Author: rhoward

From the October 2019 issue of Journal of Chemical Theory & Computation (v. 15 pp. 6752–6759):

InfleCS: clustering free energy landscapes with Gaussian mixtures

Annie M. Westerlund, Lucie Delemotte

Free energy landscapes provide insights into conformational ensembles of biomolecules. In order to analyze these landscapes and elucidate mechanisms underlying conformational changes, there is a need to extract metastable states with limited noise. This has remained a formidable task, despite a plethora of existing clustering methods. We present InfleCS, a novel method for extracting well-defined core states from free energy landscapes. The method is based on a Gaussian mixture free energy estimator and exploits the shape of the estimated density landscape. The core states that naturally arise from the clustering allow for detailed characterization of the conformational ensemble. The clustering quality is evaluated on three toy models with different properties, where the method is shown to consistently outperform other conventional and state-of-the-art clustering methods. Finally, the method is applied to a temperature enhanced molecular dynamics simulation of Ca2+ -bound Calmodulin. Through the free energy landscape, we discover a pathway between a canonical and a compact state, revealing conformational changes driven by electrostatic interactions.

Read the full publication here.

Published 17 September 2019 in the Journal of Chemical Information and Modeling (v. 59 pp. 4093–4099):

Sharing data from molecular simulations

Mark Abraham, Rossen Apostolov, Jonathan Barnoud, Paul Bauer, Christian Blau, Alexandre MJJ Bonvin, Matthieu Chavent, John Chodera, Karmen Čondić-Jurkić, Lucie Delemotte, Helmut Grubmüller, Rebecca J Howard, E Joseph Jordan, Erik Lindahl, OH Samuli Ollila, Jana Selent, Daniel GA Smith, Phillip J Stansfeld, Johanna KS Tiemann, Mikael Trellet, Christopher Woods, Artem Zhmurov

Given the need for modern researchers to produce open, reproducible scientific output, the lack of standards and best practices for sharing data and workflows used to produce and analyze molecular dynamics (MD) simulations has become an important issue in the field. There are now multiple well-established packages to perform molecular dynamics simulations, often highly tuned for exploiting specific classes of hardware, each with strong communities surrounding them, but with very limited interoperability/transferability options. Thus, the choice of the software package often dictates the workflow for both simulation production and analysis. The level of detail in documenting the workflows and analysis code varies greatly in published work, hindering reproducibility of the reported results and the ability for other researchers to build on these studies. An increasing number of researchers are motivated to make their data available, but many challenges remain in order to effectively share and reuse simulation data. To discuss these and other issues related to best practices in the field in general, we organized a workshop in November 2018. Here, we present a brief overview of this workshop and topics discussed. We hope this effort will spark further conversation in the MD community to pave the way toward more open, interoperable, and reproducible outputs coming from research studies using MD simulations.

Read the full publication here.

For the November 2019 issue of Neuron (v. 104 pp. 501–511.e6):

Agonist selectivity and ion permeation in the α3β4 ganglionic nicotinic receptor

Anant Gharpure, Jinfeng Teng, Yuxuan Zhuang, Colleen M. Noviello, Richard M. Walsh Jr., Rico Cabuco, Rebecca J. Howard, Nurulain T. Zaveri, Erik Lindahl, Ryan E. Hibbs

Nicotinic acetylcholine receptors are pentameric ion channels that mediate fast chemical neurotransmission. The α3β4 nicotinic receptor subtype forms the principal relay between the central and peripheral nervous systems in the autonomic ganglia. This receptor is also expressed focally in brain areas that affect reward circuits and addiction. Here, we present structures of the α3β4 nicotinic receptor in lipidic and detergent environments, using functional reconstitution to define lipids appropriate for structural analysis. The structures of the receptor in complex with nicotine, as well as the α3β4-selective ligand AT-1001, complemented by molecular dynamics, suggest principles of agonist selectivity. The structures further reveal much of the architecture of the intracellular domain, where mutagenesis experiments and simulations define residues governing ion conductance.

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From the June 2019 issue of Nature Structural & Molecular Biology (v. 26 pp. 528–530):

Outlining the proton-conduction pathway in otopetrin channels

Lucie Delemotte

New structural work sheds light on the architecture of otopetrin channels, offering insights into the mechanisms for proton permeation in this family.

Read the full News & Views article here, referencing the publication Structures of the otopetrin proton channels Otop1 and Otop3 by Saotome & colleagues.

Accepted manuscript, posted ahead of online November 9, 2018 in eLife (v. 7 art. e42166):

New tools for automated high-resolution cryo-EM structure determination in RELION-3

Jasenko Zivanov, Takanori Nakane, Björn O Forsberg, Dari Kimanius, Wim JH Hagen, Erik Lindahl & Sjors HW Scheres

Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher-resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2–0.7 Å compared to previous RELION versions.

Read the full publication here.

From the October 16, 2018 issue of Proceedings of the National Academy of Sciences of the USA (v. 115 pp. 10672–10677):

Allosteric potentiation of a ligand-gated ion channel is mediated by access to a deep membrane-facing cavity

Stephanie A Heusser, Marie Lycksell, Xueqing Wang, Sarah E McComas, Rebecca J Howard & Erik Lindahl

Molecular mechanisms of general anesthetic modulation in pentameric ligand-gated ion channels remain controversial. Here we present molecular simulations and functional data that reveal correlations between dynamic differences in a membrane-accessible cavity and dramatic anesthetic effects, separate inhibitory and potentiating effects within the same electrophysiology recordings, and support a model for communication between the lipid bilayer and ion channel pore. In particular, enhanced electrostatic interactions in the membrane-accessible site were associated with a unique mode of anesthetic potentiation, persisting tens of minutes after washout. These results offer a bridge between lipid- and receptor-based theories of anesthesia, with the potential to inform both mechanistic understanding and drug development.

Read the full publication here.

From the September 25, 2018 issue of Scientific Reports (v. 8 art. 14324):

Uptake dynamics in the Lactose permease (LacY) membrane protein transporter

Dari Kimanius, Erik Lindahl & Magnus Andersson

The sugar transporter Lactose permease (LacY) of Escherichia coli has become a prototype to understand the underlying molecular details of membrane transport. Crystal structures have trapped the protein in sugar-bound states facing the periplasm, but with narrow openings unable to accommodate sugar. Therefore, the molecular details of sugar uptake remain elusive. In this work, we have used extended simulations and metadynamics sampling to explore a putative sugar-uptake pathway and associated free energy landscape. We found an entrance at helix-pair 2 and 11, which involved lipid head groups and residues Gln 241 and Gln 359. Furthermore, the protein displayed high flexibility on the periplasmic side of Phe 27, which is located at the narrowest section of the pathway. Interactions to Phe 27 enabled passage into the binding site, which was associated with a 24 ± 4 kJ/mol binding free energy in excellent agreement with an independent binding free energy calculation and experimental data. Two free energy minima corresponding to the two possible binding poses of the lactose analog β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG) were aligned with the crystal structure-binding pocket. This work outlines the chemical environment of a putative periplasmic sugar pathway and paves way for understanding substrate affinity and specificity in LacY.

Corresponding author and former group member Magnus Andersson can now be reached at Umeå University. Read the full publication here.

Commentary for the October 1, 2018 issue of the Journal of General Physiology (v. 150 art. 1356):

Opening leads to closing: Allosteric crosstalk between the activation and inactivation gates in KcsA

Lucie Delemotte

Voltage-gated potassium (Kv) channels control a number of different physiological processes, including the firing rate in axons. Such K+ channels display a reduction of conductance after exposure to a prolonged activating stimulus. This process, referred to as inactivation, causes repolarization of the cell membrane after the depolarizing phase of an action potential. The transient openings that result from it also allow neurons to readily fire a new action potential. Two types of inactivation mechanisms have been described in Kv channels (Hoshi et al., 1990). Fast inactivation, also called N-type inactivation, results from a mechanism that has been ascribed to pore blocking by a N-terminal peptide. Slow inactivation, or C-type inactivation, is revealed upon suppression of fast inactivation and is thought to be due to a conformational change occurring within the pore of the channel. While the structural basis of C-type inactivation appears to have been established, how it is dynamically coupled to channel activation remains to be understood in detail. In the Journal of General Physiology, a new study (see Li et al. 2018) proposes an intriguing mechanism for the allosteric control of C-type inactivation by the activation gate in the bacterial K+ channel KcsA.

Read the full commentary here.

From the August 30, 2018 issue of Physical Review E (v. 98 art. 023312):

Riemann metric approach to optimal sampling of multidimensional free-energy landscapes

Viveca Lindahl, Jack Lidmar & Berk Hess

Exploring the free-energy landscape along reaction coordinates or system parameters λ is central to many studies of high-dimensional model systems in physics, e.g., large molecules or spin glasses. In simulations this usually requires sampling conformational transitions or phase transitions, but efficient sampling is often difficult to attain due to the roughness of the energy landscape. For Boltzmann distributions, crossing rates decrease exponentially with free-energy barrier heights. Thus, exponential acceleration can be achieved in simulations by applying an artificial bias along λ tuned such that a flat target distribution is obtained. A flat distribution is, however, an ambiguous concept unless a proper metric is used and is generally suboptimal. Here we propose a multidimensional Riemann metric, which takes the local diffusion into account, and redefine uniform sampling such that it is invariant under nonlinear coordinate transformations. We use the metric in combination with the accelerated weight histogram method, a free-energy calculation and sampling method, to adaptively optimize sampling toward the target distribution prescribed by the metric. We demonstrate that for complex problems, such as molecular dynamics simulations of DNA base-pair opening, sampling uniformly according to the metric, which can be calculated without significant computational overhead, improves sampling efficiency by 50%–70%.

Read the full publication here.

From the June 1, 2018 release of eLife (v. 7 art. e36861):

Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION

Takanori Nakane, Dari Kimanius, Erik Lindahl & Sjors HW Scheres

Macromolecular complexes that exhibit continuous forms of structural flexibility pose a challenge for many existing tools in cryo-EM single-particle analysis. We describe a new tool, called multi-body refinement, which models flexible complexes as a user-defined number of rigid bodies that move independently from each other. Using separate focused refinements with iteratively improved partial signal subtraction, the new tool generates improved reconstructions for each of the defined bodies in a fully automated manner. Moreover, using principal component analysis on the relative orientations of the bodies over all particle images in the data set, we generate movies that describe the most important motions in the data. Our results on two test cases, a cytoplasmic ribosome from Plasmodium falciparum, and the spliceosomal B-complex from yeast, illustrate how multi-body refinement can be useful to gain unique insights into the structure and dynamics of large and flexible macromolecular complexes.

Read the full publication here.

From the June 2018 release of The Journal of Membrane Biology (v. 251 pp. 419–430):

Exploring the viral channel Kcv(PBCV-1) function via computation

Alma EV Andersson, Marina A Kasimova & Lucie Delemotte

Viral potassium channels (Kcv) are homologous to the pore module of complex K⁺-selective ion channels of cellular organisms. Due to their relative simplicity, they have attracted interest towards understanding the principles of K⁺ conduction and channel gating. In this work, we construct a homology model of the Kcv(PBCV-1) open state, which we validate by studying the binding of known blockers and by monitoring ion conduction through the channel. Molecular dynamics simulations of this model reveal that the re-orientation of selectivity filter carbonyl groups coincides with the transport of potassium ions, suggesting a possible mechanism for fast gating. In addition, we show that the voltage sensitivity of this mechanism can originate from the relocation of potassium ions inside the selectivity filter. We also explore the interaction of Kcv(PBCV-1) with the surrounding bilayer and observe the binding of lipids in the area between two adjacent subunits. The model is available to the scientific community to further explore the structure/function relationship of Kcv channels.

Read the full publication here.

Featured on the April 24, 2018 cover of Cell Reports (v. 23 pp. 993–1004):

Structural basis for a bimodal allosteric mechanism of general anesthetic modulation in pentameric ligand-gated ion channels

Zaineb Fourati*, Rebecca J Howard*, Stephanie A Heusser, Haidai Hu, Reinis R Ruza, Ludovic Sauguet, Erik Lindahl** & Marc Delarue**

*Equal contributions; **senior authors

Ion channel modulation by general anesthetics is a vital pharmacological process with implications for receptor biophysics and drug development. Functional studies have implicated conserved sites of both potentiation and inhibition in pentameric ligand-gated ion channels, but a detailed structural mechanism for these bimodal effects is lacking. The prokaryotic model protein GLIC recapitulates anesthetic modulation of human ion channels, and it is accessible to structure determination in both apparent open and closed states. Here, we report ten X-ray structures and electrophysiological characterization of GLIC variants in the presence and absence of general anesthetics, including the surgical agent propofol. We show that general anesthetics can allosterically favor closed channels by binding in the pore or favor open channels via various subsites in the transmembrane domain. Our results support an integrated, multi-site mechanism for allosteric modulation, and they provide atomic details of both potentiation and inhibition by one of the most common general anesthetics.

Read the full publication here.

From the April 2018 release of Biochimica et Biophysica Acta – Biomembranes (v. 1860 pp. 927–942):

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport

Rebecca J Howard, Vincenzo Carnevale, Lucie Delemotte, Ute A Hellmich & Brad S Rothberg

Ion translocation across biological barriers is a fundamental requirement for life. In many cases, controlling this process—for example with neuroactive drugs—demands an understanding of rapid and reversible structural changes in membrane-embedded proteins, including ion channels and transporters. Classical approaches to electrophysiology and structural biology have provided valuable insights into several such proteins over macroscopic, often discontinuous scales of space and time. Integrating these observations into meaningful mechanistic models now relies increasingly on computational methods, particularly molecular dynamics simulations, while surfacing important challenges in data management and conceptual alignment. Here, we seek to provide contemporary context, concrete examples, and a look to the future for bridging disciplinary gaps in biological ion transport. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Read the full review article here.

From the April 3, 2018 release of PLoS Computational Biology (v. 14 art. e1006072):

Effect of Ca²⁺ on the promiscuous target-protein binding of calmodulin

Annie M Westerlund & Lucie Delemotte

Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca²⁺-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca²⁺-bound and Ca²⁺-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca²⁺ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.

Read the full publication here.