Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR

NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of metabolic enzymes. In the endoplasmic reticulum of liver cells, such enzymes metabolize ∼75% of the pharmaceuticals in use today. It is known that the transmembrane domain of CYPOR plays a crucial role in aiding the formation of a complex between CYPOR and cyt P450. Here we present the transmembrane structure, topology, and dynamics of the FMN binding domain of CYPOR in a native membrane-like environment. Our solid-state NMR results reveal that the N-terminal transmembrane domain of CYPOR adopts an α-helical conformation in the lipid membrane environment. Most notably, we also show that the transmembrane helix is tilted ∼13° from the lipid bilayer normal, and exhibits motions on a submillisecond timescale including rotational diffusion of the whole helix and fluctuation of the helical director axis. The approaches and the information reported in this study would enable further investigations on the structure and dynamics of the full-length NADPH-cytochrome P450 oxidoreductase and its interaction with other membrane proteins in a membrane environment.     

Structure and Function of an NADPH-Cytochrome P450 Oxidoreductase in an Open Conformation Capable of Reducing Cytochrome P450

NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner.NADPH-cytochrome P450 oxidoreductase (CYPOR)⁴ is a ∼78-kDa, multidomain, microsomal diflavin protein that shuttles electrons from NADPH → FAD → FMN to members of the ubiquitous cytochrome P450 superfamily (1, 2). In humans, the cytochromes P450 (cyt P450) are one of the most important families of proteins involved in the biosynthesis and degradation of a vast number of endogenous compounds and the detoxification and biodegradation of most foreign compounds. CYPOR also donates electrons to heme oxygenase (3), cytochrome b₅ (4), and cytochrome c (5).The FAD receives a hydride anion from the obligate two electron donor NADPH and passes the electrons one at a time to FMN. The FMN then donates electrons to the redox partners of CYPOR, again one electron at a time. Cyt P450 accepts electrons at two different steps in its complex reaction cycle. Ferric cyt P450 is reduced to the ferrous protein, and oxyferrous cyt P450 receives the second of the two electrons to form the peroxo (Fe⁺³OO)^2- cyt P450 intermediate (6). In vivo, CYPOR cycles between the one- and three-electron reduced forms (7, 8). Although the one-electron reduced form is an air-stable, neutral blue semiquinone (FMN_ox/sq, -110 mV), it is the FMN hydroquinone (FMN_sq/hq, -270 mV), not the semiquinone, that donates an electron to its redox partners (8–11). CYPOR is the prototype of the mammalian diflavin-containing enzyme family, which includes nitric-oxide synthase (12), methionine synthase reductase (13, 14), and a novel reductase expressed in the cytoplasm of certain cancer cells (15). CYPOR is also a target for anticancer therapy, because it reductively activates anticancer prodrugs (16).CYPOR consists of an N-terminal single α-helical transmembrane anchor (∼6 kDa) responsible for its localization to the endoplasmic reticulum and the soluble cytosolic portion (∼66 kDa) capable of reducing cytochrome c. Crystal structures of the soluble form of the wild-type and several mutant CYPORs are available (17, 18). The first ∼170 amino acids of the soluble domain are highly homologous to flavodoxin and bind FMN (FMN domain), whereas the C-terminal portion of the soluble protein consists of a FAD- and NADPH-binding domain with sequence and structural similarity to ferredoxin-NADP⁺ oxidoreductase (FAD domain). A connecting domain, possessing a unique sequence and structure, joins the FMN and FAD domains and is partly responsible for the relative orientation of the FMN and FAD domains. In the crystal structure, a convex anionic surface surrounds FMN. In the wild-type crystal structure, the two flavin isoalloxazine rings are in van der Waals contact, poised for efficient interflavin electron transfer (17). Based on the juxtaposition of the two flavins, an extrinsic electron transfer rate of ∼10¹⁰ s^-1 is predicted (19). However, the experimentally observed electron transfer rate between the two flavins is 30–55 s^-1 (20, 21). This modest rate and slowing of electron transfer in a viscous solvent (75% glycerol) suggest that interflavin electron transfer is likely conformationally gated. Moreover, the “closed” crystal structure, in which the flavins are in contact, is difficult to reconcile with mutagenesis studies that indicate the acidic amino acid residues on the surface near FMN are involved in interacting with cyt P450 (22). The first structural insight into how cyt P450 might interact with the FMN domain of CYPOR was provided by the crystal structure of a complex between the heme and FMN-containing domains of cyt P450 BM3 (23). In this complex, the methyl groups of FMN are oriented toward the heme on the proximal surface of cyt P450 BM3. Considered together, these three observations, the slow interflavin electron transfer, the mutagenesis data, and the structure of the complex between the heme and FMN domains of cyt P450 BM3, suggest that CYPOR will undergo a large conformational rearrangement in the course of shuttling electrons from NADPH to cyt P450. In addition, crystal structures of various CYPOR variants indicate that the FMN domain is highly mobile with respect to the rest of the molecule (18).Consideration of how the reductase would undergo a reorientation to interact with its redox partners led us to hypothesize the existence of a structural element in the reductase that would regulate the conformational changes and the relative dynamic motion of the domains. Our attention focused on the hinge region between the FMN and the connecting domain, because it is often disordered and highly flexible in the crystal structure (supplemental Fig. S1). The length and sequence of the hinge have been altered by site-directed mutagenesis, and the effects of the mutations on the catalytic properties of each mutant have been determined. The results demonstrate that lengthening the linker or altering its sequence do not modify the properties of CYPOR. In contrast, deletion of four amino acids markedly disrupts electron transfer from FAD to FMN, whereas the ability of the FMN domain to donate electrons to cyt P450 remains intact. The hinge deletion variant has been crystallized in three “open” conformations capable of interacting with cyt P450.     

Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase. Comparisons with NADPH-cytochrome P450 oxidoreductase

Nitric-oxide synthase (NOS) is composed of a C-terminal, flavin-containing reductase domain and an N-terminal, heme-containing oxidase domain. The reductase domain, similar to NADPH-cytochrome P450 reductase, can be further divided into two different flavin-containing domains: (a) the N terminus, FMN-containing portion, and (b) the C terminus FAD- and NADPH-binding portion. The crystal structure of the FAD/NADPH-containing domain of rat neuronal nitric-oxide synthase, complexed with NADP(+), has been determined at 1.9 A resolution. The protein is fully capable of reducing ferricyanide, using NADPH as the electron donor. The overall polypeptide fold of the domain is very similar to that of the corresponding module of NADPH-cytochrome P450 oxidoreductase (CYPOR) and consists of three structural subdomains (from N to C termini): (a) the connecting domain, (b) the FAD-binding domain, and (c) the NADPH-binding domain. A comparison of the structure of the neuronal NOS FAD/NADPH domain and CYPOR reveals the strict conservation of the flavin-binding site, including the tightly bound water molecules, the mode of NADP(+) binding, and the aromatic residue that lies at the re-face of the flavin ring, strongly suggesting that the hydride transfer mechanisms in the two enzymes are very similar. In contrast, the putative FMN domain-binding surface of the NOS protein is less positively charged than that of its CYPOR counterpart, indicating a different nature of interactions between the two flavin domains and a different mode of regulation in electron transfer between the two flavins involving the autoinhibitory element and the C-terminal 33 residues, both of which are absent in CYPOR.     

Association of multiple developmental defects and embryonic lethality with loss of microsomal NADPH-cytochrome P450 oxidoreductase.

The microsomal flavoprotein NADPH-cytochrome P450 oxidoreductase (CYPOR) is believed to function as the primary, if not sole, electron donor for the microsomal cytochrome P450 mixed-function oxidase system. Development of the mammalian embryo is dependent upon temporally and spatially regulated expression of signaling factors, many of which are synthesized and/or degraded via the cytochromes P450 and other pathways involving NADPH-cytochrome P450 oxidoreductase as the electron donor. Expression of CYPOR as early as the two-cell stage of embryonic development (The Institute for Genomic Research Mouse Gene Index, version 5.0, www.tigr.org/tdb/mgi) suggests that CYPOR is essential for normal cellular functions and/or early embryogenesis. Targeted deletion of the translation start site and membrane-binding domain of CYPOR abolished microsomal CYPOR expression and led to production of a truncated, 66-kDa protein localized to the cytoplasm. Although early embryogenesis was not affected, a variety of embryonic defects was observable by day 10.5 of gestation, leading to lethality by day 13.5. Furthermore, a deficiency of heterozygotes was observed in 2-week-old mice as well as late gestational age embryos, suggesting that loss of one CYPOR allele produced some embryonic lethality. CYPOR -/- embryos displayed a marked friability, consistent with defects in cell adhesion. Ninety percent of CYPOR -/- embryos isolated at days 10.5 or 11.5 of gestation could be classified as either Type I, characterized by grossly normal somite formation but having neural tube, cardiac, eye, and limb abnormalities, or Type II, characterized by a generalized retardation of development after approximately day 8.5 of gestation. No CYPOR -/- embryos were observed after day 13.5 of gestation. These studies demonstrate that loss of microsomal CYPOR does not block early embryonic development but is essential for progression past mid-gestation.     

Mutations of human cytochrome P450 reductase differentially modulate heme oxygenase-1 activity and oligomerization

Genetic variations in POR, encoding NADPH-cytochrome P450 oxidoreductase (CYPOR), can diminish the function of numerous cytochromes P450, and also have the potential to block degradation of heme by heme oxygenase-1 (HO-1). Purified full-length human CYPOR, HO-1, and biliverdin reductase were reconstituted in lipid vesicles and assayed for NADPH-dependent conversion of heme to bilirubin. Naturally-occurring human CYPOR variants queried were: WT, A115V, Y181D, P228L, M263V, A287P, R457H, Y459H, and V492E. All CYPOR variants exhibited decreased bilirubin production relative to WT, with a lower apparent affinity of the CYPOR–HO-1 complex than WT. Addition of FMN or FAD partially restored the activities of Y181D, Y459H, and V492E. When mixed with WT CYPOR, only the Y181D CYPOR variant inhibited heme degradation by sequestering HO-1, whereas Y459H and V492E were unable to inhibit HO-1 activity suggesting that CYPOR variants might have differential binding affinities with redox partners. Titrating the CYPOR–HO-1 complex revealed that the optimal CYPOR:HO-1 ratio for activity was 1:2, lending evidence in support of productive HO-1 oligomerization, with higher ratios of CYPOR:HO-1 showing decreased activity. In conclusion, human POR mutations, shown to impact P450 activities, also result in varying degrees of diminished HO-1 activity, which may further complicate CYPOR deficiency.     

Organization of Cytochrome P450 Enzymes Involved in Sex Steroid Synthesis: PROTEIN-PROTEIN INTERACTIONS IN LIPID MEMBRANES*

Mounting evidence underscores the importance of protein-protein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17α-hydroxylase/17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET)³ in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline ± cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17- or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom.     

Shallow Boomerang-shaped Influenza Hemagglutinin G13A Mutant Structure Promotes Leaky Membrane Fusion

Our previous studies showed that an angled boomerang-shaped structure of the influenza hemagglutinin (HA) fusion domain is critical for virus entry into host cells by membrane fusion. Because the acute angle of ∼105° of the wild-type fusion domain promotes efficient non-leaky membrane fusion, we asked whether different angles would still support fusion and thus facilitate virus entry. Here, we show that the G13A fusion domain mutant produces a new leaky fusion phenotype. The mutant fusion domain structure was solved by NMR spectroscopy in a lipid environment at fusion pH. The mutant adopted a boomerang structure similar to that of wild type but with a shallower kink angle of ∼150°. G13A perturbed the structure of model membranes to a lesser degree than wild type but to a greater degree than non-fusogenic fusion domain mutants. The strength of G13A binding to lipid bilayers was also intermediate between that of wild type and non-fusogenic mutants. These membrane interactions provide a clear link between structure and function of influenza fusion domains: an acute angle is required to promote clean non-leaky fusion suitable for virus entry presumably by interaction of the fusion domain with the transmembrane domain deep in the lipid bilayer. A shallower angle perturbs the bilayer of the target membrane so that it becomes leaky and unable to form a clean fusion pore. Mutants with no fixed boomerang angle interacted with bilayers weakly and did not promote any fusion or membrane perturbation.     

Chimeric enzymes of cytochrome P450 oxidoreductase and neuronal nitric-oxide synthase reductase domain reveal structural and functional differences

The nitric-oxide synthases (NOSs) are comprised of an oxygenase domain and a reductase domain bisected by a calmodulin (CaM) binding region. The NOS reductase domains share approximately 60% sequence similarity with the cytochrome P450 oxidoreductase (CYPOR), which transfers electrons to microsomal cytochromes P450. The crystal structure of the neuronal NOS (nNOS) connecting/FAD binding subdomains reveals that the structure of the nNOS-connecting subdomain diverges from that of CYPOR, implying different alignments of the flavins in the two enzymes. We created a series of chimeric enzymes between nNOS and CYPOR in which the FMN binding and the connecting/FAD binding subdomains are swapped. A chimera consisting of the nNOS heme domain and FMN binding subdomain and the CYPOR FAD binding subdomain catalyzed significantly increased rates of cytochrome c reduction in the absence of CaM and of NO synthesis in its presence. Cytochrome c reduction by this chimera was inhibited by CaM. Other chimeras consisting of the nNOS heme domain, the CYPOR FMN binding subdomain, and the nNOS FAD binding subdomain with or without the tail region also catalyzed cytochrome c reduction, were not modulated by CaM, and could not transfer electrons into the heme domain. A chimera consisting of the heme domain of nNOS and the reductase domain of CYPOR reduced cytochrome c and ferricyanide at rates 2-fold higher than that of native CYPOR, suggesting that the presence of the heme domain affected electron transfer through the reductase domain. These data demonstrate that the FMN subdomain of CYPOR cannot effectively substitute for that of nNOS, whereas the FAD subdomains are interchangeable. The differences among these chimeras most likely result from alterations in the alignment of the flavins within each enzyme construct.     

Monitoring Shifts in the Conformation Equilibrium of the Membrane Protein Cytochrome P450 Reductase (POR) in Nanodiscs

Nanodiscs are self-assembled ∼50-nm² patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.     

Conformational Changes and Slow Dynamics through Microsecond Polarized Atomistic Molecular Simulation of an Integral Kv1.2 Ion Channel

Structure and dynamics of voltage-gated ion channels, in particular the motion of the S4 helix, is a highly interesting and hotly debated topic in current membrane protein research. It has critical implications for insertion and stabilization of membrane proteins as well as for finding how transitions occur in membrane proteins—not to mention numerous applications in drug design. Here, we present a full 1 µs atomic-detail molecular dynamics simulation of an integral Kv1.2 ion channel, comprising 120,000 atoms. By applying 0.052 V/nm of hyperpolarization, we observe structural rearrangements, including up to 120° rotation of the S4 segment, changes in hydrogen-bonding patterns, but only low amounts of translation. A smaller rotation (∼35°) of the extracellular end of all S4 segments is present also in a reference 0.5 µs simulation without applied field, which indicates that the crystal structure might be slightly different from the natural state of the voltage sensor. The conformation change upon hyperpolarization is closely coupled to an increase in 3₁₀ helix contents in S4, starting from the intracellular side. This could support a model for transition from the crystal structure where the hyperpolarization destabilizes S4–lipid hydrogen bonds, which leads to the helix rotating to keep the arginine side chains away from the hydrophobic phase, and the driving force for final relaxation by downward translation is partly entropic, which would explain the slow process. The coordinates of the transmembrane part of the simulated channel actually stay closer to the recently determined higher-resolution Kv1.2 chimera channel than the starting structure for the entire second half of the simulation (0.5–1 µs). Together with lipids binding in matching positions and significant thinning of the membrane also observed in experiments, this provides additional support for the predictive power of microsecond-scale membrane protein simulations.     

Cryo-EM of the ATP11C flippase reconstituted in Nanodiscs shows a distended phospholipid bilayer inner membrane around transmembrane helix 2

ATP11C is a member of the P4-ATPase flippase family that mediates translocation of phosphatidylserine (PtdSer) across the lipid bilayer. In order to characterize the structure and function of ATP11C in a model natural lipid environment, we revisited and optimized a quick procedure for reconstituting ATP11C into Nanodiscs using methyl-β-cyclodextrin as a reagent for the detergent removal. ATP11C was efficiently reconstituted with the endogenous lipid, or the mixture of endogenous lipid and synthetic dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylserine (DOPS), all of which retained the ATPase activity. We obtained 3.4 Å and 3.9 Å structures using single-particle cryo-electron microscopy (cryo-EM) of AlF- and BeF-stabilized ATP11C transport intermediates, respectively, in a bilayer containing DOPS. We show that the latter exhibited a distended inner membrane around ATP11C transmembrane helix 2, possibly reflecting the perturbation needed for phospholipid release to the lipid bilayer. Our structures of ATP11C in the lipid membrane indicate that the membrane boundary varies upon conformational changes of the enzyme and is no longer flat around the protein, a change that likely contributes to phospholipid translocation across the membrane leaflets.     

Nitric oxide metabolism in mammalian cells: substrate and inhibitor profiles of a NADPH-cytochrome P450 oxidoreductase-coupled microsomal nitric oxide dioxygenase

Human intestinal Caco-2 cells metabolize and detoxify NO via a dioxygen- and NADPH-dependent, cyanide- and CO-sensitive pathway that yields nitrate. Enzymes catalyzing NO dioxygenation fractionate with membranes and are enriched in microsomes. Microsomal NO metabolism shows apparent KM values for NO, O2, and NADPH of 0.3, 9, and 2 microM, respectively, values similar to those determined for intact or digitonin-permeabilized cells. Similar to cellular NO metabolism, microsomal NO metabolism is superoxide-independent and sensitive to heme-enzyme inhibitors including CO, cyanide, imidazoles, quercetin, and allicin-enriched garlic extract. Selective inhibitors of several cytochrome P450s and heme oxygenase fail to inhibit the activity, indicating limited roles for a subset of microsomal heme enzymes in NO metabolism. Diphenyleneiodonium and cytochrome c(III) inhibit NO metabolism, suggesting a role for the NADPH-cytochrome P450 oxidoreductase (CYPOR). Involvement of CYPOR is demonstrated by the specific inhibition of the NO metabolic activity by inhibitory anti-CYPOR IgG. In toto, the results suggest roles for a microsomal CYPOR-coupled and heme-dependent NO dioxygenase in NO metabolism, detoxification, and signal attenuation in mammalian cells.     

A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes

Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes.     

Secondary structure and membrane topology of cytochrome P450s

The secondary structure prediction of 19 microsomal cytochrome P450s from two different families was made on the basis of their amino acid sequences. It was shown that there is structural similarity between the heme-binding sites in these enzymes and those in the bacterial P450cam. An average predicted secondary structure of cytochrome P450 proteins with 70% accuracy contains about 46% alpha-helices, 12% beta-sheets, 9% beta-turns, and 33% random coils. In the region of residues 35-120 in microsomal P450s two adjacent beta alpha beta-units (the Rossmann domain), were recognized and may be available to interact with the NADPH-cytochrome P450 reductase. Using the procedure for identification of hydrophobic and membrane-associated alpha-helical segments, only one N-terminal transmembrane anchor was predicted. Also the heme-binding site may include the surface-bound helix. A model for vertebrate microsomal P450s having an amphipathic membrane protein located on the cytoplasmic side of the endoplasmic reticulum membrane, with their active center lying outside or on the bilayer border, is proposed.     

Conformational Changes in Talin on Binding to Anionic Phospholipid Membranes Facilitate Signaling by Integrin Transmembrane Helices

Integrins are heterodimeric (αβ) cell surface receptors that are activated to a high affinity state by the formation of a complex involving the α/β integrin transmembrane helix dimer, the head domain of talin (a cytoplasmic protein that links integrins to actin), and the membrane. The talin head domain contains four sub-domains (F0, F1, F2 and F3) with a long cationic loop inserted in the F1 domain. Here, we model the binding and interactions of the complete talin head domain with a phospholipid bilayer, using multiscale molecular dynamics simulations. The role of the inserted F1 loop, which is missing from the crystal structure of the talin head, PDB:3IVF, is explored. The results show that the talin head domain binds to the membrane predominantly via cationic regions on the F2 and F3 subdomains and the F1 loop. Upon binding, the intact talin head adopts a novel V-shaped conformation which optimizes its interactions with the membrane. Simulations of the complex of talin with the integrin α/β TM helix dimer in a membrane, show how this complex promotes a rearrangement, and eventual dissociation of, the integrin α and β transmembrane helices. A model for the talin-mediated integrin activation is proposed which describes how the mutual interplay of interactions between transmembrane helices, the cytoplasmic talin protein, and the lipid bilayer promotes integrin inside-out activation.     

Mosquito NADPH‐cytochrome P450 oxidoreductase: kinetics and role of phenylalanine amino acid substitutions at leu86 and leu219 in CYP6AA3‐mediated deltamethrin metabolism

The NADPH‐cytochrome P450 oxidoreductase (CYPOR) enzyme is a membrane‐bound protein and contains both FAD and FMN cofactors. The enzyme transfers two electrons, one at a time, from NADPH to cytochrome P450 enzymes to function in the enzymatic reactions. We previously expressed in Escherichia coli the membrane‐bound CYPOR (flAnCYPOR) from Anopheles minimus mosquito. We demonstrated the ability of flAnCYPOR to support the An. minimus CYP6AA3 enzyme activity in deltamethrin degradation in vitro. The present study revealed that the flAnCYPOR purified enzyme, analyzed by a fluorometric method, readily lost its flavin cofactors. When supplemented with exogenous flavin cofactors, the activity of flAnCYPOR‐mediated cytochrome c reduction was increased. Mutant enzymes containing phenylalanine substitutions at leucine residues 86 and 219 were constructed and found to increase retention of FMN cofactor in the flAnCYPOR enzymes. Kinetic study by measuring cytochrome c–reducing activity indicated that the wild‐type and mutant flAnCYPORs followed a non‐classical two‐site Ping‐Pong mechanism, similar to rat CYPOR. The single mutant (L86F or L219F) and double mutant (L86F/L219F) flAnCYPOR enzymes, upon reconstitution with the An. minimus cytochrome P450 CYP6AA3 and a NADPH‐regenerating system, increased CYP6AA3‐mediated deltamethrin degradation compared to the wild‐type flAnCYPOR enzyme. The increased enzyme activity could illustrate a more efficient electron transfer of AnCYPOR to CYP6AA3 cytochrome P450 enzyme. Addition of extra flavin cofactors could increase CYP6AA3‐mediated activity supported by wild‐type and mutant flAnCYPOR enzymes. Thus, both leucine to phenylalanine substitutions are essential for flAnCYPOR enzyme in supporting CYP6AA3‐mediated metabolism. © 2010 Wiley Periodicals, Inc.     

The Transmembrane Helices of Beef Heart Cytochrome Oxidase

The locations of the transmembrane helices in the 12 subunits of beef heart cytochrome oxidase were predicted with a modified form of the von Heijne-Blomberg hydrophobicity scale. Based on ~20 residues per transmembrane helix, about 480 of the estimated 660 helical residues (36.8% of 1,793 total residues) are expected to be in transmembrane helices that have their axes tilted by a small angle α from the normal to the plane of the membrane. This angle is calculated to be ~30°, based on the observed overall tilt angle θ of 39° obtained from circular dichroism (CD) measurements on multilamellar films, or about 25°, based on the observed tilt angle θ of 36° obtained from the infrared linear dichroism of films. For 21 residues per transmembrane helix, the calculated values of α become 32° and 28°, respectively, depending upon the value of θ used. Thus, a transmembrane helical tilt angle of ~30° accounts for the predicted transmembrane stretches in cytochrome oxidase if 20-21 residues are sufficient to span the membrane. Additional helical residues in the lipid head region may deviate by a larger angle from the normal to the plane of the membrane in cytochrome oxidase.     

Geometrical Membrane Curvature as an Allosteric Regulator of Membrane Protein Structure and Function

Transmembrane proteins are embedded in cellular membranes of varied lipid composition and geometrical curvature. Here, we studied for the first time the allosteric effect of geometrical membrane curvature on transmembrane protein structure and function. We used single-channel optical analysis of the prototypic transmembrane β-barrel α-hemolysin (α-HL) reconstituted on immobilized single small unilamellar liposomes of different diameter and therefore curvature. Our data demonstrate that physiologically abundant geometrical membrane curvatures can enforce a dramatic allosteric regulation (1000-fold inhibition) of α-HL permeability. High membrane curvatures (1/diameter ∼1/40 nm⁻¹) compressed the effective pore diameter of α-HL from 14.2 ± 0.8 Å to 11.4 ± 0.6 Å. This reduction in effective pore area (∼40%) when combined with the area compressibility of α-HL revealed an effective membrane tension of ∼50 mN/m and a curvature-imposed protein deformation energy of ∼7 k_BT. Such substantial energies have been shown to conformationally activate, or unfold, β-barrel and α-helical transmembrane proteins, suggesting that membrane curvature could likely regulate allosterically the structure and function of transmembrane proteins in general.     

NMR-Based Simulation Studies of Pf1 Coat Protein in Explicit Membranes

As time- and ensemble-averaged measures, NMR observables contain information about both protein structure and dynamics. This work represents a computational study to extract such information for membrane proteins from orientation-dependent NMR observables: solid-state NMR chemical shift anisotropy and dipolar coupling, and solution NMR residual dipolar coupling. We have performed NMR-restrained molecular dynamics simulations to refine the structure of the membrane-bound form of Pf1 coat protein in explicit lipid bilayers using the recently measured chemical shift anisotropy, dipolar coupling, and residual dipolar coupling data. From the simulations, we have characterized detailed protein-lipid interactions and explored the dynamics. All simulations are stable and the NMR restraints are well satisfied. The C-terminal transmembrane (TM) domain of Pf1 finds its optimal position in the membrane quickly (within 6 ns), illustrating efficient solvation of TM domains in explicit bilayer environments. Such rapid convergence also leads to well-converged interaction patterns between the TM helix and the membrane, which clearly show the interactions of interfacial membrane-anchoring residues with the lipids. For the N-terminal periplasmic helix of Pf1, we identify a stable, albeit dynamic, helix orientation parallel to the membrane surface that satisfies the amphiphatic nature of the helix in an explicit lipid bilayer. Such detailed information cannot be obtained solely from NMR observables. Therefore, the present simulations illustrate the usefulness of NMR-restrained MD refinement of membrane protein structure in explicit membranes.     

High‐resolution analysis of the conformational transition of pro‐apoptotic Bak at the lipid membrane

Permeabilization of the outer mitochondrial membrane by pore‐forming Bcl2 proteins is a crucial step for the induction of apoptosis. Despite a large set of data suggesting global conformational changes within pro‐apoptotic Bak during pore formation, high‐resolution structural details in a membrane environment remain sparse. Here, we used NMR and HDX‐MS (Hydrogen deuterium exchange mass spectrometry) in lipid nanodiscs to gain important high‐resolution structural insights into the conformational changes of Bak at the membrane that are dependent on a direct activation by BH3‐only proteins. Furthermore, we determined the first high‐resolution structure of the Bak transmembrane helix. Upon activation, α‐helix 1 in the soluble domain of Bak dissociates from the protein and adopts an unfolded and dynamic potentially membrane‐bound state. In line with this finding, comparative protein folding experiments with Bak and anti‐apoptotic BclxL suggest that α‐helix 1 in Bak is a metastable structural element contributing to its pro‐apoptotic features. Consequently, mutagenesis experiments aimed at stabilizing α‐helix 1 yielded Bak variants with delayed pore‐forming activity. These insights will contribute to a better mechanistic understanding of Bak‐mediated membrane permeabilization.