RESUMEN
Based on the crystal structures, three possible sequence determinants have been suggested as the cause of a 285 mV increase in reduction potential of the rubredoxin domain of rubrerythrin over rubredoxin by modulating the polar environment around the redox site. Here, electrostatic calculations of crystal structures of rubredoxin and rubrerythrin and molecular dynamics simulations of rubredoxin wild-type and mutants are used to elucidate the contributions to the increased reduction potential. Asn(160) and His(179) in rubrerythrin versus valines in rubredoxins are predicted to be the major contributors, as the polar side chains contribute significantly to the electrostatic potential in the redox site region. The mutant simulations show both side chains rotating on a nanosecond timescale between two conformations with different electrostatic contributions. Reduction also causes a change in the reduction energy that is consistent with a linear response due to the interesting mechanism of shifting the relative populations of the two conformations. In addition to this, a simulation of a triple mutant indicates the side-chain rotations are approximately anticorrelated so whereas one is in the high potential conformation, the other is in the low potential conformation. However, Ala(176) in rubrerythrin versus a leucine in rubredoxin is not predicted to be a large contributor, because the solvent accessibility increases only slightly in mutant simulations and because it is buried in the interface of the rubrerythrin homodimer.
Asunto(s)
Hemeritrina/química , Rubredoxinas/química , Secuencia de Aminoácidos , Clostridium , Cristalografía por Rayos X , Desulfovibrio vulgaris , Hemeritrina/genética , Hemeritrina/metabolismo , Simulación de Dinámica Molecular , Mutación , Oxidación-Reducción , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Rubredoxinas/genética , Rubredoxinas/metabolismo , Homología de Secuencia de Aminoácido , Electricidad Estática , Factores de TiempoRESUMEN
Unlike human thrombin, murine thrombin lacks Na+ activation due to the charge reversal substitution D222K in the Na+ binding loop. However, the enzyme is functionally stabilized in a Na+-bound form and is highly active toward physiologic substrates. The structural basis of this peculiar property is unknown. Here, we present the 2.2 A resolution x-ray crystal structure of murine thrombin in the absence of inhibitors and salts. The enzyme assumes an active conformation, with Ser-195, Glu-192, and Asp-189 oriented as in the Na+-bound fast form of human thrombin. Lys-222 completely occludes the pore of entry to the Na+ binding site and positions its side chain inside the pore, with the Nzeta atom H-bonded to the backbone oxygen atoms of Lys-185, Asp-186b, and Lys-186d. The same architecture is observed in the 1.75 A resolution structure of a thrombin chimera in which the human enzyme carries all residues defining the Na+ pore in the murine enzyme. These findings demonstrate that Na+ activation in thrombin is linked to the architecture of the Na+ pore. The molecular strategy of Na+ activation mimicry unraveled for murine thrombin is relevant to serine proteases and enzymes activated by monovalent cations in general.
Asunto(s)
Sodio/química , Trombina/química , Sustitución de Aminoácidos , Animales , Sitios de Unión/genética , Cationes Monovalentes , Cristalografía por Rayos X , Activación Enzimática , Humanos , Ratones , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Unión Proteica/genética , Estructura Secundaria de Proteína , Sodio/metabolismo , Especificidad de la Especie , Relación Estructura-Actividad , Trombina/genética , Trombina/metabolismoRESUMEN
Molecular dynamics simulations based on a 0.95-A resolution crystal structure of Pyrococcus furiosus have been performed to elucidate the effects of the environment on the structure of rubredoxin, and proteins in general. Three 1-ns simulations are reported here: two crystalline state simulations at 123 and 300 K, and a solution state simulation at 300 K. These simulations show that temperature has a greater impact on the protein structure than the close molecular contacts of the crystal matrix in rubredoxin, although both have an effect on its dynamic properties. These results indicate that differences between NMR solution structures and X-ray crystal structures will be relatively minor if they are done at similar temperatures. In addition, the crystal simulations appears to mimic previous crystallographic experiments on the effects of cryo-temperature on temperature factors, and might provide a useful tool in the structural analysis of protein structures solved at cryo-temperatures.
Asunto(s)
Proteínas Bacterianas/química , Pyrococcus furiosus/metabolismo , Rubredoxinas/química , Simulación por Computador , Cristalografía por Rayos X , Ambiente , Cinética , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
Predicting the effects of mutation on the reduction potential of proteins is crucial in understanding how reduction potentials are modulated by the protein environment. Previously, we proposed that an alanine vs. a valine at residue 44 leads to a 50-mV difference in reduction potential found in homologous rubredoxins because of a shift in the polar backbone relative to the iron site due to the different side-chain sizes. Here, the aim is to determine the effects of mutations to glycine, isoleucine, and leucine at residue 44 on the structure and reduction potential of rubredoxin, and if the effects are proportional to side-chain size. Crystal structure analysis, molecular mechanics simulations, and experimental reduction potentials of wild-type and mutant Clostridium pasteurianum rubredoxin, along with sequence analysis of homologous rubredoxins, indicate that the backbone position relative to the redox site as well as solvent penetration near the redox site are both structural determinants of the reduction potential, although not proportionally to side-chain size. Thus, protein interactions are too complex to be predicted by simple relationships, indicating the utility of molecular mechanics methods in understanding them.