RESUMEN
Brr2 is an essential Ski2-like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N-terminal helicase cassette and a structurally similar but enzymatically inactive C-terminal helicase cassette connected by a linker region. Both cassettes contain a nucleotide-binding pocket, but it is unclear whether nucleotide binding in these two pockets is related. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and determine whether occupancy of one nucleotide-binding site may influence nucleotide binding at the other cassette. Our results show that Brr2 exhibits high specificity for adenine nucleotides, with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C-terminal cassette is more than two orders of magnitude higher than that of the active N-terminal cassette, as determined by slow nucleotide release. Mutations at the intercassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Moreover, we found that abrogation of nucleotide binding at the C-terminal cassette reduces nucleotide binding at the N-terminal cassette 70 Å away. Molecular dynamics simulations identified structural communication lines that likely mediate these long-range allosteric effects, predominantly across the intercassette interface. Together, our results reveal intricate networks of intramolecular interactions in the complex Brr2 RNA helicase, which fine-tune its nucleotide affinities and which could be exploited to regulate enzymatic activity during splicing.
Asunto(s)
Nucleótidos de Adenina/metabolismo , ARN Helicasas/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Aminoácidos/metabolismo , Sitios de Unión , Humanos , Cinética , Simulación de Dinámica Molecular , Mutación/genética , Dominios Proteicos , Ribonucleoproteínas Nucleares Pequeñas/química , Especificidad por SustratoRESUMEN
Proton transfer in cytochrome c oxidase (CcO) from the cellular inside to the binuclear redox centre as well as proton pumping through the membrane takes place through proton entrance via two distinct pathways, the D- and K-channel. Both channels show a dependence of their hydration level on the protonation states of their key residues, K362 for the K-channel, and E286 or D132 for the D-channel. In the oxidative half of CcO's catalytic cycle the D-channel is the proton-conducting path. For this channel, an interplay of protonation state of the D-channel residues with the water and hydrogen-bond dynamics has been observed in molecular dynamics simulations of the CcO protein, embedded in a lipid bi-layer, modelled in different protonation states. Protonation of residue E286 at the end of the D-channel results in a hydrogen-bonded network pointing from E286 to N139, that is against proton transport, and favouring N139 conformations which correspond to a closed asparagine gate (formed by residues N121 and N139). Consequently, the hydration level is lower than with unprotonated E286. In those models, the Asn gate is predominantly open, allowing water molecules to pass and thus increase the hydration level. The hydrogen-bonded network in these states exhibits longer life times of the Asn residues with water than other models and shows the D-channel to be traversable from the entrance, D132, to exit, E286. The D-channel can thus be regarded as auto-regulated with respect to proton transport, allowing proton passage only when required, that is the proton is located at the lower part of the D-channel (D132 to Asn gate) and not at the exit (E286).
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Agua/metabolismo , Complejo IV de Transporte de Electrones/química , Enlace de Hidrógeno , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Simulación de Dinámica Molecular , Conformación Proteica , ProtonesRESUMEN
Proton transfer in cytochrome c oxidase from the cellular inside to the binuclear redox center (BNC) can occur through two distinct pathways, the D- and K-channels. For the protein to function as both a redox enzyme and a proton pump, proton transfer into the protein toward the BNC or toward a proton loading site (and ultimately through the membrane) must be highly regulated. The PR â F transition is the first step in a catalytic cycle that requires proton transfer from the bulk at the N-side to the BNC. Molecular dynamics simulations of the PR â F intermediate of this transition, with 16 different combinations of protonation states of key residues in the D- and K-channel, show the impact of the K-channel on the D-channel to be protonation-state dependent. Strength as well as means of communication, correlations in positions, or communication along the hydrogen-bonded network depends on the protonation state of the K-channel residue K362. The conformational and hydrogen-bond dynamics of the D-channel residue N139 is regulated by an interplay of protonation in the D-channel and K362. N139 thus assumes a gating function by which proton passage through the D-channel toward E286 is likely facilitated for states with protonated K362 and unprotonated E286. In contrast, proton passage through the D-channel is hindered by N139's preference for a closed conformation in situations with protonated E286.
Asunto(s)
Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/metabolismo , Simulación de Dinámica Molecular , Protones , Enlace de Hidrógeno , Oxidación-Reducción , Conformación ProteicaRESUMEN
Proton transfer in cytochrome c oxidase from the cellular inside to the binuclear redox center (BNC) can occur through two distinct pathways, the D- and K-channels. For the protein to function as both redox enzyme and proton pump, proton transfer out of either of the channels toward the BNC or into the protein toward a proton loading site, and ultimately through the membrane, must be highly regulated. The OâE intermediate of cytochrome c oxidase is the first redox state in its catalytic cycle, where proton transfer through the K-channel, from K362 to Y288 at the BNC, is important. Molecular dynamics simulations of this intermediate with 16 different combinations of protonation states of key residues in the D- and K-channel show the mutual impact of the two proton-conducting channels to be protonation state-dependent. Strength as well as means of communication, correlations in positions, or connections along the hydrogen-bonded network, change with the protonation state of the K-channel residue K362. The conformational and hydrogen-bond dynamics of the D-channel residue N139 regulated by an interplay of protonation in the D-channel and K362. N139 thus assumes a gating function by which proton passage through the D-channel toward E286 is likely facilitated for states with protonated K362 and unprotonated E286, which would in principle allow proton transfer to the BNC, but no proton pumping until a proton has reached E286.
Asunto(s)
Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/metabolismo , Protones , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
The width of the DNA minor groove varies with sequence and can be a major determinant of DNA shape recognition by proteins. For example, the minor groove within the center of the Fis-DNA complex narrows to about half the mean minor groove width of canonical B-form DNA to fit onto the protein surface. G/C base pairs within this segment, which is not contacted by the Fis protein, reduce binding affinities up to 2000-fold over A/T-rich sequences. We show here through multiple X-ray structures and binding properties of Fis-DNA complexes containing base analogs that the 2-amino group on guanine is the primary molecular determinant controlling minor groove widths. Molecular dynamics simulations of free-DNA targets with canonical and modified bases further demonstrate that sequence-dependent narrowing of minor groove widths is modulated almost entirely by the presence of purine 2-amino groups. We also provide evidence that protein-mediated phosphate neutralization facilitates minor groove compression and is particularly important for binding to non-optimally shaped DNA duplexes.
Asunto(s)
ADN/química , Factor Proteico para Inverción de Estimulación/química , Emparejamiento Base , ADN/metabolismo , Factor Proteico para Inverción de Estimulación/metabolismo , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Fosfatos/química , Unión Proteica , Purinas/químicaRESUMEN
We present a method and web server for predicting DNA structural features in a high-throughput (HT) manner for massive sequence data. This approach provides the framework for the integration of DNA sequence and shape analyses in genome-wide studies. The HT methodology uses a sliding-window approach to mine DNA structural information obtained from Monte Carlo simulations. It requires only nucleotide sequence as input and instantly predicts multiple structural features of DNA (minor groove width, roll, propeller twist and helix twist). The results of rigorous validations of the HT predictions based on DNA structures solved by X-ray crystallography and NMR spectroscopy, hydroxyl radical cleavage data, statistical analysis and cross-validation, and molecular dynamics simulations provide strong confidence in this approach. The DNAshape web server is freely available at http://rohslab.cmb.usc.edu/DNAshape/.
Asunto(s)
ADN/química , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN , Programas Informáticos , Genómica , Internet , Conformación de Ácido NucleicoRESUMEN
We present a combined investigation of the dynamics and optics of triplex DNA, based on classical molecular dynamics and time-dependent density functional theory. Our approach is devised to include the effects of conformational fluctuations on the electronic structure and optical excitations of the system. We find that the structural flexibility has a strong role in the determination of the optical signals. Our results allow us to unravel the peculiar fingerprints of Watson-Crick and Hoogsteen H-bonding in the optical absorption spectra. We find a specific optical absorption feature that is due to the simultaneous presence of the two H-bonding patterns in C(+)GC triplets. While this peculiar triplet signal is wiped out in some structures that are representative of the finite-temperature dynamics, it can be recovered in an average view, so that it is a pristine result of this work.