RESUMEN
CYP119, a bacterial thermophilic protein from the cytochrome P450 superfamily, has previously been observed in three different conformations with different inhibitors bound using X-ray crystallography. The significance of these states in solution and in the function of the enzyme is not well-known. Double electron-electron resonance (DEER) was used to measure distances and distance distributions between spin-labels for populated conformational states in solution. DEER spectroscopy and molecular dynamics for the ligand-free enzyme suggest that the G helix is in a slightly different conformation than seen previously by crystallography, with the F/G loop in a slightly open conformation. Inhibitor-bound samples showed that this conformation remains as the predominant form, but partial conversion is indicated to a more closed conformation of the F/G loop. However, when the enzyme binds to lauric acid, the proposed substrate, it induces the conversion to a state that is characterized by increased disorder. We propose that similar to recent results with soluble CYP3A4, binding of the inhibitor to CYP119 is accompanied by only small changes in the enzyme structure, but substrate binding results in greater heterogeneity in the structure of the F/G loop region.
Asunto(s)
Proteínas Arqueales/química , Sistema Enzimático del Citocromo P-450/química , Electrones , Sulfolobus solfataricus/enzimología , Proteínas Arqueales/aislamiento & purificación , Proteínas Arqueales/metabolismo , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Ácidos Láuricos/farmacología , Modelos Moleculares , Conformación Proteica , Soluciones , Especificidad por SustratoRESUMEN
Putidaredoxin (Pdx) is the exclusive reductase and a structural effector for P450cam (CYP101A1). However, the mechanism of how Pdx modulates the conformational states of P450cam remains elusive. Here we report a putative communication pathway for the Pdx-induced conformational change in P450cam using results of double electron-electron resonance (DEER) spectroscopy and molecular dynamics simulations. Use of solution state DEER measurements allows us to observe subtle conformational changes in the internal helices in P450cam among closed, open, and P450cam-Pdx complex states. Molecular dynamics simulations and dynamic network analysis suggest that Pdx binding is coupled to small coordinated movements of several regions of P450cam, including helices C, B', I, G, and F. These changes provide a linkage between the Pdx binding site on the proximal side of the enzyme and helices F/G on the distal side and the site of the largest movement resulting from the Pdx-induced closed-to-open transition. This study provides a detailed rationale for how Pdx exerts its long-recognized effector function at the active site from its binding site on the opposite face of the enzyme.
Asunto(s)
Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Ferredoxinas/metabolismo , Pseudomonas putida/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Ferredoxinas/química , Ferredoxinas/genética , Simulación de Dinámica MolecularRESUMEN
Cytochrome P450cam is an archetypal example of the vast family of heme monooxygenases and serves as a model for an enzyme that is highly specific for both its substrate and reductase. During catalysis, it undergoes significant conformational changes of the F and G helices upon binding its substrate and redox partner, putidaredoxin (Pdx). Recent studies have shown that Pdx binding to the closed camphor-bound form of ferric P450cam results in its conversion to a fully open state. However, during catalytic turnover, it remains unclear whether this same conformational change also occurs or whether it is coupled to the formation of the critical compound I intermediate. Here, we have examined P450cam bound simultaneously by camphor, CN-, and Pdx as a mimic of the catalytically competent ferrous oxy-P450cam-Pdx state. The combined use of double electron-electron resonance and molecular dynamics showed direct observation of intermediate conformational states of the enzyme upon CN- and subsequent Pdx binding. This state is coupled to the movement of the I helix and residues at the active site, including Arg-186, Asp-251, and Thr-252. These movements enable occupation of a water molecule that has been implicated in proton delivery and peroxy bond cleavage to give compound I. These findings provide a detailed understanding of how the Pdx-induced conformational change may sequentially promote compound I formation followed by product release, while retaining stereoselective hydroxylation of the substrate of this highly specific monooxygenase.
Asunto(s)
Proteínas Bacterianas/química , Alcanfor 5-Monooxigenasa/química , Ferredoxinas/química , Simulación de Dinámica Molecular , Conformación Proteica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biocatálisis , Alcanfor 5-Monooxigenasa/genética , Alcanfor 5-Monooxigenasa/metabolismo , Dominio Catalítico , Ferredoxinas/metabolismo , Oxidación-Reducción , Unión Proteica , Pseudomonas putida/enzimología , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Especificidad por SustratoRESUMEN
Human cytochrome P450 3A4 (CYP3A4) is a membrane-associated monooxygenase that is responsible for metabolizing >50% of the pharmaceuticals in the current market, so studying its chemical mechanism and structural changes upon ligand binding will help provide deeper insights into drug metabolism and further drug development. The best-characterized cytochrome P450 is a bacterial form, P450cam, which undergoes significant conformational changes upon binding substrate and its redox partner, putidaredoxin. In contrast, most crystal structures of CYP3A4 with or without ligands have shown few changes, although allosteric effects and multiple-substrate binding in solution are well-documented. In this study, we use double electron-electron resonance (DEER) to measure distances between spatially separated spin-labels on CYP3A4 and molecular dynamics to interpret the DEER data. These methods were applied to a soluble N-terminally truncated CYP3A4 form, and the results show that there are few changes in the average structure upon binding ketoconazole, ritonavir, or midazolam. However, binding of midazolam, but not ketoconazole or ritonavir, resulted in a significant change in the motion and/or disorder in the F/G helix region near the substrate binding pocket. These results suggest that soluble CYP3A4 behaves in a unique way in response to inhibitor and substrate binding.
Asunto(s)
Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Simulación de Dinámica Molecular , Espectroscopía de Resonancia por Spin del Electrón/métodos , Humanos , Cetoconazol/química , Cetoconazol/metabolismo , Ligandos , Unión Proteica/fisiología , Conformación Proteica , Estructura Secundaria de Proteína , Ritonavir/química , Ritonavir/metabolismoRESUMEN
Many genomes contain families of paralogs--proteins with divergent function that evolved from a common ancestral gene after a duplication event. To understand how paralogous transcription factors evolve divergent DNA specificities, we examined how the glucocorticoid receptor and its paralogs evolved to bind activating response elements [(+)GREs] and negative glucocorticoid response elements (nGREs). We show that binding to nGREs is a property of the glucocorticoid receptor (GR) DNA-binding domain (DBD) not shared by other members of the steroid receptor family. Using phylogenetic, structural, biochemical, and molecular dynamics techniques, we show that the ancestral DBD from which GR and its paralogs evolved was capable of binding both nGRE and (+)GRE sequences because of the ancestral DBD's ability to assume multiple DNA-bound conformations. Subsequent amino acid substitutions in duplicated daughter genes selectively restricted protein conformational space, causing this dual DNA-binding specificity to be selectively enhanced in the GR lineage and lost in all others. Key substitutions that determined the receptors' response element-binding specificity were far from the proteins' DNA-binding interface and interacted epistatically to change the DBD's function through DNA-induced allosteric mechanisms. These amino acid substitutions subdivided both the conformational and functional space of the ancestral DBD among the present-day receptors, allowing a paralogous family of transcription factors to control disparate transcriptional programs despite high sequence identity.
Asunto(s)
ADN/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismo , Regulación Alostérica , Sustitución de Aminoácidos , Secuencia de Bases , Células HeLa , Humanos , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Elementos de Respuesta/genética , Especificidad por Sustrato , Factores de Transcripción/químicaRESUMEN
The urokinase-type plasminogen activator receptor (uPAR) is an essential regulator for cell signaling in tumor cell proliferation, adhesion, and metastasis. The ubiquitous nature of uPAR in many aggressive cancer types makes uPAR an attractive target for immunotherapy. Here, we present a rapid and successful workflow for developing cross-reactive anti-uPAR recombinant antibodies (rAbs) using high-throughput optofluidic screening of single B-cells from human uPAR-immunized mice. A total of 80 human and cynomolgus uPAR cross-reactive plasma cells were identified, and selected mouse VH/VL domains were linked to the trastuzumab (Herceptin®) constant domains for the expression of mouse-human chimeric antibodies. The resulting rAbs were characterized by their tumor-cell recognition, binding activity, and cell adhesion inhibition on triple-negative breast cancer cells. In addition, the rAbs were shown to enact antibody-dependent cellular cytotoxicity (ADCC) in the presence of either human natural killer cells or peripheral blood mononuclear cells, and were evaluated for the potential use of uPAR-targeting antibody-drug conjugates (ADCs). Three lead antibodies (11857, 8163, and 3159) were evaluated for their therapeutic efficacy in vivo and were shown to suppress tumor growth. Finally, the binding epitopes of the lead antibodies were characterized, providing information on their unique binding modes to uPAR. Altogether, the strategy identified unique cross-reactive antibodies with ADCC, ADC, and functional inhibitory effects by targeting cell-surface uPAR, that can be tested in safety studies and serve as potential immunotherapeutics.
Asunto(s)
Leucocitos Mononucleares , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Humanos , Animales , Ratones , Anticuerpos , Transducción de Señal , Linfocitos BRESUMEN
In vitro biopanning platforms using synthetic phage display antibody libraries have enabled the identification of antibodies against antigens that were once thought to be beyond the scope of immunization. Applying these methods against challenging targets remains a critical challenge. Here, we present a new biopanning pipeline, RAPID (Rare Antibody Phage Isolation and Discrimination), for the identification of rare high-affinity antibodies against challenging targets. RAPID biopanning uses fluorescent labeled phage displayed fragment antigen-binding (Fab) antibody libraries for the isolation of high-affinity binders with fluorescent activated sorting. Subsequently, discriminatory hit screening is performed with a biolayer interferometry (BLI) method, BIAS (Biolayer Interferometry Antibody Screen), where candidate binders are ranked and prioritized according to their estimated kinetic off rates. Previously reported antibodies were used to develop the methodology, and the RAPID biopanning pipeline was applied to three challenging targets (CHIP, Gαq, and CS3D), enabling the identification of high-affinity antibodies.
Asunto(s)
Bacteriófagos , Biblioteca de Péptidos , Bioprospección , Anticuerpos/genética , AntígenosRESUMEN
Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection.
Asunto(s)
Infecciones por Citomegalovirus , Citomegalovirus , Cisteína , Citomegalovirus/fisiología , Humanos , Péptido Hidrolasas , Proteasas ViralesRESUMEN
This study based on the synthesis, characterization, and structural calculation of small molecular potassium-encapsulated arsenic-dithiolato compounds will provide fundamental knowledge about arsenic metabolism behavior in biological system. Two novel air-stable potassium-encapsulated arsenic-dithiolato compounds, [K@As(2)(L1)(3)](BF(4)) (1) and [K@As(2)(L2)(3)](BF(4)) (2), were prepared using deprotonated 2,6-bis(mercaptomethyl)pyridine (L1H(2)) and 1,3-dimercapto-m-xylene (L2H(2)) to react with AsCl(3) in the presence of potassium cation. Compounds 1 and 2 have been characterized by electrospray ionization-mass spectra, nuclear magnetic resonance spectra, and elemental microanalysis. Density functional theory calculation also supports the formation and binding properties of the potassium-encapsulated arsenic-dithiolato compounds.