RESUMEN
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.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Citocromos b5/química , NADPH-Ferrihemoproteína Reductasa/química , Sistema Enzimático del Citocromo P-450/metabolismo , Citocromos b5/metabolismo , Humanos , Modelos Moleculares , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxidación-Reducción , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromos b5/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Animales , Hidrocarburo de Aril Hidroxilasas/química , Benzfetamina/química , Hidroxitolueno Butilado/química , Ciclohexanos/química , Familia 2 del Citocromo P450/química , Familia 2 del Citocromo P450/metabolismo , Citocromos b5/química , Ligandos , Metoxiflurano/química , Modelos Químicos , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , NADPH-Ferrihemoproteína Reductasa/química , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Conejos , Ratas , Especificidad por SustratoRESUMEN
Structural interactions that enable electron transfer to cytochrome-P450 (CYP450) from its redox partner CYP450-reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane-bound functional complex to reveal interactions between the full-length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome-b5 (cyt-b5 ), Arg 125 on the C-helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein-protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Mononucleótido de Flavina/metabolismo , Nanoestructuras/química , Péptidos/química , Sistema Enzimático del Citocromo P-450/química , Mononucleótido de Flavina/química , Modelos Moleculares , Oxidación-ReducciónRESUMEN
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.
Asunto(s)
Citocromos b5/química , Citocromos c/química , Electrones , Membrana Dobles de Lípidos/química , Animales , Simulación de Dinámica Molecular , Unión Proteica , ConejosRESUMEN
Protein misfolding results in the accumulation of aggregated ß-sheet-rich structures in Parkinson's disease (PD) and Alzheimer's disease. The toxic oligomer hypothesis stipulates that prefibrillar assemblies, such as soluble oligomers or protofibrils, are responsible for the poor prognosis of these diseases. Previous studies demonstrated that a small molecule related to the natural compound orcein, O4, directly binds to amyloid-ß fibrils and stabilizes them, accelerating the formation of end-stage mature fibrils. Here we demonstrate a similar phenomenon during O4 treatment of α-synuclein (αsyn) aggregates, the protein responsible for PD pathology. While the drug did not change the kinetics of aggregate formation as measured by the amyloidophilic dye thioflavin T, O4 depleted αsyn oligomers and promoted the formation of sodium dodecyl sulfate and proteinase K resistant aggregates consisting of large fibril clusters. These fibril clusters exhibited reduced toxicity to human neuronal model cells and reduced seeding activity in vitro. The effectiveness of O4 decreased when it was added at later points in the αsyn aggregation pathway, which suggests that the incorporation of O4 into fibril assemblies stabilizes them against chemical, enzymatic, and mechanic degradation. These findings suggest that small molecules, which stabilize amyloid fibrils, can prevent fibril fragmentation and seeding and consequently prevent prion-like replication of misfolded αsyn. Inhibiting prion replication by fibril stabilization could thus be a therapeutic strategy for PD.