Modelling the active SARS-CoV-2 helicase complex as a basis for structure-based inhibitor design.

The RNA helicase (non-structural protein 13, NSP13) of SARS-CoV-2 is essential for viral replication, and it is highly conserved among the coronaviridae family, thus a prominent drug target to treat COVID-19. We present here structural models and dynamics of the helicase in complex with its native substrates based on thorough analysis of homologous sequences and existing experimental structures. We performed and analysed microseconds of molecular dynamics (MD) simulations, and our model provides valuable insights to the binding of the ATP and ssRNA at the atomic level. We identify the principal motions characterising the enzyme and highlight the effect of the natural substrates on this dynamics. Furthermore, allosteric binding sites are suggested by our pocket analysis. Our obtained structural and dynamical insights are important for subsequent studies of the catalytic function and for the development of specific inhibitors at our characterised binding pockets for this promising COVID-19 drug target.

A novel RNA pol II CTD interaction site on the mRNA capping enzyme is essential for its allosteric activation.

Recruitment of the mRNA capping enzyme (CE/RNGTT) to the site of transcription is essential for the formation of the 5' mRNA cap, which in turn ensures efficient transcription, splicing, polyadenylation, nuclear export and translation of mRNA in eukaryotic cells. The CE GTase is recruited and activated by the Serine-5 phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. Through the use of molecular dynamics simulations and enhanced sampling techniques, we provide a systematic and detailed characterization of the human CE-CTD interface, describing the effect of the CTD phosphorylation state, length and orientation on this interaction. Our computational analyses identify novel CTD interaction sites on the human CE GTase surface and quantify their relative contributions to CTD binding. We also identify, for the first time, allosteric connections between the CE GTase active site and the CTD binding sites, allowing us to propose a mechanism for allosteric activation. Through binding and activity assays we validate the novel CTD binding sites and show that the CDS2 site is essential for CE GTase activity stimulation. Comparison of the novel sites with cocrystal structures of the CE-CTD complex in different eukaryotic taxa reveals that this interface is considerably more conserved than previous structures have indicated.

A two-lane mechanism for selective biological ammonium transport.

The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+ transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea. The pathway underpins a mechanism by which charged H+ and neutral NH3 are carried separately across the membrane after NH4+ deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process.

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.

On the role of subunit M in cytochrome cbb3 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 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 fourth 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.

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.

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.

Molecular dynamics simulations reveal proton transfer pathways in cytochrome C-dependent nitric oxide reductase.

Nitric oxide reductases (NORs) are membrane proteins that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N(2)O), which is a critical step of the nitrate respiration process in denitrifying bacteria. Using the recently determined first crystal structure of the cytochrome c-dependent NOR (cNOR) [Hino T, Matsumoto Y, Nagano S, Sugimoto H, Fukumori Y, et al. (2010) Structural basis of biological N2O generation by bacterial nitric oxide reductase. Science 330: 1666-70.], we performed extensive all-atom molecular dynamics (MD) simulations of cNOR within an explicit membrane/solvent environment to fully characterize water distribution and dynamics as well as hydrogen-bonded networks inside the protein, yielding the atomic details of functionally important proton channels. Simulations reveal two possible proton transfer pathways leading from the periplasm to the active site, while no pathways from the cytoplasmic side were found, consistently with the experimental observations that cNOR is not a proton pump. One of the pathways, which was newly identified in the MD simulation, is blocked in the crystal structure and requires small structural rearrangements to allow for water channel formation. That pathway is equivalent to the functional periplasmic cavity postulated in cbb(3) oxidase, which illustrates that the two enzymes share some elements of the proton transfer mechanisms and confirms a close evolutionary relation between NORs and C-type oxidases. Several mechanisms of the critical proton transfer steps near the catalytic center are proposed.

Structural insights into electron transfer in caa3-type cytochrome oxidase.

Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme's function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36 Å resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain, which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.

Crystal structure of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus.

The structure of quinol-dependent nitric oxide reductase (qNOR) from G. stearothermophilus, which catalyzes the reduction of NO to produce the major ozone-depleting gas N(2)O, has been characterized at 2.5 Å resolution. The overall fold of qNOR is similar to that of cytochrome c-dependent NOR (cNOR), and some structural features that are characteristic of cNOR, such as the calcium binding site and hydrophilic cytochrome c domain, are observed in qNOR, even though it harbors no heme c. In contrast to cNOR, structure-based mutagenesis and molecular dynamics simulation studies of qNOR suggest that a water channel from the cytoplasm can serve as a proton transfer pathway for the catalytic reaction. Further structural comparison of qNOR with cNOR and aerobic and microaerobic respiratory oxidases elucidates their evolutionary relationship and possible functional conversions.

Enzyme millisecond conformational dynamics do not catalyze the chemical step.

The idea that enzymes catalyze reactions by dynamical coupling between the conformational motions and the chemical coordinates has recently attracted major experimental and theoretical interest. However, experimental studies have not directly established that the conformational motions transfer energy to the chemical coordinate, and simulating enzyme catalysis on the relevant timescales has been impractical. Here, we introduce a renormalization approach that transforms the energetics and dynamics of the enzyme to an equivalent low-dimensional system, and allows us to simulate the dynamical coupling on a ms timescale. The simulations establish, by means of several independent approaches, that the conformational dynamics is not remembered during the chemical step and does not contribute significantly to catalysis. Nevertheless, the precise nature of this coupling is a question of great importance.

Electrostatic basis for the unidirectionality of the primary proton transfer in cytochrome c oxidase.

Gaining detailed understanding of the energetics of the proton-pumping process in cytochrome c oxidase (CcO) is one of the challenges of modern biophysics. Despite promising mechanistic proposals, most works have not related the activation barriers of the different assumed steps to the protein structure, and there has not been a physically consistent model that reproduced the barriers needed to create a working pump. This work reevaluates the activation barriers for the primary proton transfer (PT) steps by calculations that reflect all relevant free energy contributions, including the electrostatic energies of the generated charges, the energies of water insertion, and large structural rearrangements of the donor and acceptor. The calculations have reproduced barriers that account for the directionality and sequence of events in the primary PT in CcO. It has also been found that the PT from Glu-286 (E) to the propionate of heme a(3) (Prd) provides a gate for an initial back leakage from the high pH side of the membrane. Interestingly, the rotation of E that brings it closer to Prd appears to provide a way for blocking competing pathways in the primary PT. Our study elucidates and quantifies the nature of the control of the directionality in the primary PT in CcO and provides instructive insight into the role of the water molecules in biological PT, showing that "bridges" of several water molecules in hydrophobic regions present a problem (rather than a solution) that is minimized in the primary PT.

The energetics of the primary proton transfer in bacteriorhodopsin revisited: it is a sequential light-induced charge separation after all.

The light-induced proton transport in bacteriorhodopsin has been considered as a model for other light-induced proton pumps. However, the exact nature of this process is still unclear. For example, it is not entirely clear what the driving force of the initial proton transfer is and, in particular, whether it reflects electrostatic forces or other effects. The present work simulates the primary proton transfer (PT) by a specialized combination of the EVB and the QCFF/PI methods. This combination allows us to obtain sufficient sampling and a quantitative free energy profile for the PT at different protein configurations. The calculated profiles provide new insight about energetics of the primary PT and its coupling to the protein conformational changes. Our finding confirms the tentative analysis of an earlier work (A. Warshel, Conversion of light energy to electrostatic energy in the proton pump of Halobacterium halobium, Photochem. Photobiol. 30 (1979) 285-290) and determines that the overall PT process is driven by the energetics of the charge separation between the Schiff base and its counterion Asp85. Apparently, the light-induced relaxation of the steric energy of the chromophore leads to an increase in the ion-pair distance, and this drives the PT process. Our use of the linear response approximation allows us to estimate the change in the protein conformational energy and provides the first computational description of the coupling between the protein structural changes and the PT process. It is also found that the PT is not driven by twist-modulated changes of the Schiff base's pKa, changes in the hydrogen bond directionality, or other non-electrostatic effects. Overall, based on a consistent use of structural information as the starting point for converging free energy calculations, we conclude that the primary event should be described as a light-induced formation of an unstable ground state, whose relaxation leads to charge separation and to the destabilization of the ion-pair state. This provides the driving force for the subsequent PT steps.

Polarizable Force Fields: History, Test Cases, and Prospects.

A consistent treatment of electrostatic energies is arguably the most important requirement for the realistic modeling of biological systems. An important part of electrostatic modeling is the ability to account for the polarizability of the simulated system. This can be done both macroscopically and microscopically, but the use of macroscopic models may lead to conceptual traps, which do not exist in the microscopic treatments. The present work describes the development of microscopic polarizable force fields starting with the introduction of these powerful tools and following some of the subsequent developments in the field. Special effort has been made to review a wide range of applications and emphasize cases when the use of polarizable force fields is important. Finally, a brief perspective is given on the future of this rapidly growing field.

Two-dimensional optical three-pulse photon echo spectroscopy. I. Nonperturbative approach to the calculation of spectra.

The nonperturbative approach to the calculation of nonlinear optical spectra of Seidner et al. [J. Chem. Phys. 103, 3998 (1995)] is extended to describe four-wave mixing experiments. The system-field interaction is treated nonperturbatively in the semiclassical dipole approximation, enabling a calculation of third order nonlinear spectroscopic signals directly from molecular dynamics and an efficient modeling of multilevel systems exhibiting relaxation and transfer phenomena. The method, coupled with the treatment of dynamics within the Bloch model, is illustrated by calculations of the two-dimensional three-pulse photon echo spectra of a simple model system-a two-electronic-level molecule. The nonperturbative calculations reproduce well-known results obtained by perturbative methods. Technical limitations of the nonperturbative approach in dealing with a dynamic inhomogeneity are discussed, and possible solutions are suggested. An application of the approach to an excitonically coupled dimer system with emphasis on the manifestation of complex exciton dynamics in two-dimensional optical spectra is presented in paper II Pisliakov et al. [J. Chem. Phys. 124, 234505 (2006), following paper].

Two-dimensional optical three-pulse photon echo spectroscopy. II. Signatures of coherent electronic motion and exciton population transfer in dimer two-dimensional spectra.

Using the nonperturbative approach to the calculation of nonlinear optical spectra developed in a foregoing paper [Mancal et al., J. Chem. Phys. 124, 234504 (2006), preceding paper], calculations of two-dimensional electronic spectra of an excitonically coupled dimer model system are presented. The dissipative exciton transfer dynamics is treated within the Redfield theory and energetic disorder within the molecular ensemble is taken into account. The manner in which the two-dimensional spectra reveal electronic couplings in the aggregate system and the evolution of the spectra in time is studied in detail. Changes in the intensity and shape of the peaks in the two-dimensional relaxation spectra are related to the coherent and dissipative dynamics of the system. It is shown that coherent electronic motion, an electronic analog of a vibrational wave packet, can manifest itself in two-dimensional optical spectra of molecular aggregate systems as a periodic modulation of both the diagonal and off-diagonal peaks.

The barrier for proton transport in aquaporins as a challenge for electrostatic models: the role of protein relaxation in mutational calculations.

The origin of the barrier for proton transport through the aquaporin channel is a problem of general interest. It is becoming increasingly clear that this barrier is not attributable to the orientation of the water molecules across the channel but rather to the electrostatic penalty for moving the proton charge to the center of the channel. However, the reason for the high electrostatic barrier is still rather controversial. It has been argued by some workers that the barrier is due to the so-called NPA motif and/or to the helix macrodipole or to other specific elements. However, our works indicated that the main reason for the high barrier is the loss of the generalized solvation upon moving the proton charge from the bulk to the center of the channel and that this does not reflect a specific repulsive electrostatic interaction but the absence of sufficient electrostatic stabilization. At this stage it seems that the elucidation and clarification of the origin of the electrostatic barrier can serve as an instructive test case for electrostatic models. Thus, we reexamine the free-energy surface for proton transport in aquaporins using the microscopic free-energy perturbation/umbrella sampling (FEP/US) and the empirical valence bond/umbrella sampling (EVB/US) methods as well as the semimacroscopic protein dipole Langevin dipole model in its linear response approximation version (the PDLD/S-LRA). These extensive studies help to clarify the nature of the barrier and to establish the "reduced solvation effect" as the primary source of this barrier. That is, it is found that the barrier is associated with the loss of the generalized solvation energy (which includes of course all electrostatic effects) upon moving the proton charge from the bulk solvent to the center of the channel. It is also demonstrated that the residues in the NPA region and the helix dipole cannot be considered as the main reasons for the electrostatic barrier. Furthermore, our microscopic and semimacroscopic studies clarify the problems with incomplete alternative calculations, illustrating that the effects of various electrostatic elements are drastically overestimated by macroscopic calculations that use a low dielectric constant and do not consider the protein reorganization. Similarly, it is pointed out that microscopic potential of mean force calculations that do not evaluate the electrostatic barrier relative to the bulk water cannot be used to establish the origin of the electrostatic barrier. The relationship between the present study and calculations of pK(a)s in protein interiors is clarified, pointing out that approaches that are applied to study the aquaporin barrier should be validated by pK(a)s calculations. Such calculations also help to clarify the crucial role of solvation energies in establishing the barrier in aquaporins.

Transient phenomena in time- and frequency-gated spontaneous emission.

The effect of overlapping pump and gate pulses on time- and frequency-gated spontaneous emission spectra is explored for a model of material dynamics that accounts for strong nonadiabatic and electron-vibrational coupling effects, vibrational relaxation, and optical dephasing, thus representing characteristic features of photoinduced excited-state dynamics in large molecules in the gas phase or in condensed phases. The behaviors of the sequential, coherent, and doorway-window contributions to the spontaneous emission spectrum are studied separately. The interrelation between the sequential and coherent contributions is demonstrated to be sensitive to the carrier frequencies of the pump and gate pulses and also to the optical dephasing rate, opening the possibility of an experimental determination of the latter. The coherent contribution is shown to dominate the spectrum at specific emission frequencies.