In vertebrates, skin upholds homeostasis by preventing body water loss. The skin’s permeability barrier is located intercellularly in stratum corneum and consists of stacked lipid lamellae composed of ceramides, cholesterol and free fatty acids. We have combined cryo-EM with molecular dynamics modelling and EM-simulation in our analysis of the lamellae’s formation, a maturation process beginning in stratum granulosum and ending in stratum corneum. Previously, we have revealed the lipid lamellae’s initial- and end-stage molecular organizations. Here, we reveal two cryo-EM patterns representing intermediate stages in the lamellae’s maturation process: a single-band pattern with 2.0-2.5 nm periodicity and a two-band pattern with 5.5-6.0 nm periodicity, that may be derived from lamellar lipid structures with 4.0-5.0 nm and 5.5-6.0 nm periodicity, respectively. Based on the analysis of the data now available on the four maturation stages identified, we can present a tentative molecular model for the complete skin barrier formation process.
Many membrane proteins are modulated by external stimuli, such as small molecule binding or change in pH, transmembrane voltage, or temperature. This modulation typically occurs at sites that are structurally distant from the functional site. Revealing the communication, known as allostery, between these two sites is key to understanding the mechanistic details of these proteins. Residue interaction networks of isolated proteins are commonly used to this end. Membrane proteins, however, are embedded in a lipid bilayer, which may contribute to allosteric communication. The fast diffusion of lipids hinders direct use of standard residue interaction networks. Here, we present an extension that includes cofactors such as lipids and small molecules in the network. The novel framework is applied to three membrane proteins: a voltage-gated ion channel (KCNQ1), a G-protein coupled receptor (GPCR—β2 adrenergic receptor), and a pH-gated ion channel (KcsA). Through systematic analysis of the obtained networks and their components, we demonstrate the importance of lipids for membrane protein allostery. Finally, we reveal how small molecules may stabilize different protein states by allosterically coupling and decoupling the protein from the membrane.
The introduction of accelerator devices such as graphics processing units (GPUs) has had profound impact on molecular dynamics simulations and has enabled order-of-magnitude performance advances using commodity hardware. To fully reap these benefits, it has been necessary to reformulate some of the most fundamental algorithms, including the Verlet list, pair searching, and cutoffs. Here, we present the heterogeneous parallelization and acceleration design of molecular dynamics implemented in the GROMACS codebase over the last decade. The setup involves a general cluster-based approach to pair lists and non-bonded pair interactions that utilizes both GPU and central processing unit (CPU) single instruction, multiple data acceleration efficiently, including the ability to load-balance tasks between CPUs and GPUs. The algorithm work efficiency is tuned for each type of hardware, and to use accelerators more efficiently, we introduce dual pair lists with rolling pruning updates. Combined with new direct GPU–GPU communication and GPU integration, this enables excellent performance from single GPU simulations through strong scaling across multiple GPUs and efficient multi-node parallelization.
Since its initiation by the Royal Swedish Academy of Sciences in 2011, the Young Academy has provided an independent, interdisciplinary platform for the most prominent younger researchers in Sweden. Its ~35 members meet regularly across institutional and disciplinary borders during their 5-year terms to discuss research, science policy, and related topics.
The pyruvate dehydrogenase complex (PDC) is a multienzyme complex central to aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Similar to the E3-binding protein (E3BP) of mammalian PDC, PX selectively recruits E3 to the fungal PDC, but its divergent sequence suggests a distinct structural mechanism. Here, we report reconstructions of PDC from the filamentous fungus Neurospora crassa by cryo-electron microscopy, where we find protein X (PX) interior to the PDC core as opposed to substituting E2 core subunits as in mammals. Steric occlusion limits PX binding, resulting in predominantly tetrahedral symmetry, explaining previous observations in Saccharomyces cerevisiae. The PX-binding site is conserved in (and specific to) fungi, and complements possible C-terminal binding motifs in PX that are absent in mammalian E3BP. Consideration of multiple symmetries thus reveals a differential structural basis for E3BP-like function in fungal PDC.
Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.
As we return to the (semi-) normal academic year, Molecular Biophysics Stockholm also celebrated PhD student Yuxuan Zhuang‘s successful completion of a competitive summer internship contributing to MDAnalysis, a freely available, open-source object-oriented Python library to analyze trajectories from molecular dynamics simulations.
Zhuang’s internship was awarded through the Google Summer of Code, focused on bringing more student developers into open source software development. Just past its 15th year, the global program has produced over 38 million lines of code for 715 open source organizations.
Read Zhuang’s final remarks on his summer project, Serialization of the MDAnalysis-Universe for Parallelism, here.
Pulsed electric fields are increasingly used in medicine to transiently increase the cell membrane permeability via electroporation to deliver therapeutic molecules into the cell. One type of event that contributes to this increase in membrane permeability is the formation of pores in the membrane lipid bilayer. However, electrophysiological measurements suggest that membrane proteins are affected as well, particularly voltage-gated ion channels (VGICs). The molecular mechanisms by which the electric field could affects these molecules remain unidentified. In this study, we used molecular dynamics simulations to unravel the molecular events that take place in different VGICs when exposing them to electric fields mimicking electroporation conditions. We show that electric fields can induce pores in the voltage-sensor domains (VSDs) of different VGICs and that these pores form more easily in some channels than in others. We demonstrate that poration is more likely in VSDs that are more hydrated and are electrostatically more favorable for the entry of ions. We further show that pores in VSDs can expand into so-called complex pores, which become stabilized by lipid headgroups. Our results suggest that such complex pores are considerably more stable than conventional lipid pores, and their formation can lead to severe unfolding of VSDs from the channel. We anticipate that such VSDs become dysfunctional and unable to respond to changes in transmembrane voltage, which is in agreement with previous electrophysiological measurements showing a decrease in the voltage-dependent transmembrane ionic currents after pulse treatment. Finally, we discuss the possibility of activation of VGICs by submicrosecond-duration pulses. Overall, our study reveals a new, to our knowledge, mechanism of electroporation through membranes containing VGICs.
We use large-scale molecular dynamics to study the dynamics at the three-phase contact line in electrowetting of water and electrolytes on no-slip substrates. Under the applied electrostatic potential the line friction at the contact line is diminished. The effect is consistent for droplets of different sizes as well as for both pure water and electrolyte solution droplets. We analyze the electric field at the contact line to show how it assists ions and dipolar molecules to advance the contact line. Without an electric field, the interaction between a substrate and a liquid has a very short range, mostly affecting the bottom, immobilized layer of liquid molecules which leads to high friction since mobile molecules are not pulled towards the surface. In electrowetting, the electric field attracts charged and polar molecules over a longer range, which diminishes the friction.
Pentameric ligand-gated ion channels (pLGICs) are allosteric receptors that mediate rapid electrochemical signal transduction in the animal nervous system through the opening of an ion pore upon binding of neurotransmitters. Orthologs have been found and characterized in prokaryotes and they display highly similar structure–function relationships to eukaryotic pLGICs; however, they often encode greater architectural diversity involving additional amino-terminal domains (NTDs). Here we report structural, functional, and normal-mode analysis of two conformational states of a multidomain pLGIC, called DeCLIC, from a Desulfofustis deltaproteobacterium, including a periplasmic NTD fused to the conventional ligand-binding domain (LBD). X-ray structure determination revealed an NTD consisting of two jelly-roll domains interacting across each subunit interface. Binding of Ca2+ at the LBD subunit interface was associated with a closed transmembrane pore, with resolved monovalent cations intracellular to the hydrophobic gate. Accordingly, DeCLIC-injected oocytes conducted currents only upon depletion of extracellular Ca2+; these were insensitive to quaternary ammonium block. Furthermore, DeCLIC crystallized in the absence of Ca2+ with a wide-open pore and remodeled periplasmic domains, including increased contacts between the NTD and classic LBD agonist-binding sites. Functional, structural, and dynamical properties of DeCLIC paralleled those of sTeLIC, a pLGIC from another symbiotic prokaryote. Based on these DeCLIC structures, we would reclassify the previous structure of bacterial ELIC (the first high-resolution structure of a pLGIC) as a “locally closed” conformation. Taken together, structures of DeCLIC in multiple conformations illustrate dramatic conformational state transitions and diverse regulatory mechanisms available to ion channels in pLGICs, particularly involving Ca2+ modulation and periplasmic NTDs.
While many seminars in our science communities have been suspended this spring, EU-funded Centre of Excellence BioExcel has stepped up its promotion of educational webinars for computational biomolecular research.
In support of this effort, Molecular Biophysics Stockholm (MBS) members delivered two presentations in the spring series.
Conceived in 2016, BioExcel webinars cover broad topics related to the latest development of major software packages; their application to modeling and simulation; best practices for performance tuning and efficient usage on HPC and novel architectures; introductory tutorials for novel users; and much more. Prior to this spring, MBS members also contributed regularly to the series on optimizing molecular dynamics simulations in GROMACS.
Webinar slides and video recordings are freely available from BioExcel; for updates and registration on upcoming events, subscribe to the community newsletter.
Three remarkable members of Molecular Biophysics Stockholm celebrated completion of their doctoral degrees this spring.
Though their presentations had to be revised to meet public-health recommendations, including video-casting to off-site opponents and committee members, all three delivered polished, informative defenses of their respective theses to enthusiastic remote audiences.
Like so many of our colleagues worldwide, Molecular Biophysics Stockholm (MBS) has been engaged in various efforts to navigate and combat the novel coronavirus. Although the group’s past research rarely intersected with virology or clinical work, and none of us are are experts in the field, we hope to help where we can.
Members of the GROMACS team and BioExcel Centre also contribute to Exscalate4CoV, a consortium awarded €3 million by the European Commission for research on COVID-19 vaccines, treatment, and diagnostics. Key tasks at MBS include deployment of molecular-dynamics code and free-energy calculations for coronavirus protein simulations and drug-candidate scoring. For an overview of relevant targets, data repositories, and community collaborations, watch Lindahl’s presentation to the Centre Européen de Calcul Atomique et Moléculaire (English, 4:13–36:04).
On the clinical side, members of the Ligand-Gated Ion Channels (LGICs) team have worked with fellow chemists at Stockholm University to alleviate urgent needs for medical supplies, helping coordinate donations of masks, gloves, and other personal protective equipment from SciLifeLab to local hospitals. With reagents contributed by academic groups, the Museum of Natural History, and companies including GE Health, Petrolia, Runa and Absolut Vodka, we also assisted in the department’s production and distribution of over 20,000 liters of hand sanitizer to medical and care facilities. Read more in recent coverage by the university (English) and national (Swedish) media.
We are deeply grateful for the dedication and compassion of all MBS members, who continue to find creative paths to productivity even at a distance. Many have managed unprecedented disruptions to academic, research, and development work, including DISstudents Jojo Scott and Phaedra Robinson, whose time abroad was truncated months early by US travel restrictions. We so look forward to collaborating again at higher density on the other side of this pandemic.
Published 1 April 2020 in Acta Crystallographica Section D (v. 76 pp. 350–356):
Development of basic building blocks for cryo-EM: the emcore and emvis software libraries
José Miguel de la Rosa-Trevín, Pedro Alberto Hernández Viga, Joaquín Otónc, Erik Lindahl
Image-processing software has always been an integral part of structure determination by cryogenic electron microscopy (cryo-EM). Recent advances in hardware and software are recognized as one of the key factors in the so-called cryo-EM resolution revolution. Increasing computational power has opened many possibilities to consider more demanding algorithms, which in turn allow more complex biological problems to be tackled. Moreover, data processing has become more accessible to many experimental groups, with computations that used to last for many days at supercomputing facilities now being performed in hours on personal workstations. All of these advances, together with the rapid expansion of the community, continue to pose challenges and new demands on the software-development side. In this article, the development of emcore and emvis, two basic software libraries for image manipulation and data visualization in cryo-EM, is presented. The main goal is to provide basic functionality organized in modular components that other developers can reuse to implement new algorithms or build graphical applications. An additional aim is to showcase the importance of following established practices in software engineering, with the hope that this could be a first step towards a more standardized way of developing and distributing software in the field.
Agonist binding to G protein-coupled receptors (GPCRs) leads to conformational changes in the transmembrane region that activate cytosolic signaling pathways. Although high-resolution structures of different receptor states are available, atomistic details of allosteric signaling across the membrane remain elusive. We calculated free energy landscapes of β2 adrenergic receptor activation using atomistic molecular dynamics simulations in an optimized string of swarms framework, which shed new light on how microswitches govern the equilibrium between conformational states. Contraction of the extracellular binding site in the presence of the agonist BI-167107 is obligatorily coupled to conformational changes in a connector motif located in the core of the transmembrane region. The connector is probabilistically coupled to the conformation of the intracellular region. An active connector promotes desolvation of a buried cavity, a twist of the conserved NPxxY motif, and an interaction between two conserved tyrosines in transmembrane helices 5 and 7 (Y–Y motif), which lead to a larger population of active-like states at the G protein binding site. This coupling is augmented by protonation of the strongly conserved Asp792.50. The agonist binding site hence communicates with the intracellular region via a cascade of locally connected microswitches. Characterization of these can be used to understand how ligands stabilize distinct receptor states and contribute to development drugs with specific signaling properties. The developed simulation protocol can likely be transferred to other class A GPCRs.
Biomolecular simulations are intrinsically high dimensional and generate noisy data sets of ever-increasing size. Extracting important features from the data is crucial for understanding the biophysical properties of molecular processes, but remains a big challenge. Machine learning (ML) provides powerful dimensionality reduction tools. However, such methods are often criticized as resembling black boxes with limited human-interpretable insight. We use methods from supervised and unsupervised ML to efficiently create interpretable maps of important features from molecular simulations. We benchmark the performance of several methods, including neural networks, random forests, and principal component analysis, using a toy model with properties reminiscent of macromolecular behavior. We then analyze three diverse biological processes: conformational changes within the soluble protein calmodulin, ligand binding to a G protein-coupled receptor, and activation of an ion channel voltage-sensor domain, unraveling features critical for signal transduction, ligand binding, and voltage sensing. This work demonstrates the usefulness of ML in understanding biomolecular states and demystifying complex simulations.
Elucidating the mechanism of sugar import requires a molecular understanding of how transporters couple sugar binding and gating events. Whereas mammalian glucose transporters (GLUTs) are specialists1, the hexose transporter from the malaria parasite Plasmodium falciparum PfHT12,3 has acquired the ability to transport both glucose and fructose sugars as efficiently as the dedicated glucose (GLUT3) and fructose (GLUT5) transporters. Here, to establish the molecular basis of sugar promiscuity in malaria parasites, we determined the crystal structure of PfHT1 in complex with D-glucose at a resolution of 3.6 Å. We found that the sugar-binding site in PfHT1 is very similar to those of the distantly related GLUT3 and GLUT5 structures4,5. Nevertheless, engineered PfHT1 mutations made to match GLUT sugar-binding sites did not shift sugar preferences. The extracellular substrate-gating helix TM7b in PfHT1 was positioned in a fully occluded conformation, providing a unique glimpse into how sugar binding and gating are coupled. We determined that polar contacts between TM7b and TM1 (located about 15 Å from D-glucose) are just as critical for transport as the residues that directly coordinate D-glucose, which demonstrates a strong allosteric coupling between sugar binding and gating. We conclude that PfHT1 has achieved substrate promiscuity not by modifying its sugar-binding site, but instead by evolving substrate-gating dynamics.
In contrast to most voltage-gated ion channels, hyperpolarization- and cAMP gated (HCN) ion channels open on hyperpolarization. Structure-function studies show that the voltage-sensor of HCN channels are unique but the mechanisms that determine gating polarity remain poorly understood. All-atom molecular dynamics simulations (~20 μs) of HCN1 channel under hyperpolarization reveals an initial downward movement of the S4 voltage-sensor but following the transfer of last gating charge, the S4 breaks into two sub-helices with the lower sub-helix becoming parallel to the membrane. Functional studies on bipolar channels show that the gating polarity strongly correlates with helical turn propensity of the substituents at the breakpoint. Remarkably, in a proto-HCN background, the replacement of breakpoint serine with a bulky hydrophobic amino acid is sufficient to completely flip the gating polarity from inward to outward-rectifying. Our studies reveal an unexpected mechanism of inward rectification involving a linker sub-helix emerging from HCN S4 during hyperpolarization.
Members of Molecular Biophysics Stockholm celebrated Berk Hess‘ promotion to full Professor in Applied Physics at KTH Royal Institute of Technology, 15 November 2019, conferred by President Sigbritt Karlsson at Konserthuset in Stockholm, Sweden. With funding recently awarded by Vetenskapsrådet to the INTERFACE project, Hess’ team stands poised grow in size and contribution; check back soon for new openings!
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.
Twenty-three members of Molecular Biophysics Stockholm gathered for our fall off-site retreat, 24–25 October 2019 at historic Vår Gård in Saltsjöbaden, Sweden, workshopping topics related to individual core competencies, academic and industry CVs, group networking, and team communication. Thanks to co-organizers Annie, Joe, Lucie, Reba, and Urška, and to Vår Gård for a beautiful autumn getaway!
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.
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.
Four current and former members of Molecular Biophysics Stockholm, along with multiple collaborators, presented their research at the 2019 Jacques Monod Conference Canaux ioniques activés par les ligands: de la structure atomique à la transmission synaptique (Ligand-gated ion channels from atomic structure to synaptic transmission) 24–29 May 2019 in Roscoff, France. Among others, Reba Howard gave an invited talk on Biochemical and simulation studies of allosteric mechanisms in a model Cys-loop receptor.