EGFR stability, EGF-stimulated receptor dimerization, and autophosphorylation activity. Two peptides were synthesized as follows: one containing the wild-type sequence of the eight-amino acid segment, which we call Disruptin; and one with the scrambled sequence. Disruptin inhibits Hsp90 binding to the EGFR and causes slow degradation of the EGFR in two EGFR-dependent cancer cell lines, whereas the scrambled peptide is inactive. This effect is specific for EGFR versus other Hsp90 client proteins. In the presence of EGF, Disruptin, but not the scrambled peptide, inhibits EGFR dimerization and causes rapid degradation of the EGFR. In contrast to the Hsp90 inhibitor geldanamycin, Disruptin inhibits cancer cell growth by a nonapoptotic mechanism. Disruptin provides proof of concept for the development of a new class of anti-tumor drugs that specifically cause EGFR degradation.

A General Method to Screen Nanobodies for Cytochrome P450 Enzymes from a Yeast Surface Display Library.

The availability of yeast surface display nanobody (Nb) libraries offers a convenient way to acquire antigen-specific nanobodies that may be useful for protein structure-function studies and/or therapeutic applications, complementary to the conventional method of acquiring nanobodies through immunization in camelids. In this study, we developed a general approach to select nanobodies for cytochrome P450 enzymes from a highly diverse yeast display library. We tested our method on three P450 enzymes including CYP102A1, neuronal nitric oxide synthase (nNOS), and the complex of CYP2B4:POR, using a novel streamlined approach where biotinylated P450s were bound to fluorescent-labeled streptavidin for Nb screening. The Nb-antigen binders were selectively enriched using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). After two rounds of MACS, the population of positive binders was enriched by >5-fold compared to the naïve library. The subsequent FACS selection, with a gating of 0.1%, identified 634, 270, and 215 positive binders for CYP102A1, nNOS, and CYP2B4:POR, respectively. The positive binders for CYP102A1 were further triaged based on EC50 determined at various antigen concentrations. DNA sequencing of the top 30 binders of CYP102A1 resulted in 26 unique clones, 8 of which were selected for over-expression and characterization. They were found to inhibit CYP102A1-catalyzed oxidation of omeprazole with IC50 values in the range of 0.16-2.8 µM. These results validate our approach and may be applied to other protein targets for the effective selection of specific nanobodies.

Potent mechanism-based inactivation of cytochrome P450 2B4 by 9-ethynylphenanthrene: implications for allosteric modulation of cytochrome P450 catalysis.

The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and k(inact) are 0.2 and 0.25 min(-1), respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and an S(50) of 4.5 µM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by electrospray ionization liquid chromatography and mass spectrometry, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206, leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low-affinity site being the catalytic site and the high-affinity site on the protein periphery. Computer-aided docking and molecular dynamics simulations with one or two ligands bound revealed that the high-affinity site is situated at the entrance of a substrate access channel surrounded by the F' helix, ß1-ß2 loop, and ß4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302.

Modulation of heme/substrate binding cleft of neuronal nitric-oxide synthase (nNOS) regulates binding of Hsp90 and Hsp70 proteins and nNOS ubiquitination.

Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the Hsp90/Hsp70-based chaperone machinery, which regulates signaling proteins by modulating ligand binding clefts (Pratt, W. B., Morishima, Y., and Osawa, Y. (2008) J. Biol. Chem. 283, 22885-22889). We have previously shown that nNOS turnover is due to Hsp70/CHIP-dependent ubiquitination and proteasomal degradation. In this work, we use an intracellular cross-linking approach to study both chaperone binding and nNOS ubiquitination in intact HEK293 cells. Treatment of cells with N(G)-nitro-L-arginine, a slowly reversible competitive inhibitor that stabilizes nNOS, decreases both nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP. Treatment with the calcium ionophore A23187, which increases Ca(2+)-calmodulin binding to nNOS, increases nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP in a manner that is specific for changes in the heme/substrate binding cleft. Both Hsp90 and Hsp70 are bound to the expressed nNOS oxygenase domain, which contains the heme/substrate binding cleft, but not to the reductase domain, and binding is increased to an expressed fragment containing both the oxygenase domain and the calmodulin binding site. Overexpression of Hsp70 promotes nNOS ubiquitination and decreases nNOS protein, and overexpression of Hsp90 inhibits nNOS ubiquitination and increases nNOS protein, showing the opposing effects of the two chaperones as they participate in nNOS quality control in the cell. These observations support the notion that changes in the state of the heme/substrate binding cleft affect chaperone binding and thus nNOS ubiquitination.

Ubiquitination of neuronal nitric-oxide synthase in the calmodulin-binding site triggers proteasomal degradation of the protein.

Nitric-oxide synthase, a cytochrome P450-like hemoprotein enzyme, catalyzes the synthesis of nitric oxide, a critical signaling molecule in a variety of physiological processes. Our laboratory has discovered that certain drugs suicide-inactivate neuronal nitric-oxide synthase (nNOS) and lead to the preferential ubiquitination of the inactivated nNOS by an Hsp70- and CHIP (C terminus of Hsc70-interacting protein)-dependent process. To further understand the process by which altered nNOS is recognized, ubiquitinated, and proteasomally degraded, we examined the sites of ubiquitination on nNOS. We utilized an in vitro ubiquitination system containing purified E1, E2 (UbcH5a), Hsp70, and CHIP that recapitulates the ability of the cells to selectively recognize and ubiquitinate the altered forms of nNOS. LC-MS/MS analysis of the tryptic peptides obtained from the in vitro ubiquitinated nNOS identified 12 ubiquitination sites. Nine of the sites were within the oxygenase domain and two were in the calmodulin-binding site, which links the oxygenase and reductase domains, and one site was in the reductase domain. Mutational analysis of the lysines in the calmodulin-binding site revealed that Lys-739 is a major site for poly-ubiquitination of nNOS in vitro and regulates, in large part, the CHIP-dependent degradation of nNOS in HEK293 cells, as well as in in vitro studies with fraction II. Elucidating the exact site of ubiquitination is an important step in understanding how chaperones recognize and trigger degradation of nNOS.