Insights into proton translocation in cbb3 oxidase from MD simulations

Heme-copper oxidases are membrane protein complexes that catalyse the final step of the aerobic respiration, namely the reduction of oxygen to water. The energy released during catalysis is coupled to the active translocation of protons across the membrane, which contributes to the establishment of an electrochemical gradient that is used for ATP synthesis. The distinctive C-type (or cbb3) cytochrome c oxidases, which are mostly present in proteobacteria, exhibit a number of unique structural and functional features, including high catalytic activity at low oxygen concentrations. At the moment, the functioning mechanism of C-type oxidases, in particular the proton transfer/pumping mechanism presumably via a single proton channel, is still poorly understood. In this work we used all-atom molecular dynamics simulations and continuum electrostatics calculations to obtain atomic-level insights into the hydration and dynamics of a cbb3 oxidase. We provide the details of the water dynamics and proton transfer pathways for both the “chemical” and “pumped” protons, and show that formation of protonic connections is strongly affected by the protonation state of key residues, namely H243, E323 and H337

On the role of subunit M in cytochrome <i>cbb₃</i>oxidase

Cytochrome cbb3 (or C-type) oxidases are a highly divergent group and the least studied members of the heme-copper oxidases (HCOs) superfamily. HCOs couple the reduction of oxygen at the end of the respiratory chain to the active proton translocation across the membrane, contributing to establishment of an electrochemical gradient essential for ATP synthesis. Cbb3 oxidases exhibit unique structural and functional features and have an essential role in the metabolism of many clinically relevant human pathogens. Such characteristics make them a promising therapeutic target. Three subunits, N, O and P, comprise the core cbb3 complex, with N, the catalytic subunit, being highly conserved among all members of the HCO superfamily, including the A-type (aa3, mitochondrial-like) oxidases. An additional fourth subunit containing of a single transmembrane (TM) helix was present in the first crystal structure of cbb3. This TM segment was recently proposed to be part of a novel protein CcoM, which was shown to have a putative role in the complex stability and assembly. In this work, we performed large-scale all-atom molecular dynamics simulations of the CcoNOPM complex to further characterize the interactions between subunit M and the core subunits and to determine whether the presence of the 4th subunit influences the water/proton channels previously described for the core complex. The previously proposed putative CcoNOPH complex is also assessed, and the potential functional redundancy of CcoM and CcoQ is discussed.<br/

Mechanism of allosteric activation of human mRNA cap methyltransferase (RNMT) by RAM:Insights from accelerated molecular dynamics simulations

The RNA guanine-N7 methyltransferase (RNMT) in complex with RNMT-Activating Miniprotein (RAM) catalyses the formation of a N7-methylated guanosine cap structure on the 5’ end of nascent RNA polymerase II transcripts. The mRNA cap protects the primary transcript from exonucleases and recruits cap-binding complexes that mediate RNA processing, export and translation. By using microsecond standard and accelerated molecular dynamics simulations, we provide for the first time a detailed molecular mechanism of allosteric regulation of RNMT by RAM. We show that RAM selects the RNMT active site conformations that are optimal for binding of substrates (AdoMet and the cap), thus enhancing their affinity. Furthermore, our results strongly suggest the likely scenario in which the cap binding promotes the subsequent AdoMet binding, consistent with the previously suggested cooperative binding model. By employing the network community analyses, we revealed the underlying long-range allosteric networks and paths that are crucial for allosteric regulation by RAM. Our findings complement and explain previous experimental data on RNMT activity. Moreover, this study provides the most complete description of the cap and AdoMet binding poses and interactions within the enzyme’s active site. This information is critical for the drug discovery efforts that consider RNMT as a promising anti-cancer target

Intracellular Transfer of Na+ in an Active State G Protein Coupled Receptor

Playing a central role in cell signaling, G-protein-coupled receptors (GPCRs) are the largest superfamily of membrane proteins and form the majority of drug targets in humans. How extracellular agonist binding triggers the activation of GPCRs and associated intracellular effector proteins remains, however, poorly understood. Structural studies have revealed that inactive class A GPCRs harbor a conserved binding site for Na+ ions in the center of their transmembrane domain, accessible from the extracellular space. Here, we show that the opening of a conserved hydrated channel in the activated state receptors allows the Na+ ion to egress from its binding site into the cytosol. Coupled with protonation changes, this ion movement occurs without significant energy barriers, and can be driven by physiological transmembrane ion and voltage gradients. We propose that Na+ ion exchange with the cytosol is a key step in GPCR activation. Further, we hypothesize that this transition locks receptors in long-lived active-state conformations. Vickery et al. present molecular dynamics simulations and free energy calculations, which suggest that a key step in class A GPCR activation is the exchange of a Na+ ion from an extracellular binding pocket to the cytoplasm.</p

Theorie zeit- und frequenzaufgelöster spontaner Emissionsspektren

Theory of time- and frequency-gated spontaneous emission spectra is elaborated. The formulation establishes the validity of certain commonly assumed approximations and yields explicit expressions which are suitable for performing calculations for nontrivial systems. The spontaneous emission spectra are calculated for a class of model electron-transfer systems, describing by two electronically coupled diabatic states which are strongly coupled to a reaction mode, wich is weakly coupled to a dissipative environment, whose influence is described in the framework of Redfield theory. For suitably chosen parameters, the spectra are shown to reflect interesting features of ultrafast electron-transfer dynamics, such as electronic beatings (due to electronic coherence) and step-like electronic population decay (due to vibraltional coherence). The model explains general trends in the behavior of the time-resolved flourescence signals which have been in the recent literature.Zentrales Thema der vorliegenden Arbeit ist die Theorie der Femtosekundenspektroskopie molekularer Systeme. Im Mittelpunkt stand dabei die theoretische Beschreibung zeit- und frequenzaufgelöster spontaner Emissionsspektren. Die dabei entwickelte Theorie erlaubt es einerseits, häufig verwendete Näherungverfahren zu testen, andereseits wurden praktisch verwendbare Rechenverfahren entwickelt. Als Anwendung wurden spontane Emmisionsspektren von Elektrontransfer-Systemen simuliert. Die Modelle beinhalten dabei zwei diabatische Zustände, die stark an ein Reaktionsmode koppeln, welche mit einer dissipativen Umgebung wechselwirkt. Die Spektren zeigen eine Reihe interessanter Effekte in der Elektrontransfer-Dynamik, insbesondere durch elektronische Kohärenzen verursachte Oszillationen, sowie auf Schwingungskohärenzen zurückzuführende stufenartige Strukturen in der Populationsdynamik. Anhand der Modellstudien konnten auch eine Reihe von exprerimentellen Fluoreszenzspektren interpretiert werden

Parametric Projection Operator Technique for Second Order Non-linear Response

We demonstrate the application of the recently introduced parametric projector operator technique to a calculation of the second order non-linear optical response of a multilevel molecular system. We derive a parametric quantum master equation (QME) for the time evolution of the excited state of an excitonic system after excitation by the first two pulses in the usual spectroscopic four-wave-mixing scheme. This master equation differs from the usual QME by a correction term which depends on the delay \tau between the pulses. In the presence of environmental degrees of freedom with finite bath correlation time and in the presence of intramolecular vibrations we find distinct dynamics of both the excite state populations and the electronic coherence for different delays \tau.Comment: 15 pages, 8 figure

Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM

Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation

Capturing the essence of folding and functions of biomolecules using Coarse-Grained Models

The distances over which biological molecules and their complexes can function range from a few nanometres, in the case of folded structures, to millimetres, for example during chromosome organization. Describing phenomena that cover such diverse length, and also time scales, requires models that capture the underlying physics for the particular length scale of interest. Theoretical ideas, in particular, concepts from polymer physics, have guided the development of coarse-grained models to study folding of DNA, RNA, and proteins. More recently, such models and their variants have been applied to the functions of biological nanomachines. Simulations using coarse-grained models are now poised to address a wide range of problems in biology.Comment: 37 pages, 8 figure

Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase

Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis. Two isotopic hybrids were prepared; one with the mobile N-terminal segment containing heavy isotopes (2H, 13C, 15N) and the remainder of the protein with natural isotopic abundance, and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only a physical step of catalysis, whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling, which is difficult to obtain with other studies, and helps define the dynamic networks of intramolecular interactions central to enzyme catalysis

Electronic Coherence Dephasing in Excitonic Molecular Complexes: Role of Markov and Secular Approximations

We compare four different types of equations of motion for reduced density matrix of a system of molecular excitons interacting with thermodynamic bath. All four equations are of second order in the linear system-bath interaction Hamiltonian, with different approximations applied in their derivation. In particular we compare time-nonlocal equations obtained from so-called Nakajima-Zwanzig identity and the time-local equations resulting from the partial ordering prescription of the cummulant expansion. In each of these equations we alternatively apply secular approximation to decouple population and coherence dynamics from each other. We focus on the dynamics of intraband electronic coherences of the excitonic system which can be traced by coherent two-dimensional spectroscopy. We discuss the applicability of the four relaxation theories to simulations of population and coherence dynamics, and identify features of the two-dimensional coherent spectrum that allow us to distinguish time-nonlocal effects.Comment: 14 pages, 8 figure