Tracking protein-protein interactions by NMR: conformational selection in human steroidogenic cytochrome P450 CYP17A1 induced by cytochrome b5.

The human steroidogenic cytochrome P450 CYP17A1 catalyzes two types of reactions in the biosynthetic pathway leading from pregnenolone to testosterone and several other steroid hormones. The first is the hydroxylation of pregnenolone or progesterone to the corresponding 17α-hydroxy steroid, followed by a lyase reaction that converts these 17α-hydroxy intermediates to the androgens dehydroepiandrosterone and androstenedione, respectively. cytochrome b5 (cytb5) is known to act as both an effector and electron donor for the lyase oxidations, markedly stimulating the rate of the lyase reaction in its presence relative to the rate in its absence. Extensive sequential backbone 1H,15N and 13C nuclear magnetic resonance assignments have now been made for oxidized CYP17A1 bound to the prostate cancer drug and inhibitor abiraterone. This is the first eukaryotic P450 for which such assignments are now available. These assignments allow more complete interpretation of the structural perturbations observed upon cytb5 addition. Possible mechanism(s) for the effector activity of cytb5 are discussed in light of this new information.

Direct interaction between the transmembrane helices stabilize cytochrome P450 2B4 and cytochrome b5 redox complex.

The catalytic activity of cytochrome P450 2B4 (CYP2B4) is moderated by its cognate redox partner cytochrome b5 (Cyt-b5). The endoplasmic reticulum (ER) membrane and intermolecular transmembrane (TM) interaction between CYP2B4 and Cyt-b5 regulate the substrate catalysis and the reaction rate. This emphasizes the significance of elucidating the molecular basis of CYP2B4 and Cyt-b5 complexation in a membrane environment to better understand the enzymatic activity of CYP2B4. Our previous solid-state NMR studies revealed the membrane topology of the transmembrane domains of these proteins in the free and complex forms. Here, we show the cross-angle complex formation by the single-pass TM domains of CYP2B4 and Cyt-b5, which is mainly driven by several salt-bridges (E2-R128, R21-D104 and K25-D104), using a multi-microsecond molecular dynamic simulation. Additionally, the leucine-zipper residues (L8, L12, L15, L18 and L19 from CYP2B4) and π-stacking between H23 and F20 residues of CYP2B4 and W110 of Cyt-b5 are identified to stabilize the TM-TM complex in the ER membrane. The simulated tilts of the helices in the free and in the complex are in excellent agreement with solid-state NMR results. The TM-TM packing influences a higher order structural stability when compared to the complex formed by the truncated soluble domains of these two proteins. MM/PBSA based binding free energy estimates nearly 100-fold higher binding affinity (ΔG = -2810.68 ± 696.44 kJ/mol) between the soluble domains of the full-length CYP2B4 and Cyt-b5 when embedded in lipid membrane as compared to the TM-domain-truncated soluble domains (ΔG = -27.406 ± 10.32 kJ/mol). The high-resolution full-length CYP2B4-Cyt-b5 complex structure and its dynamics in a native ER membrane environment reported here could aid in the development of approaches to effectively modulate the drug-metabolism activity of CYP2B4.

Mechanism of electron transfers mediated by cytochromes c and b5 in mitochondria and endoplasmic reticulum: classical and murburn perspectives.

We explore the mechanism of electron transfers mediated by cytochrome c, a soluble protein involved in mitochondrial oxidative phosphorylation and cytochrome b5, a microsomal membrane protein acting as a redox aide in xenobiotic metabolism. We found minimal conservation in the sequence and surface amino acid residues of cytochrome c/b5 proteins among divergent species. Therefore, we question the evolutionary logic for electron transfer (ET) occurring through affinity binding via recognition of specific surface residues/topography. Also, analysis of putative protein-protein interactions in the crystal structures of these proteins and their redox partners did not point to any specific interaction logic. A comparison of the kinetic and thermodynamic constants of wildtype vs. mutants did not provide strong evidence to support the binding-based ET paradigm, but indicated support for diffusible reactive species (DRS)-mediated process. Topographically divergent cytochromes from one species have been substituted for reaction with proteins from other species, implying the involvement of non-specific interactions. We provide a viable alternative (murburn concept) to classical protein-protein binding-based long range ET mechanism. To account for the promiscuity of interactions and solvent-accessible hemes, we propose that the two proteins act as non- specific redox capacitors, mediating one-electron redox equilibriums involving DRS and unbound ions.Communicated by Ramaswamy H. Sarma.

Substrate-Specific Allosteric Effects on the Enhancement of CYP17A1 Lyase Efficiency by Cytochrome b5.

CYP17A1 is an essential human steroidogenic enzyme, which catalyzes two sequential reactions leading to the formation of androstenedione from progesterone and dehydroepiandrosterone from pregnenolone. The second reaction is the C17-C20 bond scission, which is strongly dependent on the presence of cytochrome b5 and displays a heretofore unexplained more pronounced acceleration when 17OH-progesteone (17OH-PROG) is a substrate. The origin of the stimulating effect of cytochrome b5 on C-C bond scission catalyzed by CYP17A1 is still debated as mostly due to either the acceleration of the electron transfer to the P450 oxy complex or allosteric effects of cytochrome b5 favoring active site conformations that promote lyase activity. Using resonance Raman spectroscopy, we compared the effect of Mn-substituted cytochrome b5 (Mn-Cytb5) on the oxy complex of CYP17A1 with both proteins co-incorporated in lipid nanodiscs. For CYP17A1 with 17OH-PROG, a characteristic shift of the Fe-O mode is observed in the presence of Mn-b5, indicating reorientation of a hydrogen bond between the 17OH group of the substrate from the terminal to the proximal oxygen atom of the Fe-O-O moiety, a configuration favorable for the lyase catalysis. For 17OH-pregnenolone, no such shift is observed, the favorable H-bonding orientation being present even without Mn-Cytb5. These new data provide a precise allosteric interpretation for the more pronounced acceleration seen for the 17OH-PROG substrate.

Structure of cytochrome b5 unique to tardigrades.

Cytochrome b5 is an essential electron transfer protein, which is ubiquitously found in living systems and involved in wide variety of biological processes. Tardigrades (also known as water bears), some of which are famous for desiccation resistance, have many proteins unique to them. Here, we report spectroscopic and structural characterization of a cytochrome b5 like protein from one of the desiccation-tolerant tardigrades, Ramazzottius varieornatus strain YOKOZUNA-1 (RvCytb5 ). A 1.4 Å resolution crystal structure revealed that RvCytb5 is a new cytochrome b5 protein specific to tardigrades.

Detergent-free extraction, reconstitution and characterization of membrane-anchored cytochrome-b5 in native lipids.

Despite their denaturing properties, detergents are used to purify and study membrane proteins. Herein, we demonstrated a polymer-based detergent-free extraction of the membrane protein cytochrome-b5 along with E. coli lipids. Nuclear magnetic resonance experiments revealed the suitability of using nanodiscs for high-resolution studies and revealed the types of native lipids associated with the protein.

Expression, purification, and functional reconstitution of 19F-labeled cytochrome b5 in peptide nanodiscs for NMR studies.

Microsomal cytochrome b5 (cytb5) is a membrane-bound protein capable of donating the second electron to cytochrome P450s (cytP450s) in the cytP450s monooxygenase reactions. Recent studies have demonstrated the importance of the transmembrane domain of cytb5 in the interaction with cytP450 by stabilizing its monomeric structure. While recent NMR studies have provided high-resolution insights into the structural interactions between the soluble domains of ~16-kDa cytb5 and ~57-kDa cytP450 in a membrane environment, there is need for studies to probe the residues in the transmembrane region as well as to obtain intermolecular distance constraints to better understand the very large size cytb5-cytP450 complex structure in a near native membrane environment. In this study, we report the expression, purification, functional reconstitution of 19F-labeled full-length rabbit cytb5 in peptide based nanodiscs for structural studies using NMR spectroscopy. Size exclusion chromatography, dynamic light scattering, transmission electron microscopy, and NMR experiments show a stable reconstitution of cytb5 in 4F peptide-based lipid-nanodiscs. The reported results demonstrate the use of 19F NMR experiments to study 19F-labeled (with 5-fluorotryptophan (5FW)) cytb5 reconstituted in peptide-nanodiscs and the detection of residues from the transmembrane domain by solution 19F NMR experiments. 19F NMR results revealing the interaction of the transmembrane domain of cytb5 with the full-length rabbit cytochrome P450 2B4 (CYP2B4) are also presented. We expect the results presented in this study to be useful to devise approaches to probe the structure, dynamics and functional roles of transmembrane domains of a membrane protein, and also to measure intermolecular 19F-19F distance constraints to determine the structural interactions between the transmembrane domains.

Probing protein-protein and protein-substrate interactions in the dynamic membrane-associated ternary complex of cytochromes P450, b5, and reductase.

Cytochrome P450 (cytP450) interacts with two redox partners, cytP450 reductase and cytochrome-b5, to metabolize substrates. Using NMR, we reveal changes in the dynamic interplay when all three proteins are incorporated into lipid nanodiscs in the absence and presence of substrates.

Crystal structures of the naturally fused CS and cytochrome b5 reductase (b5R) domains of Ncb5or reveal an expanded CS fold, extensive CS-b5R interactions and productive binding of the NAD(P)+ nicotinamide ring.

Ncb5or (NADH-cytochrome b5 oxidoreductase), a cytosolic ferric reductase implicated in diabetes and neurological diseases, comprises three distinct domains, cytochrome b5 (b5) and cytochrome b5 reductase (b5R) domains separated by a CHORD-Sgt1 (CS) domain, and a novel 50-residue N-terminal region. Understanding how interdomain interactions in Ncb5or facilitate the shuttling of electrons from NAD(P)H to heme, and how the process compares with the microsomal b5 (Cyb5A) and b5R (Cyb5R3) system, is of interest. A high-resolution structure of the b5 domain (PDB entry 3lf5) has previously been reported, which exhibits substantial differences in comparison to Cyb5A. The structural characterization of a construct comprising the naturally fused CS and b5R domains with bound FAD and NAD+ (PDB entry 6mv1) or NADP+ (PDB entry 6mv2) is now reported. The structures reveal that the linker between the CS and b5R cores is more ordered than predicted, with much of it extending the ß-sandwich motif of the CS domain. This limits the flexibility between the two domains, which recognize one another via a short ß-sheet motif and a network of conserved side-chain hydrogen bonds, salt bridges and cation-π interactions. Notable differences in FAD-protein interactions in Ncb5or and Cyb5R3 provide insight into the selectivity for docking of their respective b5 redox partners. The structures also afford a structural explanation for the unusual ability of Ncb5or to utilize both NADH and NADPH, and represent the first examples of native, fully oxidized b5R family members in which the nicotinamide ring of NAD(P)+ resides in the active site. Finally, the structures, together with sequence alignments, show that the b5R domain is more closely related to single-domain Cyb5R proteins from plants, fungi and some protists than to Cyb5R3 from animals.

Ligand accessibility to heme cytochrome b5 coordinating sphere and enzymatic activity enhancement upon tyrosine ionization.

Recently, we observed that at extreme alkaline pH, cytochrome b5 (Cb5) acquires a peroxidase-like activity upon formation of a low spin hemichrome associated with a non-native state. A functional characterization of Cb5, in a wide pH range, shows that oxygenase/peroxidase activities are stimulated in alkaline media, and a correlation between tyrosine ionization and the attained enzymatic activities was noticed, associated with an altered heme spin state, when compared to acidic pH values at which the heme group is released. In these conditions, a competitive assay between imidazole binding and Cb5 endogenous heme ligands revealed the appearance of a binding site for this exogenous ligand that promotes a heme group exposure to the solvent upon ligation. Our results shed light on the mechanism behind Cb5 oxygenase/peroxidase activity stimulation in alkaline media and reveal a role of tyrosinate anion enhancing Cb5 enzymatic activities on the distorted protein before maximum protein unfolding.

The use of tail-anchored protein chimeras to enhance liposomal cargo delivery.

BACKGROUND: Liposomes are employed as drug delivery vehicles offering a beneficial pharmacokinetic/distribution mechanism for in vivo therapeutics. Therapeutic liposomes can be designed to target specific cell types through the display of epitope-specific targeting peptides on their surface. The majority of peptides are currently attached by chemical modification of lipid constituents. Here we investigate an alternative and novel method of decorating liposomes with targeting ligand, using remotely and spontaneously inserting chimeric tail-anchored membrane (TA) proteins to drug loaded liposomes. METHODS AND RESULTS: An artificial TA protein chimera containing the transmembrane domain from the spontaneously inserting TA protein cytochrome b5 (Cytb5) provided a robust membrane tether for the incorporation of three different targeting moieties into preformed liposomes. The moieties investigated were the transactivator of transcription (TAT) peptide, the EGF-receptor binding sequence GE11 and the placental and tumour homing ligand CCGKRK. In all cases, TA protein insertion neither significantly altered the size of the liposomes nor reduced drug loading. The efficacy of this novel targeted delivery system was investigated using two human cell lines, HeLa M and BeWo. Short term incubation with one ligand-modified TA chimera, incorporating the TAT peptide, significantly enhanced liposomal delivery of the encapsulated carboxyfluorescein reporter. CONCLUSION: The Cytb5 TA was successfully employed as a membrane anchor for the incorporation of the desired peptide ligands into a liposomal drug delivery system, with minimal loss of cargo during insertion. This approach therefore provides a viable alternative to chemical conjugation and its potential to accommodate a wider range of targeting ligands may provide an opportunity for enhancing drug delivery.

Substrate mediated redox partner selectivity of cytochrome P450.

Investigating the interplay between cytochrome-P450 and its redox partners (CPR and cytochrome-b5) is vital for understanding the metabolism of most hydrophobic drugs. Dynamic structural interactions with the ternary complex, with and without substrates, captured by NMR reveal a gating mechanism for redox partners to promote P450 function.

Discrimination between the endoplasmic reticulum and mitochondria by spontaneously inserting tail-anchored proteins.

Tail-anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein-free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5-ER and b5-RR, which differ only in the charge of the C-terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell-free and in cellula results, we analyzed targeting in digitonin-permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C-terminal negative charge of b5-ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ESI ) reduced insertion of b5-ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ESI , Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C-terminal charge, and reveal a novel, substrate-specific ER-targeting pathway.

Structural and enzymatic analysis of the cytochrome b5 reductase domain of Ulva prolifera nitrate reductase.

Rapid accumulations of unattached green macroalgae, referred to as blooms, constitute ecological disasters and occur in many coastal regions. Ulva are a major cause of blooms, owing to their high nitrogen utilization capacity, which requires nitrate reductase (NR) activity; however, molecular characterization of Ulva NR remains lacking. Herein we determined the crystal structure and performed an enzymatic analysis of the cytochrome b5 reductase domain of Ulva prolifera NR (UpCbRNR). The structural analysis revealed an N-terminal FAD-binding domain primarily consisting of six antiparallel ß strands, a C-terminal NADH-binding domain forming a Rossmann fold, and a three ß-stranded linker region connecting these two domains. The FAD cofactor was located in the cleft between the two domains and interacted primarily with the FAD-binding domain. UpCbRNR shares similarities in overall structure and cofactor interactions with homologs, and its catalytic ability is comparable to that of higher plant CbRNRs. Structure and sequence comparisons of homologs revealed two regions of sequence length variation potentially useful for phylogenetic analysis: one in the FAD-binding domain, specific to U. prolifera, and another in the linker region that may be used to differentiate between plant, fungi, and animal homologs. Our data will facilitate molecular-level understanding of nitrate assimilation in Ulva.

Peroxidase-like activity of cytochrome b5 is triggered upon hemichrome formation in alkaline pH.

In alkaline media (pH12) a catalytic peroxidase activity of cytochrome b5 was found associated to a different conformational state. Upon incubation at this pH, cytochrome b5 electronic absorption spectrum was altered, with disappearance of characteristic bands of cytochrome b5 at pH7.0. The appearance of new electronic absorption bands and EPR measurements support the formation of a cytochrome b5 class B hemichrome with an acquired ability to bind polar ligands. This hemichrome is characterized by a negative formal redox potential and the same folding properties than cytochrome b5 at pH7. The acquired peroxidase-like activity of cytochrome b5 found at pH12, driven by a hemichrome formation, suggests a role of this protein in peroxidation products propagation.

Topography of human cytochrome b5/cytochrome b5 reductase interacting domain and redox alterations upon complex formation.

Cytochrome b5 is the main electron acceptor of cytochrome b5 reductase. The interacting domain between both human proteins has been unidentified up to date and very little is known about its redox properties modulation upon complex formation. In this article, we characterized the protein/protein interacting interface by solution NMR and molecular docking. In addition, upon complex formation, we measured an increase of cytochrome b5 reductase flavin autofluorescence that was dependent upon the presence of cytochrome b5. Data analysis of these results allowed us to calculate a dissociation constant value between proteins of 0.5±0.1µM and a 1:1 stoichiometry for the complex formation. In addition, a 30mV negative shift of cytochrome b5 reductase redox potential in presence of cytochrome b5 was also measured. These experiments suggest that the FAD group of cytochrome b5 reductase increase its solvent exposition upon complex formation promoting an efficient electron transfer between the proteins.

Membrane environment drives cytochrome P450's spin transition and its interaction with cytochrome b5.

Heme's spin-multiplicity is key in determining the enzymatic function of cytochrome P450 (cytP450). The origin of the low-spin state in ferric P450 is still under debate. Here, we report the first experimental demonstration of P450's membrane interaction altering its spin equilibrium which is accompanied by a stronger affinity for cytochrome b5. These results highlight the importance of lipid membrane for the function of P450.

Structural and functional effects of cytochrome b5 interactions with human cytochrome P450 enzymes.

The small heme-containing protein cytochrome b5 can facilitate, inhibit, or have no effect on cytochrome P450 catalysis, often in a P450-dependent and substrate-dependent manner that is not well understood. Herein, solution NMR was used to identify b5 residues interacting with different human drug-metabolizing P450 enzymes. NMR results revealed that P450 enzymes bound to either b5 α4-5 (CYP2A6 and CYP2E1) or this region and α2-3 (CYP2D6 and CYP3A4) and suggested variation in the affinity for b5 Mutations of key b5 residues suggest not only that different b5 surfaces are responsible for binding different P450 enzymes, but that these different complexes are relevant to the observed effects on P450 catalysis.

Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c.

Cytochrome b 5 (cytb 5) is a membrane protein vital for the regulation of cytochrome P450 (cytP450) metabolism and is capable of electron transfer to many redox partners. Here, using cyt c as a surrogate for cytP450, we report the effect of membrane on the interaction between full-length cytb 5 and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cytb 5 - cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cytb 5-cyt c binding interface. Our experimental results identify differences in the binding epitope of cytb 5 in the presence and absence of membrane. Notably, in the presence of membrane, cytb 5 only engaged cyt c at its lower and upper clefts while the membrane-free cytb 5 also uses a distal region. Using restraints generated from both cytb 5 and cyt c, a complex structure was generated and a potential electron transfer pathway was identified. These results demonstrate the importance of studying protein-protein complex formation in membrane mimetic systems. Our results also demonstrate the successful preparation of novel peptide-based lipid nanodiscs, which are detergent-free and possesses size flexibility, and their use for NMR structural studies of membrane proteins.

Engineering Aromatic-Aromatic Interactions To Nucleate Folding in Intrinsically Disordered Regions of Proteins.

Aromatic interactions are an important force in protein folding as they combine the stability of a hydrophobic interaction with the selectivity of a hydrogen bond. Much of our understanding of aromatic interactions comes from "bioinformatics" based analyses of protein structures and from the contribution of these interactions to stabilizing secondary structure motifs in model peptides. In this study, the structural consequences of aromatic interactions on protein folding have been explored in engineered mutants of the molten globule protein apo-cytochrome b5. Structural changes from disorder to order due to aromatic interactions in two variants of the protein, viz., WF-cytb5 and FF-cytb5, result in significant long-range secondary and tertiary structure. The results show that 54 and 52% of the residues in WF-cytb5 and FF-cytb5, respectively, occupy ordered regions versus 26% in apo-cytochrome b5. The interactions between the aromatic groups are offset-stacked and edge-to-face for the Trp-Phe and Phe-Phe mutants, respectively. Urea denaturation studies indicate that both mutants have a Cm higher than that of apo-cytochrome b5 and are more stable to chaotropic agents than apo-cytochrome b5. The introduction of these aromatic residues also results in "trimer" interactions with existing aromatic groups, reaffirming the selectivity of the aromatic interactions. These studies provide insights into the aromatic interactions that drive disorder-to-order transitions in intrinsically disordered regions of proteins and will aid in de novo protein design beyond small peptide scaffolds.