Different responses of mice and rats hippocampus CA1 pyramidal neurons to in vitro and in vivo-like inputs.

The fundamental role of any neuron within a network is to transform complex spatiotemporal synaptic input patterns into individual output spikes. These spikes, in turn, act as inputs for other neurons in the network. Neurons must execute this function across a diverse range of physiological conditions, often based on species-specific traits. Therefore, it is crucial to determine the extent to which findings can be extrapolated between species and, ultimately, to humans. In this study, we employed a multidisciplinary approach to pinpoint the factors accounting for the observed electrophysiological differences between mice and rats, the two species most used in experimental and computational research. After analyzing the morphological properties of their hippocampal CA1 pyramidal cells, we conducted a statistical comparison of rat and mouse electrophysiological features in response to somatic current injections. This analysis aimed to uncover the parameters underlying these distinctions. Using a well-established computational workflow, we created ten distinct single-cell computational models of mouse CA1 pyramidal neurons, ready to be used in a full-scale hippocampal circuit. By comparing their responses to a variety of somatic and synaptic inputs with those of rat models, we generated experimentally testable hypotheses regarding species-specific differences in ion channel distribution, kinetics, and the electrophysiological mechanisms underlying their distinct responses to synaptic inputs during the behaviorally relevant Gamma and Sharp-Wave rhythms.

Discrimination between cyclic nucleotides in a cyclic nucleotide-gated ion channel.

Cyclic nucleotide-gated ion channels are crucial in many physiological processes such as vision and pacemaking in the heart. SthK is a prokaryotic homolog with high sequence and structure similarities to hyperpolarization-activated and cyclic nucleotide-modulated and cyclic nucleotide-gated channels, especially at the level of the cyclic nucleotide binding domains (CNBDs). Functional measurements showed that cyclic adenosine monophosphate (cAMP) is a channel activator while cyclic guanosine monophosphate (cGMP) barely leads to pore opening. Here, using atomic force microscopy single-molecule force spectroscopy and force probe molecular dynamics simulations, we unravel quantitatively and at the atomic level how CNBDs discriminate between cyclic nucleotides. We find that cAMP binds to the SthK CNBD slightly stronger than cGMP and accesses a deep-bound state that a cGMP-bound CNBD cannot reach. We propose that the deep binding of cAMP is the discriminatory state that is essential for cAMP-dependent channel activation.

Automated cryo-EM structure refinement using correlation-driven molecular dynamics.

We present a correlation-driven molecular dynamics (CDMD) method for automated refinement of atomistic models into cryo-electron microscopy (cryo-EM) maps at resolutions ranging from near-atomic to subnanometer. It utilizes a chemically accurate force field and thermodynamic sampling to improve the real-space correlation between the modeled structure and the cryo-EM map. Our framework employs a gradual increase in resolution and map-model agreement as well as simulated annealing, and allows fully automated refinement without manual intervention or any additional rotamer- and backbone-specific restraints. Using multiple challenging systems covering a wide range of map resolutions, system sizes, starting model geometries and distances from the target state, we assess the quality of generated models in terms of both model accuracy and potential of overfitting. To provide an objective comparison, we apply several well-established methods across all examples and demonstrate that CDMD performs best in most cases.

Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P.

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.

A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest.

Nascent polypeptides can induce ribosome stalling, regulating downstream genes. Stalling of ErmBL peptide translation in the presence of the macrolide antibiotic erythromycin leads to resistance in Streptococcus sanguis. To reveal this stalling mechanism we obtained 3.6-Å-resolution cryo-EM structures of ErmBL-stalled ribosomes with erythromycin. The nascent peptide adopts an unusual conformation with the C-terminal Asp10 side chain in a previously unseen rotated position. Together with molecular dynamics simulations, the structures indicate that peptide-bond formation is inhibited by displacement of the peptidyl-tRNA A76 ribose from its canonical position, and by non-productive interactions of the A-tRNA Lys11 side chain with the A-site crevice. These two effects combine to perturb peptide-bond formation by increasing the distance between the attacking Lys11 amine and the Asp10 carbonyl carbon. The interplay between drug, peptide and ribosome uncovered here also provides insight into the fundamental mechanism of peptide-bond formation.

Dynamic contact network between ribosomal subunits enables rapid large-scale rotation during spontaneous translocation.

During ribosomal translation, the two ribosomal subunits remain associated through intersubunit bridges, despite rapid large-scale intersubunit rotation. The absence of large barriers hindering rotation is a prerequisite for rapid rotation. Here, we investigate how such a flat free-energy landscape is achieved, in particular considering the large shifts the bridges undergo at the periphery. The dynamics and energetics of the intersubunit contact network are studied using molecular dynamics simulations of the prokaryotic ribosome in intermediate states of spontaneous translocation. Based on observed occupancies of intersubunit contacts, residues were grouped into clusters. In addition to the central contact clusters, peripheral clusters were found to maintain strong steady interactions by changing contacts in the course of rotation. The peripheral B1 bridges are stabilized by a changing contact pattern of charged residues that adapts to the rotational state. In contrast, steady strong interactions of the B4 bridge are ensured by the flexible helix H34 following the movement of protein S15. The tRNAs which span the subunits contribute to the intersubunit binding enthalpy to an almost constant degree, despite their different positions in the ribosome. These mechanisms keep the intersubunit interaction strong and steady during rotation, thereby preventing dissociation and enabling rapid rotation.

Energy barriers and driving forces in tRNA translocation through the ribosome.

During protein synthesis, tRNAs move from the ribosome's aminoacyl to peptidyl to exit sites. Here we investigate conformational motions during spontaneous translocation, using molecular dynamics simulations of 13 intermediate-translocation-state models obtained by combining Escherichia coli ribosome crystal structures with cryo-EM data. Resolving fast transitions between states, we find that tRNA motions govern the transition rates within the pre- and post-translocation states. Intersubunit rotations and L1-stalk motion exhibit fast intrinsic submicrosecond dynamics. The L1 stalk drives the tRNA from the peptidyl site and links intersubunit rotation to translocation. Displacement of tRNAs is controlled by 'sliding' and 'stepping' mechanisms involving conserved L16, L5 and L1 residues, thus ensuring binding to the ribosome despite large-scale tRNA movement. Our results complement structural data with a time axis, intrinsic transition rates and molecular forces, revealing correlated functional motions inaccessible by other means.

Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor.

Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially visible in the 30S IC, but in the 70S IC, it stabilizes interactions between IF2 and the L7/L12 stalk of the 50S, and on its deletion, proper N-formyl-methionyl(fMet)-tRNA(fMet) positioning and efficient transpeptidation are affected. Accordingly, fast kinetics and single-molecule fluorescence data indicate that the N terminus promotes 70S IC formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining. Together, our data highlight the dynamics of IF2-dependent ribosomal subunit joining and the role played by the N terminus of IF2 in this process.

Stochastic gating and drug-ribosome interactions.

Gentamicin is a potent antibiotic that is used in combination therapy for inhalation anthrax disease. The drug is also often used in therapy for methicillin-resistant Staphylococcusaureus. Gentamicin works by flipping a conformational switch on the ribosome, disrupting the reading head (i.e., 16S ribosomal decoding bases 1492-1493) used for decoding messenger RNA. We use explicit solvent all-atom molecular simulation to study the thermodynamics of the ribosomal decoding site and its interaction with gentamicin. The replica exchange molecular dynamics simulations used an aggregate sampling of 15 mus when summed over all replicas, allowing us to explicitly calculate the free-energy landscape, including a rigorous treatment of enthalpic and entropic effects. Here, we show that the decoding bases flip on a timescale faster than that of gentamicin binding, supporting a stochastic gating mechanism for antibiotic binding, rather than an induced-fit model where the bases only flip in the presence of a ligand. The study also allows us to explore the nonspecific binding landscape near the binding site and reveals that, rather than a two-state bound/unbound scenario, drug dissociation entails shuttling between many metastable local minima in the free-energy landscape. Special care is dedicated to validation of the obtained results, both by direct comparison to experiment and by estimation of simulation convergence.

Spontaneous Formation of KCl Aggregates in Biomolecular Simulations: A Force Field Issue?

Realistic all-atom simulation of biological systems requires accurate modeling of both the biomolecules and their ionic environment. Recently, ion nucleation phenomena leading to the rapid growth of KCl or NaCl clusters in the vicinity of biomolecular systems have been reported. To better understand this phenomenon, molecular dynamics simulations of KCl aqueous solutions at three (1.0, 0.25, and 0.10 M) concentrations were performed. Two popular water models (TIP3P and SPC/E) and two Lennard-Jones parameter sets (AMBER and Dang) were combined to produce a total of 80 ns of molecular dynamics trajectories. Results suggest that the use of the Dang cation Lennard-Jones parameters instead of those adopted by the AMBER force-field produces a more accurate description of the ionic solution. In the later case, formation of salt aggregates is probably indicative of an artifact resulting from misbalanced force-field parameters. Because similar results were obtained with two different water parameter sets, the simulations exclude a water model dependency in the formation of anomalous ionic clusters. Overall, the results strongly suggest that for accurate modeling of ions in biomolecular systems, great care should be taken in choosing balanced ionic parameters even when using the most popular force-fields. These results invite a reexamination of older data obtained using available force-fields and a thorough check of the quality of current parameters sets by performing simulations at finite (>0.25 M) instead of minimal salt conditions.

Fluorescence quenching of dyes by tryptophan: interactions at atomic detail from combination of experiment and computer simulation.

Fluorescence spectroscopy and molecular dynamics (MD) simulation are combined to characterize the interaction of two organic fluorescent dyes, rhodamine 6G (R6G) and an oxazine derivative (MR121), with the amino acid tryptophan in aqueous solution. Steady-state and time-resolved fluorescence quenching experiments reveal the formation of essentially nonfluorescent ground-state dye/Trp complexes. The MD simulations are used to elucidate the molecular interaction geometries involved. The MD-derived probability distribution of the distance r between the centers of geometry of the dye and quencher ring systems, P(r), extends to higher distances for R6G than for MR121 due to population in the R6G/Trp system of fluorescent interaction geometries between Trp and the phenyl ring and ester group of the dye. The consequence of this is the experimental finding that under the conditions used in the simulations about 25% of the R6G dye is fluorescent in comparison with 10% of the MR121. Combining the above findings allows determination of the "quenching distance", r, above which no quenching occurs. r is found to be very similar (approximately 5.5 A) for both dye/Trp systems, corresponding to close to van der Waals contact. Both experimental dynamic Stern-Volmer analysis and the MD trajectories demonstrate that the main determinant of the fluorescence intensity is static quenching. The approach presented is likely to be useful in the structural interpretation of data obtained from fluorescent conjugates commonly used for monitoring the binding and dynamics of biomolecular systems.

Molecular mechanics force field parameterization of the fluorescent probe rhodamine 6G using automated frequency matching.

Novel single-molecule fluorescence experimental techniques have prompted a growing need to develop refined computational models of dye-tagged biomolecules. As a necessary first step towards useful molecular simulations of fluorescence-labeled biomolecules, we have derived a force field for the commonly used dye, rhodamine 6G (R6G). A novel automated method is used that includes fitting the molecular mechanics potential to both vibrational frequencies and eigenvector projections derived from quantum chemical calculations. The method is benchmarked on a series of aromatic molecules then applied to derive new parameters for R6G. The force field derived reproduces well the crystal structure of R6G.