RESUMEN
Conformational dynamics are important factors in the function of enzymes, including protein tyrosine phosphatases (PTPs). Crystal structures of PTPs first revealed the motion of a protein loop bearing a conserved catalytic aspartic acid, and subsequent nuclear magnetic resonance and computational analyses have shown the presence of motions, involved in catalysis and allostery, within and beyond the active site. The tyrosine phosphatase from the thermophilic and acidophilic Sulfolobus solfataricus (SsoPTP) displays motions of its acid loop together with dynamics of its phosphoryl-binding P-loop and the Q-loop, the first instance of such motions in a PTP. All three loops share the same exchange rate, implying their motions are coupled. Further evidence of conformational flexibility comes from mutagenesis, kinetics, and isotope effect data showing that E40 can function as an alternate general acid to protonate the leaving group when the conserved acid, D69, is mutated to asparagine. SsoPTP is not the first PTP to exhibit an alternate general acid (after VHZ and TkPTP), but E40 does not correspond to the sequence or structural location of the alternate general acids in those precedents. A high-resolution X-ray structure with the transition state analogue vanadate clarifies the role of the active site arginine R102, which varied in structures of substrates bound to a catalytically inactive mutant. The coordinated motions of all three functional loops in SsoPTP, together with the function of an alternate general acid, suggest that catalytically competent conformations are present in solution that have not yet been observed in crystal structures.
Asunto(s)
Proteínas Tirosina Fosfatasas/genética , Sulfolobus solfataricus/enzimología , Secuencia de Aminoácidos/genética , Catálisis , Dominio Catalítico/genética , Cristalografía por Rayos X/métodos , Humanos , Cinética , Modelos Moleculares , Movimiento (Física) , Fosforilación/genética , Conformación Proteica , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas/ultraestructura , Sulfolobus solfataricus/química , Sulfolobus solfataricus/metabolismoRESUMEN
Centralities determined from Residue Interaction Networks (RIN) in proteins have been used to predict aspects of their structure and dynamics. Here, we correlate the Eigenvector Centrality (Ec) with the rate constant for thermal denaturation (kden) of the HisF protein from Thermotoga maritima based on 12 single alanine substitution mutants. The molecular basis for this correlation was further explored by studying a mutant containing a replacement of a high Ec residue, Y182A, which displayed increased kden at 80 °C. The crystallographic structure of this mutant showed few changes, mostly in two flexible loops. The 1H-15N -HSQC showed only subtle changes of cross peak positions for residues located near the mutation site and scattered throughout the structure. However, the comparison of the RIN showed that Y182 is the vertex of a set of high centrality residues that spreads throughout the HisF structure, which is lacking in the mutant. Cross-correlation displacements of Cα calculated from a molecular dynamics simulation at different temperatures showed that the Y182A mutation reduced the correlated movements in the HisF structure above 70 °C. 1H-15N NMR chemical shift covariance using temperature as perturbation were consistent with these results. In conclusion the increase in temperature drives the structure of the mutant HisF-Y182A into a less connected state, richer in non-concerted motions, located predominantly in the C-terminal half of the protein where Y182 is placed. Conversely, wild-type HisF responds to increased temperature as a single unit. Hence the replacement of a high Ec residue alters the distribution of thermal energy through HisF structure.
Asunto(s)
Proteínas , Thermotoga maritima , Modelos Moleculares , Conformación Proteica , Thermotoga maritima/genéticaRESUMEN
Proteogenomic identification of translated small open reading frames in humans has revealed thousands of microproteins, or polypeptides of fewer than 100 amino acids, that were previously invisible to geneticists. Hundreds of microproteins have been shown to be essential for cell growth and proliferation, and many regulate macromolecular complexes. However, the vast majority of microproteins remain functionally uncharacterized, and many lack secondary structure and exhibit limited evolutionary conservation. One such intrinsically disordered microprotein is NBDY, a 68-amino acid component of membraneless organelles known as P-bodies. In this work, we show that NBDY can undergo liquid-liquid phase separation, a biophysical process thought to underlie the formation of membraneless organelles, in the presence of RNA in vitro. Phosphorylation of NBDY drives liquid phase remixing in vitro and macroscopic P-body dissociation in cells undergoing growth factor signaling and cell division. These results suggest that NBDY phosphorylation enables regulation of P-body dynamics during cell proliferation and, more broadly, that intrinsically disordered microproteins may contribute to liquid-liquid phase separation and remixing behavior to affect cellular processes.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/síntesis química , Condensados Biomoleculares , Humanos , Proteínas Intrínsecamente Desordenadas/química , Tamaño de la Partícula , FosforilaciónRESUMEN
Determining the principal energy-transfer pathways responsible for allosteric communication in biomolecules remains challenging, partially due to the intrinsic complexity of the systems and the lack of effective characterization methods. In this work, we introduce the eigenvector centrality metric based on mutual information to elucidate allosteric mechanisms that regulate enzymatic activity. Moreover, we propose a strategy to characterize the range of correlations that underlie the allosteric processes. We use the V-type allosteric enzyme imidazole glycerol phosphate synthase (IGPS) to test the proposed methodology. The eigenvector centrality method identifies key amino acid residues of IGPS with high susceptibility to effector binding. The findings are validated by solution NMR measurements yielding important biological insights, including direct experimental evidence for interdomain motion, the central role played by helix h[Formula: see text], and the short-range nature of correlations responsible for the allosteric mechanism. Beyond insights on IGPS allosteric pathways and the nature of residues that could be targeted by therapeutic drugs or site-directed mutagenesis, the reported findings demonstrate the eigenvector centrality analysis as a general cost-effective methodology to gain fundamental understanding of allosteric mechanisms at the molecular level.
Asunto(s)
Regulación Alostérica , Aminohidrolasas/química , Proteínas Bacterianas/química , Sitio Alostérico , Modelos Moleculares , Modelos Teóricos , Unión Proteica , Conformación ProteicaRESUMEN
Dynamics and conformational motions are important to the activity of enzymes, including protein tyrosine phosphatases. These motions often extend to regions outside the active site, called allosteric regions. In the tyrosine phosphatase Vaccinia H1-related (VHR) enzyme, we demonstrate the importance of the allosteric interaction between the variable insert region and the active-site loops in VHR. These studies include solution nuclear magnetic resonance, computation, steady-state, and rapid kinetic measurements. Overall, the data indicate concerted millisecond motions exist between the variable insert and the catalytic acid loop in wild-type (WT) VHR. The 150 ns computation studies show a flexible acid loop in WT VHR that opens during the simulation from its initial closed structure. Mutation of the variable insert residue, asparagine 74, to alanine results in a rigidification of the acid loop as observed by molecular dynamics simulations and a disruption of crucial active-site hydrogen bonds. Moreover, enzyme kinetic analysis shows a weakening of substrate affinity in the N74A mutant and a >2-fold decrease in substrate cleavage and hydrolysis rates. These data show that despite being nearly 20 Å from the active site, the variable insert region is linked to the acid loop by coupled millisecond motions, and that disruption of the communication between the variable insert and active site alters the normal catalytic function of VHR and perturbs the active-site environment.
Asunto(s)
Fosfatasa 3 de Especificidad Dual/metabolismo , Regulación Alostérica , Biocatálisis , Fosfatasa 3 de Especificidad Dual/química , Fosfatasa 3 de Especificidad Dual/aislamiento & purificación , Humanos , Hidrólisis , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación ProteicaRESUMEN
DNA polymerase ß (pol ß) fills single nucleotide gaps in DNA during base excision repair and non-homologous end-joining. Pol ß must select the correct nucleotide from among a pool of four nucleotides with similar structures and properties in order to maintain genomic stability during DNA repair. Here, we use a combination of X-ray crystallography, fluorescence resonance energy transfer and nuclear magnetic resonance to show that pol ß's ability to access the appropriate conformations both before and upon binding to nucleotide substrates is integral to its fidelity. Importantly, we also demonstrate that the inability of the I260Q mutator variant of pol ß to properly navigate this conformational landscape results in error-prone DNA synthesis. Our work reveals that precatalytic conformational rearrangements themselves are an important underlying mechanism of substrate selection by DNA pol ß.
Asunto(s)
Codón sin Sentido , ADN Polimerasa beta/genética , Replicación del ADN/genética , ADN/química , Inestabilidad Genómica/genética , Conformación de Ácido Nucleico , Sustitución de Aminoácidos/genética , Catálisis , Cristalografía por Rayos X , ADN/metabolismo , ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , Reparación del ADN/genética , Transferencia Resonante de Energía de Fluorescencia , Ácido Glutámico/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Isoleucina/genética , Modelos Moleculares , Nucleótidos/química , Nucleótidos/metabolismo , Unión Proteica , Especificidad por Sustrato/genética , Moldes GenéticosRESUMEN
Imidazole glycerol phosphate synthase (IGPS) is a V-type allosteric enzyme, meaning that its catalytic rate is critically dependent on activation by its allosteric ligand, N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR). The allosteric mechanism of IGPS is reliant on millisecond conformational motions for efficient catalysis. We engineered four mutants of IGPS designed to disrupt millisecond motions and allosteric coupling to identify regions that are critical to IGPS function. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments and NMR chemical shift titrations reveal diminished enzyme flexibility and a reshaping of the allosteric connectivity in each mutant construct, respectively. The functional relevance of the observed motional quenching is confirmed by significant reductions in glutaminase kinetic activity and allosteric ligand binding affinity. This work presents relevant conclusions toward the control of protein allostery and design of unique allosteric sites for potential enzyme inhibitors with regulatory or therapeutic benefit.
Asunto(s)
Aminohidrolasas/química , Proteínas Bacterianas/química , Thermotoga maritima/enzimología , Resistencia betalactámica , Regulación Alostérica , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Técnicas de Silenciamiento del Gen , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Homología Estructural de Proteína , Thermotoga maritima/genéticaRESUMEN
Active-site loops are integral to the function of numerous enzymes. They enable substrate and product binding and release, sequester reaction intermediates, and recruit catalytic groups. Here, we examine the catalytic loop in the enzyme protein tyrosine phosphatase 1B (PTP1B). PTP1B has a mobile so-called WPD loop (named for its three N-terminal residues) that initiates the dephosphorylation of phosphor-tyrosine substrates upon loop closure. We have combined X-ray crystallography, solution NMR, and pre-steady-state kinetics experiments on wild-type and five WPD loop mutants to identify the relationships between the loop structure, dynamics, and function. The motions of the WPD loop are modulated by the formation of weak molecular interactions, where perturbations of these interactions modulate the conformational equilibrium landscape. The point mutants in the WPD loop alter the loop equilibrium position from a predominantly open state (P185A) to 50:50 (F182A), 35:65 (P188A), and predominantly closed states (T177A and P188A). Surprisingly, there is no correlation between the observed catalytic rates in the loop mutants and changes to the WPD loop equilibrium position. Rather, we observe a strong correlation between the rate of dephosphorylation of the phosphocysteine enzyme intermediate and uniform millisecond motions, not only within the loop but also in the adjacent α-helical domain of PTP1B. Thus, the control of loop motion and thereby catalytic activity is dispersed and resides within not only the loop sequence but also the surrounding protein architecture. This has broad implications for the general mechanistic understanding of enzyme reactions and the role that flexible loops play in the catalytic cycle.
Asunto(s)
Proteína Tirosina Fosfatasa no Receptora Tipo 1/química , Biocatálisis , Dominio Catalítico/genética , Cristalografía por Rayos X , Humanos , Cinética , Mutación , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genéticaRESUMEN
NMR relaxation dispersion experiments play a central role in exploring molecular motion over an important range of timescales, and are an example of a broader class of multidimensional NMR experiments that probe important biomolecules. However, resolving the spectral features of these experiments using the Fourier transform requires sampling the full Nyquist grid of data, making these experiments very costly in time. Practitioners often reduce the experiment time by omitting 1D experiments in the indirectly observed dimensions, and reconstructing the spectra using one of a variety of post-processing algorithms. In prior work, we described a fast, Fourier-based reconstruction method using iterated maps according to the Difference Map algorithm of Veit Elser (DiffMap). Here we describe coDiffMap, a new reconstruction method that is based on DiffMap, but which exploits the strong correlations between 2D data slices in a pseudo-3D experiment. We apply coDiffMap to reconstruct dispersion curves from an [Formula: see text] relaxation dispersion experiment, and demonstrate that the method provides fast reconstructions and accurate relaxation curves down to very low numbers of sparsely-sampled data points.
Asunto(s)
Análisis de Fourier , Procesamiento de Imagen Asistido por Computador/métodos , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Algoritmos , Factores de TiempoRESUMEN
Many of the ubiquitous experiments of biomolecular NMR, including [Formula: see text], [Formula: see text], and CEST, involve acquiring repeated 2D spectra under slightly different conditions. Such experiments are amenable to acceleration using non-uniform sampling spectral reconstruction methods that take advantage of prior information. We previously developed one such technique, an iterated maps method (DiffMap) that we successfully applied to 2D NMR spectra, including [Formula: see text] relaxation dispersion data. In that prior work, we took a top-down approach to reconstructing the 2D spectrum with a minimal number of sparse samples, reaching an undersampling fraction that appeared to leave some room for improvement. In this study, we develop an in-depth understanding of the action of the DiffMap algorithm, identifying the factors that cause reconstruction errors for different undersampling fractions. This improved understanding allows us to formulate a bottom-up approach to finding the lowest number of sparse samples required to accurately reconstruct individual spectral features with DiffMap. We also discuss the difficulty of extending this method to reconstructing many peaks at once, and suggest a way forward.
Asunto(s)
Algoritmos , Resonancia Magnética Nuclear Biomolecular/métodos , Tamaño de la Muestra , Manejo de Especímenes/métodosRESUMEN
To study factors that affect WPD-loop motion in protein tyrosine phosphatases (PTPs), a chimera of PTP1B and YopH was created by transposing the WPD loop from PTP1B to YopH. Several subsequent mutations proved to be necessary to obtain a soluble, active enzyme. That chimera, termed chimera 3, retains productive WPD-loop motions and general acid catalysis with a pH dependency similar to that of the native enzymes. Kinetic isotope effects show the mechanism and transition state for phosphoryl transfer are unaltered. Catalysis of the chimera is slower than that of either of its parent enzymes, although its rate is comparable to those of most native PTPs. X-ray crystallography and nuclear magnetic resonance were used to probe the structure and dynamics of chimera 3. The chimera's structure was found to sample an unproductive hyper-open conformation of its WPD loop, a geometry that has not been observed in either of the parents or in other native PTPs. The reduced catalytic rate is attributed to the protein's sampling of this conformation in solution, reducing the fraction in the catalytically productive loop-closed conformation.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/química , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Yersinia/enzimología , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genética , Proteínas Tirosina Fosfatasas/genética , Proteínas Recombinantes de Fusión/genética , Homología de SecuenciaRESUMEN
Allostery is a ubiquitous biological regulatory process in which distant binding sites within a protein or enzyme are functionally and thermodynamically coupled. Allosteric interactions play essential roles in many enzymological mechanisms, often facilitating formation of enzyme-substrate complexes and/or product release. Thus, elucidating the forces that drive allostery is critical to understanding the complex transformations of biomolecules. Currently, a number of models exist to describe allosteric behavior, taking into account energetics as well as conformational rearrangements and fluctuations. In the following Review, we discuss the use of solution NMR techniques designed to probe allosteric mechanisms in enzymes. NMR spectroscopy is unequaled in its ability to detect structural and dynamical changes in biomolecules, and the case studies presented herein demonstrate the range of insights to be gained from this valuable method. We also provide a detailed technical discussion of several specialized NMR experiments that are ideally suited for the study of enzymatic allostery.
Asunto(s)
Enzimas/metabolismo , Resonancia Magnética Nuclear Biomolecular , Soluciones/química , Regulación Alostérica , Sitios de Unión , Enzimas/química , Hemoglobinas/químicaRESUMEN
Macrophage migration inhibitory factor (MIF) activates CD74, which leads to severe disorders including inflammation, autoimmune diseases and cancer under pathological conditions. Molecular dynamics (MD) simulations up to one microsecond revealed dynamical correlation between a residue located at the opening of one end of the MIF solvent channel, previously thought to be a consequence of homotrimerization, and residues in a distal region responsible for CD74 activation. Experiments verified the allosteric regulatory site and identified a pathway to this site via the MIF ß-strands. The reported findings provide fundamental insights on a dynamic mechanism that controls the MIF-induced activation of CD74.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Sitio Alostérico , Antígenos de Diferenciación de Linfocitos B/química , Antígenos de Histocompatibilidad Clase II/química , Humanos , Inflamación/metabolismo , Oxidorreductasas Intramoleculares/química , Factores Inhibidores de la Migración de Macrófagos/química , Simulación de Dinámica Molecular , Conformación Proteica en Lámina betaRESUMEN
Protein tyrosine phosphatase 1B (PTP1B) is a known regulator of the insulin and leptin signaling pathways and is an active target for the design of inhibitors for the treatment of type II diabetes and obesity. Recently, cichoric acid (CHA) and chlorogenic acid (CGA) were predicted by docking methods to be allosteric inhibitors that bind distal to the active site. However, using a combination of steady-state inhibition kinetics, solution nuclear magnetic resonance experiments, and molecular dynamics simulations, we show that CHA is a competitive inhibitor that binds in the active site of PTP1B. CGA, while a noncompetitive inhibitor, binds in the second aryl phosphate binding site, rather than the predicted benzfuran binding pocket. The molecular dynamics simulations of the apo enzyme and cysteine-phosphoryl intermediate states with and without bound CGA suggest CGA binding inhibits PTP1B by altering hydrogen bonding patterns at the active site. This study provides a mechanistic understanding of the allosteric inhibition of PTP1B.
Asunto(s)
Ácidos Cafeicos/farmacología , Ácido Clorogénico/farmacología , Inhibidores Enzimáticos/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Succinatos/farmacología , Algoritmos , Regulación Alostérica , Sitios de Unión , Unión Competitiva , Ácidos Cafeicos/química , Ácidos Cafeicos/metabolismo , Dominio Catalítico , Ácido Clorogénico/química , Ácido Clorogénico/metabolismo , Inhibidores Enzimáticos/metabolismo , Humanos , Enlace de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Unión Proteica , Dominios Proteicos , Proteína Tirosina Fosfatasa no Receptora Tipo 1/química , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Succinatos/química , Succinatos/metabolismoRESUMEN
DNA polymerase ß (Pol ß) repairs single-nucleotide gapped DNA (sngDNA) by enzymatic incorporation of the Watson-Crick partner nucleotide at the gapped position opposite the templating nucleotide. The process by which the matching nucleotide is incorporated into a sngDNA sequence has been relatively well-characterized, but the process of discrimination from nucleotide misincorporation remains unclear. We report here NMR spectroscopic characterization of full-length, uniformly labeled Pol ß in apo, sngDNA-bound binary, and ternary complexes containing matching and mismatching nucleotide. Our data indicate that, while binding of the correct nucleotide to the binary complex induces chemical shift changes consistent with the process of enzyme closure, the ternary Pol ß complex containing a mismatching nucleotide exhibits no such changes and appears to remain in an open, unstable, binary-like conformation. Our findings support an induced-fit mechanism for polymerases in which a closed ternary complex can only be achieved in the presence of matching nucleotide.
Asunto(s)
ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , Nucleótidos/metabolismo , Calorimetría , ADN/química , ADN/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
Allosteric enzymes regulate a wide range of catalytic transformations, including biosynthetic mechanisms of important human pathogens, upon binding of substrate molecules to an orthosteric (or active) site and effector ligands at distant (allosteric) sites. We find that enzymatic activity can be impaired by small molecules that bind along the allosteric pathway connecting the orthosteric and allosteric sites, without competing with endogenous ligands. Noncompetitive allosteric inhibitors disrupted allostery in the imidazole glycerol phosphate synthase (IGPS) enzyme from Thermotoga maritima as evidenced by nuclear magnetic resonance, microsecond time-scale molecular dynamics simulations, isothermal titration calorimetry, and kinetic assays. The findings are particularly relevant for the development of allosteric antibiotics, herbicides, and antifungal compounds because IGPS is absent in mammals but provides an entry point to fundamental biosynthetic pathways in plants, fungi, and bacteria.
Asunto(s)
Regulación Alostérica , Aminohidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Sitio Alostérico , Aminohidrolasas/química , Proteínas Bacterianas/química , Sitios de Unión , Biocatálisis , Calorimetría/métodos , Dominio Catalítico , Cristalografía por Rayos X , Imidazoles/química , Imidazoles/metabolismo , Ligandos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Unión Proteica , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Ribonucleótidos/química , Ribonucleótidos/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Thermotoga maritima/enzimologíaRESUMEN
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine. In addition to its known receptor-mediated biological activities, MIF possesses a catalytic site of unknown function between subunits of a homotrimer. Each subunit contributes three ß-strands to adjacent subunits to form a core seven-stranded ß-sheet for each monomer. MIF monomers, dimers, or trimers have been reported, but the active form that binds and activates the MIF receptor (CD74) is still a matter of debate. A cysteine mutant (N110C) that covalently locks MIF into a trimer by forming a disulfide with Cys-80 of an adjacent subunit is used to study this issue. Partial catalytic activity and receptor binding to CD74 are retained by N110C (locked trimer), but there is no cellular signaling. Wild-type MIF-induced cellular signaling, in vivo lung neutrophil accumulation, and alveolar permeability are inhibited with a fivefold excess of N110C. NMR and size-exclusion chromatography with light scattering reveal that N110C can form a higher-order oligomer in equilibrium with a single locked trimer. The X-ray structure confirms a local conformational change that disrupts the subunit interface and results in global changes responsible for the oligomeric form. The structure also confirms these changes are consistent for the partial catalytic and receptor binding activities. The absence of any potential monomer and the retention of partial catalytic and receptor binding activities despite changes in conformation (and dynamics) in the mutant support an endogenous MIF trimer that binds and activates CD74 at nanomolar concentrations. This conclusion has implications for therapeutic development.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Oxidorreductasas Intramoleculares/química , Oxidorreductasas Intramoleculares/genética , Factores Inhibidores de la Migración de Macrófagos/química , Factores Inhibidores de la Migración de Macrófagos/genética , Animales , Cristalografía por Rayos X , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Receptores Inmunológicos/metabolismoRESUMEN
Protein allosteric pathways are investigated in the imidazole glycerol phosphate synthase heterodimer in an effort to elucidate how the effector (PRFAR, N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide) activates glutaminase catalysis at a distance of 25 Å from the glutamine-binding site. We apply solution NMR techniques and community analysis of dynamical networks, based on mutual information of correlated protein motions in the active and inactive enzymes. We find evidence that the allosteric pathways in the PRFAR bound enzyme involve conserved residues that correlate motion of the PRFAR binding loop to motion at the protein-protein interface, and ultimately at the glutaminase active site. The imidazole glycerol phosphate synthase bienzyme is an important branch point for the histidine and nucleotide biosynthetic pathways and represents a potential therapeutic target against microbes. The proposed allosteric mechanism and the underlying allosteric pathways provide fundamental insights for the design of new allosteric drugs and/or alternative herbicides.