RESUMEN
RNAs besides tRNA and rRNA contain chemical modifications, including the recently described 5' nicotinamide-adenine dinucleotide (NAD+) RNA in bacteria. Whether 5' NAD-RNA exists in eukaryotes remains unknown. We demonstrate that 5' NAD-RNA is found on subsets of nuclear and mitochondrial encoded mRNAs in Saccharomyces cerevisiae NAD-mRNA appears to be produced cotranscriptionally because NAD-RNA is also found on pre-mRNAs, and only on mitochondrial transcripts that are not 5' end processed. These results define an additional 5' RNA cap structure in eukaryotes and raise the possibility that this 5' NAD+ cap could modulate RNA stability and translation on specific subclasses of mRNAs.
Asunto(s)
Caperuzas de ARN/metabolismo , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/metabolismo , NAD/metabolismo , Caperuzas de ARN/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Estabilidad del ARN , ARN de Hongos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mitocondrial , Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
Transcription factors (TFs) play multiple roles in development. Given this multifunctionality, it has been assumed that TFs are evolutionarily highly constrained. Here, we investigate the molecular mechanisms for the origin of a derived functional interaction between two TFs, HOXA11 and FOXO1. We have previously shown that the regulatory role of HOXA11 in mammalian endometrial stromal cells requires interaction with FOXO1, and that the physical interaction between these proteins evolved before their functional cooperativity. Here, we demonstrate that the derived functional cooperativity between HOXA11 and FOXO1 is due to derived allosteric regulation of HOXA11 by FOXO1. This study shows that TF function can evolve through changes affecting the functional output of a pre-existing protein complex.
Asunto(s)
Evolución Biológica , Proteína Forkhead Box O1/metabolismo , Proteínas de Homeodominio/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Proteína de Unión a CREB/química , Proteína de Unión a CREB/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Células HeLa , Proteínas de Homeodominio/química , Humanos , Proteínas Intrínsecamente Desordenadas/química , Ratones , Modelos Biológicos , Modelos Moleculares , Fosforilación , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Activación Transcripcional/genéticaRESUMEN
Computational design of protein-ligand interfaces finds optimal amino acid sequences within a small-molecule binding site of a protein for tight binding of a specific small molecule. It requires a search algorithm that can rapidly sample the vast sequence and conformational space, and a scoring function that can identify low energy designs. This review focuses on recent advances in computational design methods and their application to protein-small molecule binding sites. Strategies for increasing affinity, altering specificity, creating broad-spectrum binding, and building novel enzymes from scratch are described. Future prospects for applications in drug development are discussed, including limitations that will need to be overcome to achieve computational design of protein therapeutics with novel modes of action.
Asunto(s)
Biología Computacional , Descubrimiento de Drogas , Ingeniería de Proteínas , Proteínas/química , Sitios de Unión , Ligandos , Unión Proteica , Estabilidad Proteica , Proteínas/metabolismoRESUMEN
The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein-ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand. We chose the thermophilic endo-1,4-ß-xylanase from Nonomuraea flexuosa as the protein scaffold on which to perform our designs. Over the course of the study, 12 proteins derived from this scaffold were produced and assayed for binding to the target ligand. Unfortunately, none of the designed proteins displayed evidence of high-affinity binding. Structural characterization of four designed proteins revealed that although the predicted structure of the protein model was highly accurate, this structural accuracy did not translate into accurate prediction of binding affinity. Crystallographic analyses indicate that the lack of binding affinity is possibly due to unaccounted for protein dynamics in the 'thumb' region of our design scaffold intrinsic to the family 11 ß-xylanase fold. Further computational analysis revealed two specific, single amino acid substitutions responsible for an observed change in backbone conformation, and decreased dynamic stability of the catalytic cleft. These findings offer new insight into the dynamic and structural determinants of the ß-xylanase proteins.
Asunto(s)
Endo-1,4-beta Xilanasas/química , Ingeniería de Proteínas/métodos , Actinomycetales/enzimología , Actinomycetales/genética , Sitios de Unión , Biología Computacional/métodos , Simulación por Computador , Cristalografía , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Simulación de Dinámica Molecular , Método de Montecarlo , Estructura Terciaria de Proteína , Programas Informáticos , Vancomicina/química , Vancomicina/farmacologíaRESUMEN
The 1:1 complexation reaction between Ba(2+) and 18-crown-6 ether is re-examined using isothermal titration calorimetry (ITC), with the goal of clarifying previously reported discrepancies between reaction enthalpies estimated directly (calorimetric) and indirectly, from the temperature dependence of the reaction equilibrium constant K (van't Hoff). The ITC thermograms are analyzed using three different non-linear fit models based on different assumptions about the data error: constant, proportional to the heat and proportional but correlated. The statistics of the fitting indicate a preference for the proportional error model, in agreement with expectations for the conditions of the experiment, where uncertainties in the delivered titrant volume should dominate. With attention to proper procedures for propagating statistical error in the van't Hoff analysis, the differences between Delta H(cal) and Delta H(vH) are deemed statistically significant. In addition, statistically significant differences are observed for the Delta H(cal) estimates obtained for two different sources of Ba(2+), BaCl(2) and Ba(NO(3))(2). The effects are tentatively attributed to deficiencies in the standard procedure in ITC of subtracting a blank obtained for pure titrant from the thermogram obtained for the sample.
Asunto(s)
Compuestos de Bario/química , Calorimetría/métodos , Éteres Corona/química , Entropía , Éteres Cíclicos/química , Sitios de Unión , Cloruros/química , Modelos Estadísticos , Nitratos/química , Temperatura , VolumetríaRESUMEN
The extent of conformational change that calcium binding induces in EF-hand proteins is a key biochemical property specifying Ca(2+) sensor versus signal modulator function. To understand how differences in amino acid sequence lead to differences in the response to Ca(2+) binding, comparative analyses of sequence and structures, combined with model building, were used to develop hypotheses about which amino acid residues control Ca(2+)-induced conformational changes. These results were used to generate a first design of calbindomodulin (CBM-1), a calbindin D(9k) re-engineered with 15 mutations to respond to Ca(2+) binding with a conformational change similar to that of calmodulin. The gene for CBM-1 was synthesized, and the protein was expressed and purified. Remarkably, this protein did not exhibit any non-native-like molten globule properties despite the large number of mutations and the nonconservative nature of some of them. Ca(2+)-induced changes in CD intensity and in the binding of the hydrophobic probe, ANS, implied that CBM-1 does undergo Ca(2+) sensorlike conformational changes. The X-ray crystal structure of Ca(2+)-CBM-1 determined at 1.44 A resolution reveals the anticipated increase in hydrophobic surface area relative to the wild-type protein. A nascent calmodulin-like hydrophobic docking surface was also found, though it is occluded by the inter-EF-hand loop. The results from this first calbindomodulin design are discussed in terms of progress toward understanding the relationships between amino acid sequence, protein structure, and protein function for EF-hand CaBPs, as well as the additional mutations for the next CBM design.
Asunto(s)
Señalización del Calcio/fisiología , Motivos EF Hand/genética , Secuencia de Aminoácidos , Aminoácidos/química , Calbindinas , Calmodulina/química , Calmodulina/genética , Cristalografía por Rayos X , Diseño de Fármacos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Conformación Proteica , Proteína G de Unión al Calcio S100/química , Proteína G de Unión al Calcio S100/genética , Relación Estructura-ActividadRESUMEN
The ability to manipulate ligand-induced conformational change, although representing a major challenge to the protein engineer, is an essential end point in efforts to produce novel functional proteins for biotechnology and therapeutic applications. Progress towards this goal requires determining not only what factors control the fold and stability of a protein, but also how ligand binding alters the complex conformational/energetic landscape. Important strides are being made on several fronts, including understanding the origin of long-range effects and allosteric structural mechanisms, using both experimental and theoretical approaches.