A membrane-associated conveyor belt controls the rotational direction of the bacterial type 9 secretion system.

Many bacteria utilize the type 9 secretion system (T9SS) for gliding motility, surface colonization, and pathogenesis. This dual-function motor supports both gliding motility and protein secretion, where rotation of the T9SS plays a central role. Fueled by the energy of the stored proton motive force and transmitted through the torque of membrane-anchored stator units, the rotary T9SS propels an adhesin-coated conveyor belt along the bacterial outer membrane like a molecular snowmobile, thereby enabling gliding motion. However, the mechanisms controlling the rotational direction and gliding motility of T9SS remain elusive. Shedding light on this mechanism, we find that in the gliding bacterium Flavobacterium johnsoniae , deletion of the C-terminus of a conveyor belt protein controls, and in fact, reverses the rotational direction of T9SS from counterclockwise (CCW) to clockwise (CW). Largescale conformational changes at the interface of the T9SS ring with the C-terminus of the conveyer belt, as well as those of the ring protein themselves, in concert with a CW bias of the stators general rotation brings forth a 'tri-component gearset' model: the conveyor belt controls the conformation of the T9SS ring, and thereby its rotational direction. Consequently, the CW rotating stator either push the outer edge of the T9SS rings, causing its CCW rotation or press against the inner surface of the rings, resulting in CW rotation. This regulatory mechanism exemplifies how an outer membrane associated conveyor belt adjusts the rotational direction of its driver, the T9SS, thus providing adaptive sensory feedback to control the motility of a molecular snowmobile.

Structural mechanism of HP1⍺-dependent transcriptional repression and chromatin compaction.

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. Here, we present the cryoelectron microscopy (cryo-EM) structure of an HP1α dimer bound to an H2A.Z-nucleosome, revealing two distinct HP1α-nucleosome interfaces. The primary HP1α binding site is located at the N terminus of histone H3, specifically at the trimethylated lysine 9 (K9me3) region, while a secondary binding site is situated near histone H2B, close to nucleosome superhelical location 4 (SHL4). Our biochemical data further demonstrates that HP1α binding influences the dynamics of DNA on the nucleosome. It promotes DNA unwrapping near the nucleosome entry and exit sites while concurrently restricting DNA accessibility in the vicinity of SHL4. Our study offers a model for HP1α-mediated heterochromatin maintenance and gene silencing. It also sheds light on the H3K9me-independent role of HP1 in responding to DNA damage.

Finding the E-channel proton loading sites by calculating the ensemble of protonation microstates.

The aerobic electron transfer chain builds a proton gradient by proton coupled electron transfer reactions through a series of proteins. Complex I is the first enzyme in the sequence. Here transfer of two electrons from NADH to quinone yields four protons pumped from the membrane N- (negative, higher pH) side to the P- (positive, lower pH) side. Protons move through three linear antiporter paths, with a few amino acids and waters providing the route; and through the E-channel, a complex of competing paths, with clusters of interconnected protonatable residues. Proton loading sites (PLS) transiently bind protons as they are transported from N- to P-compartments. PLS can be individual residues or extended clusters of residues. The program MCCE uses Monte Carlos sampling to analyze the E-channel proton binding in equilibrium with individual Molecular Dynamics snapshots from trajectories of Thermus thermuphillus Complex I in the apo, quinone and quinol bound states. At pH 7, the five E-channel subunits (Nqo4, Nqo7, Nqo8, Nqo10, and Nqo11) take >25,000 protonation microstates, each with different residues protonated. The microstate explosion is tamed by analyzing interconnected clusters of residues along the proton transfer paths. A proton is bound and released from a cluster of five coupled residues on the protein N-side and to six coupled residues in the protein center. Loaded microstates bind protons to sites closer to the P-side in the forward pumping direction. MCCE microstate analysis identifies strongly coupled proton binding amongst individual residues in the two PLS clusters.

Protein Medium Facilitates Electron Transfer in Photosynthetic Heliobacterial Reaction Center.

This computational study addresses the question of how large membrane-bound proteins of electron transport chains facilitate fast vector-based charge transport. We study electron transfer reactions following ultrafast initial charge separation induced by absorption of light by P800 primary pair and leading to the electron localization at the A0 cofactor. Two subsequent, much slower reactions, electron transfer to the iron-sulfur cluster Fx and reduction of the menaquinone (MQ) cofactor, are studied by combining molecular dynamics simulations, electronic structure calculations, and theoretical modeling. The low value of the electronic coupling between A0 and Fx brings this reaction to the microsecond time scale even at the zero activation barrier. In contrast, A0-MQ electron transfer occurs on a subnanosecond time scale and might become the preferred route for charge transport. We elucidate mechanistic properties of the protein medium allowing fast, vectorial charge transfer. The electric field is high and inhomogeneous inside the protein and is coupled to high polarizabilities of cofactors to significantly lower the reaction barrier. The A0-MQ separation puts this reaction at the edge between the plateau characterizing the reaction dynamical control and exponential falloff due to electronic tunneling. A strong separation in relaxation times of the medium dynamics for the forward and backward reactions promotes vectorial charge transfer.

Organization of a functional glycolytic metabolon on mitochondria for metabolic efficiency.

Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, and is crucial for efficient glycolysis-oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyses reversible O-GlcNAcylation, a post-translational modification influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of the regulatory domain of HK1, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances the mitochondrial association with HK1, orchestrating glycolytic and mitochondrial ATP production. Mutation in HK1's O-GlcNAcylation site reduces ATP generation in multiple cell types, specifically affecting metabolic efficiency in neurons. This study reveals a previously unappreciated pathway that links neuronal metabolism and mitochondrial function through OGT and the formation of the glycolytic metabolon, providing potential strategies for tackling metabolic and neurological disorders.

Single-Molecule Detection of the Encounter and Productive Electron Transfer Complexes of a Photosynthetic Reaction Center.

Small, diffusible redox proteins play an essential role in electron transfer (ET) in respiration and photosynthesis, sustaining life on Earth by shuttling electrons between membrane-bound complexes via finely tuned and reversible interactions. Ensemble kinetic studies show transient ET complexes form in two distinct stages: an "encounter" complex largely mediated by electrostatic interactions, which subsequently, through subtle reorganization of the binding interface, forms a "productive" ET complex stabilized by additional hydrophobic interactions around the redox-active cofactors. Here, using single-molecule force spectroscopy (SMFS) we dissected the transient ET complexes formed between the photosynthetic reaction center-light harvesting complex 1 (RC-LH1) of Rhodobacter sphaeroides and its native electron donor cytochrome c2 (cyt c2). Importantly, SMFS resolves the distribution of interaction forces into low (∼150 pN) and high (∼330 pN) components, with the former more susceptible to salt concentration and to alteration of key charged residues on the RC. Thus, the low force component is suggested to reflect the contribution of electrostatic interactions in forming the initial encounter complex, whereas the high force component reflects the additional stabilization provided by hydrophobic interactions to the productive ET complex. Employing molecular dynamics simulations, we resolve five intermediate states that comprise the encounter, productive ET and leaving complexes, predicting a weak interaction between cyt c2 and the LH1 ring near the RC-L subunit that could lie along the exit path for oxidized cyt c2. The multimodal nature of the interactions of ET complexes captured here may have wider implications for ET in all domains of life.

Perspective on Integrative Simulations of Bioenergetic Domains.

Bioenergetic processes in cells, such as photosynthesis or respiration, integrate many time and length scales, which makes the simulation of energy conversion with a mere single level of theory impossible. Just like the myriad of experimental techniques required to examine each level of organization, an array of overlapping computational techniques is necessary to model energy conversion. Here, a perspective is presented on recent efforts for modeling bioenergetic phenomena with a focus on molecular dynamics simulations and its variants as a primary method. An overview of the various classical, quantum mechanical, enhanced sampling, coarse-grained, Brownian dynamics, and Monte Carlo methods is presented. Example applications discussed include multiscale simulations of membrane-wide electron transport, rate kinetics of ATP turnover from electrochemical gradients, and finally, integrative modeling of the chromatophore, a photosynthetic pseudo-organelle.

The electrostatic landscape of MHC-peptide binding revealed using inception networks.

Predictive modeling of macromolecular recognition and protein-protein complementarity represents one of the cornerstones of biophysical sciences. However, such models are often hindered by the combinatorial complexity of interactions at the molecular interfaces. Exemplary of this problem is peptide presentation by the highly polymorphic major histocompatibility complex class I (MHC-I) molecule, a principal component of immune recognition. We developed human leukocyte antigen (HLA)-Inception, a deep biophysical convolutional neural network, which integrates molecular electrostatics to capture non-bonded interactions for predicting peptide binding motifs across 5,821 MHC-I alleles. These predictions of generated motifs correlate strongly with experimental peptide binding and presentation data. Beyond molecular interactions, the study demonstrates the application of predicted motifs in analyzing MHC-I allele associations with HIV disease progression and patient response to immune checkpoint inhibitors. A record of this paper's transparent peer review process is included in the supplemental information.

Structural mechanism of HP1α-dependent transcriptional repression and chromatin compaction.

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. In this study, we employed a multidisciplinary approach to unravel the interactions between human HP1α and nucleosomes. We have elucidated the cryo-EM structure of an HP1α dimer bound to an H2A.Z nucleosome, revealing that the HP1α dimer interfaces with nucleosomes at two distinct sites. The primary binding site is located at the N-terminus of histone H3, specifically at the trimethylated K9 (K9me3) region, while a novel secondary binding site is situated near histone H2B, close to nucleosome superhelical location 4 (SHL4). Our biochemical data further demonstrates that HP1α binding influences the dynamics of DNA on the nucleosome. It promotes DNA unwrapping near the nucleosome entry and exit sites while concurrently restricting DNA accessibility in the vicinity of SHL4. This study offers a model that explains how HP1α functions in heterochromatin maintenance and gene silencing, particularly in the context of H3K9me-dependent mechanisms. Additionally, it sheds light on the H3K9me-independent role of HP1 in responding to DNA damage.

Structural insights into the modulation of coronavirus spike tilting and infectivity by hinge glycans.

Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectrometry. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a role of hinge glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays implicated involvement of N1242-glyan in virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.

Multidimensional quantitative phenotypic and molecular analysis reveals neomorphic behaviors of p53 missense mutants.

Mutations in the TP53 tumor suppressor gene occur in >80% of the triple-negative or basal-like breast cancer. To test whether neomorphic functions of specific TP53 missense mutations contribute to phenotypic heterogeneity, we characterized phenotypes of non-transformed MCF10A-derived cell lines expressing the ten most common missense mutant p53 proteins and observed a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and 3D mammosphere architecture. The p53 mutants R248W, R273C, R248Q, and Y220C are the most aggressive while G245S and Y234C are the least, which correlates with survival rates of basal-like breast cancer patients. Interestingly, a crucial amino acid difference at one position-R273C vs. R273H-has drastic changes on cellular phenotype. RNA-Seq and ChIP-Seq analyses show distinct DNA binding properties of different p53 mutants, yielding heterogeneous transcriptomics profiles, and MD simulation provided structural basis of differential DNA binding of different p53 mutants. Integrative statistical and machine-learning-based pathway analysis on gene expression profiles with phenotype vectors across the mutant cell lines identifies quantitative association of multiple pathways including the Hippo/YAP/TAZ pathway with phenotypic aggressiveness. Further, comparative analyses of large transcriptomics datasets on breast cancer cell lines and tumors suggest that dysregulation of the Hippo/YAP/TAZ pathway plays a key role in driving the cellular phenotypes towards basal-like in the presence of more aggressive p53 mutants. Overall, our study describes distinct gain-of-function impacts on protein functions, transcriptional profiles, and cellular behaviors of different p53 missense mutants, which contribute to clinical phenotypic heterogeneity of triple-negative breast tumors.

Adaptive Ensemble Refinement of Protein Structures in High Resolution Electron Microscopy Density Maps with Radical Augmented Molecular Dynamics Flexible Fitting.

Recent advances in cryo-electron microscopy (cryo-EM) have enabled modeling macromolecular complexes that are essential components of the cellular machinery. The density maps derived from cryo-EM experiments are often integrated with manual, knowledge-driven or artificial intelligence-driven and physics-guided computational methods to build, fit, and refine molecular structures. Going beyond a single stationary-structure determination scheme, it is becoming more common to interpret the experimental data with an ensemble of models that contributes to an average observation. Hence, there is a need to decide on the quality of an ensemble of protein structures on-the-fly while refining them against the density maps. We introduce such an adaptive decision-making scheme during the molecular dynamics flexible fitting (MDFF) of biomolecules. Using RADICAL-Cybertools, the new RADICAL augmented MDFF implementation (R-MDFF) is examined in high-performance computing environments for refinement of two prototypical protein systems, adenylate kinase and carbon monoxide dehydrogenase. For these test cases, use of multiple replicas in flexible fitting with adaptive decision making in R-MDFF improves the overall correlation to the density by 40% relative to the refinements of the brute-force MDFF. The improvements are particularly significant at high, 2-3 Å map resolutions. More importantly, the ensemble model captures key features of biologically relevant molecular dynamics that are inaccessible to a single-model interpretation. Finally, the pipeline is applicable to systems of growing sizes, which is demonstrated using ensemble refinement of capsid proteins from the chimpanzee adenovirus. The overhead for decision making remains low and robust to computing environments. The software is publicly available on GitHub and includes a short user guide to install R-MDFF on different computing environments, from local Linux-based workstations to high-performance computing environments.

Functional Organization of Glycolytic Metabolon on Mitochondria.

Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme Hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, crucial for efficient glycolysis-oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis, and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyzes reversible O-GlcNAcylation, a post-translational modification, influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of HK1's regulatory domain, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances HK1's mitochondrial association, orchestrating glycolytic and mitochondrial ATP production. Mutations in HK1's O-GlcNAcylation site reduce ATP generation, affecting synaptic functions in neurons. The study uncovers a novel pathway that bridges neuronal metabolism and mitochondrial function via OGT and the formation of the glycolytic metabolon, offering new prospects for tackling metabolic and neurological disorders.

High-resolution structures with bound Mn2+ and Cd2+ map the metal import pathway in an Nramp transporter.

Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent diseases and disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations, and by conserved networks of polar residues lining the inner and outer gates. In addition, a high-resolution Cd2+-bound structure highlights differences in how Cd2+ and Mn2+ are coordinated by DraNramp. Complementary metal binding studies using isothermal titration calorimetry with a series of mutated DraNramp proteins indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn2+ is different and more robust to perturbation than for transporting the toxic Cd2+ metal. Overall, the affinity measurements and high-resolution structural information on metal substrate binding provide a foundation for understanding the substrate selectivity of essential metal ion transporters like Nramps.

Integrated analyses reveal a hinge glycan regulates coronavirus spike tilting and virus infectivity.

Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectroscopy. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a novel role of stalk glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays support the hypothesis that this glycan-dependent motion impacts virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.

Revealing a Hidden Intermediate of Rotatory Catalysis with X-ray Crystallography and Molecular Simulations.

The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remains an unresolved puzzle in biological energy transfer. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multistep rotation process. Here, using vacuolar or V1-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography, a new intermediate or "dwell" is identified, which enables the release of an inorganic phosphate (or Pi) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transformations and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the side-chain-level interactions transcend into larger-scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. An inspection of all known rates encompassing the 4-step rotation mechanism implicates the overcoming of the ADP interactions with V1-ATPase to be the rate-limiting step of motor action.

Characterization and computational simulation of human Syx, a RhoGEF implicated in glioblastoma.

Structural discovery of guanine nucleotide exchange factor (GEF) protein complexes is likely to become increasingly relevant with the development of new therapeutics targeting small GTPases and development of new classes of small molecules that inhibit protein-protein interactions. Syx (also known as PLEKHG5 in humans) is a RhoA GEF implicated in the pathology of glioblastoma (GBM). Here we investigated protein expression and purification of ten different human Syx constructs and performed biophysical characterizations and computational studies that provide insights into why expression of this protein was previously intractable. We show that human Syx can be expressed and isolated and Syx is folded as observed by circular dichroism (CD) spectroscopy and actively binds to RhoA as determined by co-elution during size exclusion chromatography (SEC). This characterization may provide critical insights into the expression and purification of other recalcitrant members of the large class of oncogenic-Diffuse B-cell lymphoma (Dbl) homology GEF proteins. In addition, we performed detailed homology modeling and molecular dynamics simulations on the surface of a physiologically realistic membrane. These simulations reveal novel insights into GEF activity and allosteric modulation by the plekstrin homology (PH) domain. These newly revealed interactions between the GEF PH domain and the membrane embedded region of RhoA support previously unexplained experimental findings regarding the allosteric effects of the PH domain from numerous activity studies of Dbl homology GEF proteins. This work establishes new hypotheses for structural interactivity and allosteric signal modulation in Dbl homology RhoGEFs.

The ugly, bad, and good stories of large-scale biomolecular simulations.

Molecular modeling of large biomolecular assemblies exemplifies a disruptive area holding both promises and contentions. Propelled by peta and exascale computing, several simulation methodologies have now matured into user-friendly tools that are successfully employed for modeling viruses, membranous nano-constructs, and key pieces of the genetic machinery. We present three unifying biophysical themes that emanate from some of the most recent multi-million atom simulation endeavors. Despite connecting molecular changes with phenotypic outcomes, the quality measures of these simulations remain questionable. We discuss the existing and upcoming strategies for constructing representative ensembles of large systems, how new computing technologies will boost this area, and make a point that integrative modeling guided by experimental data is the future of biomolecular computations.

Energy landscape of the SARS-CoV-2 reveals extensive conformational heterogeneity.

Cryo-electron microscopy (cryo-EM) has produced a number of structural models of the SARS-CoV-2 spike, already prompting biomedical outcomes. However, these reported models and their associated electrostatic potential maps represent an unknown admixture of conformations stemming from the underlying energy landscape of the spike protein. As with any protein, some of the spike's conformational motions are expected to be biophysically relevant, but cannot be interpreted only by static models. Using experimental cryo-EM images, we present the energy landscape of the glycosylated spike protein, and identify the diversity of low-energy conformations in the vicinity of its open (so called 1RBD-up) state. The resulting atomic refinement reveal global and local molecular rearrangements that cannot be inferred from an average 1RBD-up cryo-EM model. Here we report varied degrees of "openness" in global conformations of the 1RBD-up state, not revealed in the single-model interpretations of the density maps, together with conformations that overlap with the reported models. We discover how the glycan shield contributes to the stability of these low-energy conformations. Five out of six binding sites we analyzed, including those for engaging ACE2, therapeutic mini-proteins, linoleic acid, two different kinds of antibodies, switch conformations between their known apo- and holo-conformations, even when the global spike conformation is 1RBD-up. This apo-to-holo switching is reminiscent of a conformational preequilibrium. We found only one binding site, namely that of AB-C135 remains in apo state within all the sampled free energy-minimizing models, suggesting an induced fit mechanism for the docking of this antibody to the spike.