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•Advances in MD and imaging enable structural determination of large complexes.•Large-scale MD is essential to the atomic level description of cell-scale processes.•MD enables the ...study of local and global dynamics of multi-million atom complexes.
Connecting dynamics to structural data from diverse experimental sources, molecular dynamics simulations permit the exploration of biological phenomena in unparalleled detail. Advances in simulations are moving the atomic resolution descriptions of biological systems into the million-to-billion atom regime, in which numerous cell functions reside. In this opinion, we review the progress, driven by large-scale molecular dynamics simulations, in the study of viruses, ribosomes, bioenergetic systems, and other diverse applications. These examples highlight the utility of molecular dynamics simulations in the critical task of relating atomic detail to the function of supramolecular complexes, a task that cannot be achieved by smaller-scale simulations or existing experimental approaches alone.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires ...precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array.
Lipopolysaccharide (LPS) transport to the outer membrane (OM) is a crucial step in the biogenesis of microbial surface defenses. Although many features of the translocation mechanism have been ...elucidated, molecular details of LPS insertion via the LPS transport (Lpt) OM protein LptDE remain elusive. Here, we integrate native MS with hydrogen-deuterium exchange MS and molecular dynamics simulations to investigate the influence of substrate and peptide binding on the conformational dynamics of LptDE. Our data reveal that LPS induces opening of the LptD β-taco domain, coupled with conformational changes on β-strands adjacent to the putative lateral exit gate. Conversely, an antimicrobial peptide, thanatin, stabilizes the β-taco, thereby preventing LPS transport. Our results illustrate that LPS insertion into the OM relies on concerted opening movements of both the β-barrel and β-taco domains of LptD, and suggest a means for developing antimicrobial therapeutics targeting this essential process in Gram-negative ESKAPE pathogens.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Chemotactic responses in bacteria require large, highly ordered arrays of sensory proteins to mediate the signal transduction that ultimately controls cell motility. A mechanistic understanding of ...the molecular events underlying signaling, however, has been hampered by the lack of a high-resolution structural description of the extended array. Here, we report a novel reconstitution of the array, involving the receptor signaling domain, histidine kinase CheA, and adaptor protein CheW, as well as a density map of the core-signaling unit at 11.3 Å resolution, obtained by cryo-electron tomography and sub-tomogram averaging. Extracting key structural constraints from our density map, we computationally construct and refine an atomic model of the core array structure, exposing novel interfaces between the component proteins. Using all-atom molecular dynamics simulations, we further reveal a distinctive conformational change in CheA. Mutagenesis and chemical cross-linking experiments confirm the importance of the conformational dynamics of CheA for chemotactic function.
Chemotaxis signaling pathways enable bacteria to sense and respond to their chemical environment and, in some species, are critical for lifestyle processes such as biofilm formation and pathogenesis. ...The signal transduction underlying chemotaxis behavior is mediated by large, highly ordered protein complexes known as chemosensory arrays. For nearly two decades, cryo-electron tomography (cryoET) has been used to image chemosensory arrays, providing an increasingly detailed understanding of their structure and function. In this mini-review, we provide an overview of the use of cryoET to study chemosensory arrays, including imaging strategies, key results, and outstanding questions. We further discuss the application of molecular modeling and simulation techniques to complement structure determination efforts and provide insight into signaling mechanisms. We close the review with a brief outlook, highlighting promising future directions for the field.
Methane-oxidizing bacteria play a central role in greenhouse gas mitigation and have potential applications in biomanufacturing. Their primary metabolic enzyme, particulate methane monooxygenase ...(pMMO), is housed in copper-induced intracytoplasmic membranes (ICMs), of which the function and biogenesis are not known. We show by serial cryo-focused ion beam (cryoFIB) milling/scanning electron microscope (SEM) volume imaging and lamellae-based cellular cryo-electron tomography (cryoET) that these ICMs are derived from the inner cell membrane. The pMMO trimer, resolved by cryoET and subtomogram averaging to 4.8 Å in the ICM, forms higher-order hexagonal arrays in intact cells. Array formation correlates with increased enzymatic activity, highlighting the importance of studying the enzyme in its native environment. These findings also demonstrate the power of cryoET to structurally characterize native membrane enzymes in the cellular context.
Certain inhibitor of apoptosis (IAP) family members are sentinel proteins that prevent untimely cell death by inhibiting caspases. Antagonists, including second mitochondria-derived activator of ...caspases (SMAC), regulate IAPs and drive cell death. Baculoviral IAP repeat-containing protein 6 (BIRC6), a giant IAP with dual E2 and E3 ubiquitin ligase activity, regulates programmed cell death through unknown mechanisms. We show that BIRC6 directly restricts executioner caspase-3 and -7 and ubiquitinates caspase-3, -7, and -9, working exclusively with noncanonical E1, UBA6. Notably, we show that SMAC suppresses both mechanisms. Cryo-electron microscopy structures of BIRC6 alone and in complex with SMAC reveal that BIRC6 is an antiparallel dimer juxtaposing the substrate-binding module against the catalytic domain. Furthermore, we discover that SMAC multisite binding to BIRC6 results in a subnanomolar affinity interaction, enabling SMAC to competitively displace caspases, thus antagonizing BIRC6 anticaspase function.
The rise of the computer as a powerful tool for model building and refinement has revolutionized the field of structure determination for large biomolecular systems. Despite the wide availability of ...robust experimental methods capable of resolving structural details across a range of spatiotemporal resolutions, computational hybrid methods have the unique ability to integrate the diverse data from multimodal techniques such as X-ray crystallography and electron microscopy into consistent, fully atomistic structures. Here, commonly employed strategies for computational real-space structural refinement are reviewed, and their specific applications are illustrated for several large macromolecular complexes: ribosome, virus capsids, chemosensory array, and photosynthetic chromatophore. The increasingly important role of computational methods in large-scale structural refinement, along with current and future challenges, is discussed.
Lipids play important modulatory and structural roles for membrane proteins. Molecular dynamics simulations are frequently used to provide insights into the nature of these protein–lipid ...interactions. Systematic comparative analysis requires tools that provide algorithms for objective assessment of such interactions. We introduce PyLipID, a Python package for the identification and characterization of specific lipid interactions and binding sites on membrane proteins from molecular dynamics simulations. PyLipID uses a community analysis approach for binding site detection, calculating lipid residence times for both the individual protein residues and the detected binding sites. To assist structural analysis, PyLipID produces representative bound lipid poses from simulation data, using a density-based scoring function. To estimate residue contacts robustly, PyLipID uses a dual-cutoff scheme to differentiate between lipid conformational rearrangements while bound from full dissociation events. In addition to the characterization of protein–lipid interactions, PyLipID is applicable to analysis of the interactions of membrane proteins with other ligands. By combining automated analysis, efficient algorithms, and open-source distribution, PyLipID facilitates the systematic analysis of lipid interactions from large simulation data sets of multiple species of membrane proteins.
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IJS, KILJ, NUK, PNG, UL, UM
The
chemoreceptor array is a supramolecular assembly that enables cells to respond to extracellular cues dynamically and with great precision and sensitivity. In the array, transmembrane receptors ...organized as trimers of dimers are connected at their cytoplasmic tips by hexameric rings of alternating subunits of the kinase CheA and the scaffolding protein CheW (CheA-CheW rings). Interactions of CheW molecules with the members of receptor trimers not directly bound to CheA-CheW rings may lead to the formation of hexameric CheW rings in the chemoreceptor array. Here, we detected such CheW rings with a cellular cysteine-directed cross-linking assay and explored the requirements for their formation and their participation in array assembly. We found that CheW ring formation varied with cellular CheW abundance, depended on the presence of receptors capable of a trimer-of-dimers arrangement, and did not require CheA. Cross-linking studies of a CheA~CheW fusion protein incapable of forming homomeric CheW oligomers demonstrated that CheW rings were not essential for the assembly of CheA-containing arrays. Förster resonance energy transfer (FRET)-based kinase assays of arrays containing variable amounts of CheW rings revealed that CheW rings enhanced the cooperativity and the sensitivity of the responses to attractants. We propose that six-membered CheW rings provide the additional interconnectivity required for optimal signaling and gradient tracking performance by chemosensory arrays.
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