Propelled by enormous increases in computational power, molecular dynamics (MD) simulations were first reported in 1957 by B.J. Alder and T.E. Wainwright and since then have moved from this proof of concept to routinely investigating the dynamics of complex systems composed of up to tens of millions of atoms. MD simulations are based on the idea that the equations of motion of a multi-particle system can be solved numerically within an acceptable level of accuracy; the resulting trajectory is key for calculating occupancy probabilities of distinct conformational states (sampling). In advanced protocols, enhanced sampling ensures wide exploration of the configurational space. Further, a strength of MD simulations is that by starting from descriptions of the motions of all atoms, several data learning techniques can be used to conceptualize trajectories. In ion channel biophysics, these tools are used to study ion permeation, conformational cycling, drug binding, and lipid–channel interactions.
As the first potassium channel with an x-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related, in particular, to the allosteric coupling between its gates remain open. The many currently available x-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state nuclear magnetic resonance (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible x-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.
Cannabidiol inhibits the skeletal muscle Nav1.4 by blocking its pore and by altering membrane elasticity
Mohammad-Reza Ghovanloo, Koushik Choudhury, Tagore S. Bandaru, Mohamed A. Fouda, Kaveh Rayani, Radda Rusinova, Tejas Phaterpekar, Karen Nelkenbrecher, Abeline R. Watkins, Damon Poburko, Jenifer Thewalt, Olaf S. Andersen, Lucie Delemotte, Samuel J. Goodchild, Peter C. Ruben
Cannabidiol (CBD) is the primary nonpsychotropic phytocannabinoid found in Cannabis sativa, which has been proposed to be therapeutic against many conditions, including muscle spasms. Among its putative targets are voltage-gated sodium channels (Navs), which have been implicated in many conditions. We investigated the effects of CBD on Nav1.4, the skeletal muscle Nav subtype. We explored direct effects, involving physical block of the Nav pore, as well as indirect effects, involving modulation of membrane elasticity that contributes to Nav inhibition. MD simulations revealed CBD’s localization inside the membrane and effects on bilayer properties. Nuclear magnetic resonance (NMR) confirmed these results, showing CBD localizing below membrane headgroups. To determine the functional implications of these findings, we used a gramicidin-based fluorescence assay to show that CBD alters membrane elasticity or thickness, which could alter Nav function through bilayer-mediated regulation. Site-directed mutagenesis in the vicinity of the Nav1.4 pore revealed that removing the local anesthetic binding site with F1586A reduces the block of INa by CBD. Altering the fenestrations in the bilayer-spanning domain with Nav1.4-WWWW blocked CBD access from the membrane into the Nav1.4 pore (as judged by MD). The stabilization of inactivation, however, persisted in WWWW, which we ascribe to CBD-induced changes in membrane elasticity. To investigate the potential therapeutic value of CBD against Nav1.4 channelopathies, we used a pathogenic Nav1.4 variant, P1158S, which causes myotonia and periodic paralysis. CBD reduces excitability in both wild-type and the P1158S variant. Our in vitro and in silico results suggest that CBD may have therapeutic value against Nav1.4 hyperexcitability.
A blueprint for high affinity SARS-CoV-2 Mpro inhibitors from activity-based compound library screening guided by analysis of protein dynamics
Jonas Gossen, Simone Albani, Anton Hanke, Benjamin P Joseph, Cathrine Bergh, Maria Kuzikov, Elisa Costanzi, Candida Manelfi, Paola Storici, Philip Gribbon, Andrea R Beccari, Carmine Talarico, Francesca Spyrakis, Erik Lindahl, Andrea Zaliani, Paolo Carloni, Rebecca C Wade, Francesco Musiani, Daria B Kokh, and Giulia Rossetti
The SARS-CoV-2 coronavirus outbreak continues to spread at a rapid rate worldwide. The main protease (Mpro) is an attractive target for anti-COVID-19 agents. Unexpected difficulties have been encountered in the design of specific inhibitors. Here, by analyzing an ensemble of ∼30 000 SARS-CoV-2 Mpro conformations from crystallographic studies and molecular simulations, we show that small structural variations in the binding site dramatically impact ligand binding properties. Hence, traditional druggability indices fail to adequately discriminate between highly and poorly druggable conformations of the binding site. By performing ∼200 virtual screenings of compound libraries on selected protein structures, we redefine the protein’s druggability as the consensus chemical space arising from the multiple conformations of the binding site formed upon ligand binding. This procedure revealed a unique SARS-CoV-2 Mpro blueprint that led to a definition of a specific structure-based pharmacophore. The latter explains the poor transferability of potent SARS-CoV Mpro inhibitors to SARS-CoV-2 Mpro, despite the identical sequences of the active sites. Importantly, application of the pharmacophore predicted novel high affinity inhibitors of SARS-CoV-2 Mpro, that were validated by in vitro assays performed here and by a newly solved X-ray crystal structure. These results provide a strong basis for effective rational drug design campaigns against SARS-CoV-2 Mpro and a new computational approach to screen protein targets with malleable binding sites.
Voltage-gated potassium (Kv) channels can be opened by negatively charged resin acids and their derivatives. These resin acids have been proposed to attract the positively charged voltage-sensor helix (S4) toward the extracellular side of the membrane by binding to a pocket located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. By contrast to this proposed mechanism, neutralization of the top gating charge of the Shaker KV channel increased resin-acid–induced opening, suggesting other mechanisms and sites of action. Here, we explore the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated/open state of the Shaker KV channel by a combination of in silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resin-acid–binding sites around S4: (1) the S3/S4 site previously suggested, in which positively charged residues introduced at the top of S4 are critical to keep the compound bound, (2) a site in the cleft between S4 and the pore domain (S4/pore site), in which a tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound, and (3) a site located on the extracellular side of the voltage-sensor domain, in a cleft formed by S1–S4 (the top-VSD site). The multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation make the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding dynamics and the types of interactions it can form with the protein in a state-specific manner.
Ligand binding stabilizes different G protein-coupled receptor states via a complex allosteric process that is not completely understood. Here, we have derived free energy landscapes describing activation of the β2 adrenergic receptor bound to ligands with different efficacy profiles using enhanced sampling molecular dynamics (MD) simulations. These reveal shifts towards active-like states at the G protein binding site for receptors bound to partial and full agonists and that the ligands modulate the conformational ensemble of the receptor by tuning protein microswitches. We indeed find an excellent correlation between the conformation of the microswitches close to the ligand binding site and in the transmembrane region and experimentally reported cAMP signaling responses. Dimensionality reduction further reveals the similarity between the unique conformational states induced by different ligands and examining the output of classifiers highlights two distant hotspots governing agonism on transmembrane helices 5 and 7.
Direct detection of SARS-CoV-2 using non-commercial RT-LAMP reagents on heat-inactivated samples
Alisa Alekseenko, Donal Barrett, Yerma Pareja-Sanchez, Rebecca J Howard, Emilia Strandback, Henry Ampah-Korsah, Urška Rovšnik, Silvia Zuniga-Veliz, Alexander Klenov, Jayshna Malloo, Shenglong Ye, Xiyang Liu, Björn Reinius, Simon J. Elsässer, Tomas Nyman, Gustaf Sandh, Xiushan Yin & Vicent Pelechano
RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.
Members of Molecular Biophysics Stockholm joined family and friends in celebrating Annie Westerlund‘s successful defense of her PhD thesis in Biophysics, Deciphering conformational ensembles and communication pathways in biomolecules, 18 December 2020 at the KTH Royal Institute of Technology, Stockholm, Sweden. Professor Kresten Lindorff-Larsen (University of Copenhagen, Denmark) served as opponent via videoconference. Associate Professor Lucie Delemotte (KTH) led a toast to her advisee of four years, while Delemotte group members awarded an equally merited honorary diploma from the Hogwarts School of Witchcraft and Wizardry to their Network Witch.
Calmodulin acts as a state-dependent switch to control a cardiac potassium channel opening
Po Wei Kang,* Annie M Westerlund,* Jingyi Shi, Kelli McFarland White, Alex K Dou, Amy H Cui, Jonathan R Silva, Lucie Delemotte, Jianmin Cui *contributed equally to this work
Calmodulin (CaM) and phosphatidylinositol 4,5-bisphosphate (PIP2) are potent regulators of the voltage-gated potassium channel KCNQ1 (KV7.1), which conducts the cardiac IKs current. Although cryo–electron microscopy structures revealed intricate interactions between the KCNQ1 voltage-sensing domain (VSD), CaM, and PIP2, the functional consequences of these interactions remain unknown. Here, we show that CaM-VSD interactions act as a state-dependent switch to control KCNQ1 pore opening. Combined electrophysiology and molecular dynamics network analysis suggest that VSD transition into the fully activated state allows PIP2 to compete with CaM for binding to VSD. This leads to conformational changes that alter VSD-pore coupling to stabilize open states. We identify a motif in the KCNQ1 cytosolic domain, which works downstream of CaM-VSD interactions to facilitate the conformational change. Our findings suggest a gating mechanism that integrates PIP2and CaM in KCNQ1 voltage-dependent activation, yielding insights into how KCNQ1 gains the phenotypes critical for its physiological function.
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.