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Polyamines are small, organic polycations that are ubiquitous and essential to all forms of life. Currently, how polyamines are transported across membranes is not understood. Recent studies have suggested that ATP13A2 and its close homologs, collectively known as P5B-ATPases, are polyamine transporters at endo-/lysosomes. Loss-of-function mutations of ATP13A2 in humans cause hereditary early-onset Parkinson's disease. To understand the polyamine transport mechanism of ATP13A2, we determined high-resolution cryoelectron microscopy (cryo-EM) structures of human ATP13A2 in five distinct conformational intermediates, which together, represent a near-complete transport cycle of ATP13A2. The structural basis of the polyamine specificity was revealed by an endogenous polyamine molecule bound to a narrow, elongated cavity within the transmembrane domain. The structures show an atypical transport path for a water-soluble substrate, in which polyamines may exit within the cytosolic leaflet of the membrane. Our study provides important mechanistic insights into polyamine transport and a framework to understand the functions and mechanisms of P5B-ATPases. [Display omitted] • High-resolution cryo-EM structures of human ATP13A2 in five distinct conformations • Unique features of ATP13A2 in comparison to other P-type ATPases • Structural basis of polyamine substrate binding to the membrane domain of ATP13A2 • Conformational changes along the transport cycle and a model for polyamine transport The P-type ATPase ATP13A2 is an endo-/lysosomal polyamine transporter, defects of which are implicated in hereditary forms of Parkinson disease. Sim et al. report structures of human ATP13A2 in five distinct conformations, representing a near-complete catalytic cycle. This work reveals the mechanistic basis of polyamine recognition and transport by ATP13A2. [ABSTRACT FROM AUTHOR]

Most membrane proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, shielding potentially large fractions of protein surface. High glycan conformational freedom hinders complete structural elucidation of glycoproteins. Computer simulations may be used to model glycosylated proteins but require hundreds of thousands of computing hours on supercomputers, thus limiting routine use. Here, we describe GlycoSHIELD, a reductionist method that can be implemented on personal computers to graft realistic ensembles of glycan conformers onto static protein structures in minutes. Using molecular dynamics simulation, small-angle X-ray scattering, cryoelectron microscopy, and mass spectrometry, we show that this open-access toolkit provides enhanced models of glycoprotein structures. Focusing on N-cadherin, human coronavirus spike proteins, and gamma-aminobutyric acid receptors, we show that GlycoSHIELD can shed light on the impact of glycans on the conformation and activity of complex glycoproteins. [Display omitted] • GlycoSHIELD predicts morphology and span of glycan shields on protein surface • Predicts glycan impact on protein conformation and surface accessibility • GlycoSHIELD models are consistent with cryo-EM datasets • Enhances the interpretation of experimental density maps GlycoSHIELD and its associated library of precomputed glycan conformers enable the generation of realistic glycosylated protein structure models at a fraction of the computing cost of conventional molecular dynamics simulations. [ABSTRACT FROM AUTHOR]

Sensation of light is essential for all organisms. The eye-less nematode Caenorhabditis elegans detects UV and blue light to evoke escape behavior. The photosensor LITE-1 absorbs UV photons with an unusually high extinction coefficient, involving essential tryptophans. Here, we modeled the structure and dynamics of LITE-1 using AlphaFold2-multimer and molecular dynamics (MD) simulations and performed mutational and behavioral assays in C. elegans to characterize its function. LITE-1 resembles olfactory and gustatory receptors from insects, recently shown to be tetrameric ion channels. We identified residues required for channel gating, light absorption, and mechanisms of photo-oxidation, involving a likely binding site for the peroxiredoxin PRDX-2. Furthermore, we identified the binding pocket for a putative chromophore. Several residues lining this pocket have previously been established as essential for LITE-1 function. A newly identified critical cysteine pointing into the pocket represents a likely chromophore attachment site. We derived a model for how photon absorption, via a network of tryptophans and other aromatic amino acids, induces an excited state that is transferred to the chromophore. This evokes conformational changes in the protein, possibly leading to a state receptive to oxidation of cysteines and, jointly, to channel gating. Electrophysiological data support the idea that LITE-1 is a photon and H 2 O 2 -coincidence detector. Other proteins with similarity to LITE-1, specifically C. elegans GUR-3, likely use a similar mechanism for photon detection. Thus, a common protein fold and assembly, used for chemoreception in insects, possibly by binding of a particular compound, may have evolved into a light-activated ion channel. [Display omitted] • LITE-1 is a C. elegans UV/blue light receptor of unknown structure and mechanism • AlphaFold2 multimer and MD simulation suggest LITE-1 to be a tetrameric ion channel • A putative aromatic amino acid network and a chromophore may jointly detect photons • Light detection and photo-oxidation may cooperate to gate the nociceptive channel C. elegans senses noxious UV/blue light via LITE-1 photoreceptors. Based on AlphaFold2 multimer models and MD simulations, Hanson et al. suggest that LITE-1 is a light- and oxidation-gated ion channel. Mutagenesis identifies residues needed for channel gating, detecting photons and photo-oxidation products, and binding to a putative chromophore. [ABSTRACT FROM AUTHOR]

Respiratory complex I is a multicomponent enzyme conserved between eukaryotic cells and many bacteria, which couples oxidation of electron donors and quinone reduction with proton pumping. Here, we report that protein transport via the Cag type IV secretion system, a major virulence factor of the Gram-negative bacterial pathogen Helicobacter pylori , is efficiently impeded by respiratory inhibition. Mitochondrial complex I inhibitors, including well-established insecticidal compounds, selectively kill H. pylori , while other Gram-negative or Gram-positive bacteria, such as the close relative Campylobacter jejuni or representative gut microbiota species, are not affected. Using a combination of different phenotypic assays, selection of resistance-inducing mutations, and molecular modeling approaches, we demonstrate that the unique composition of the H. pylori complex I quinone-binding pocket is the basis for this hypersensitivity. Comprehensive targeted mutagenesis and compound optimization studies highlight the potential to develop complex I inhibitors as narrow-spectrum antimicrobial agents against this pathogen. [Display omitted] • T4SS reporter screening identifies species-specific H. pylori growth inhibitors • Inhibitors specifically target NuoB and NuoD of H. pylori 's respiratory complex I • A unique quinone-binding pocket explains H. pylori 's extreme inhibitor sensitivity • Molecular modeling of the quinone-binding site enables rational inhibitor design Lettl et al. report that the unique composition of respiratory complex I in H. pylori makes these bacteria extremely susceptible to small-molecule inhibitors targeting the complex I quinone-binding pocket, while intestinal bacteria remain unaffected, providing an opportunity to develop selective antibacterial drugs. [ABSTRACT FROM AUTHOR]

Summary Maintaining a fluid bilayer is essential for cell signaling and survival. Lipid saturation is a key factor determining lipid packing and membrane fluidity, and it must be tightly controlled to guarantee organelle function and identity. A dedicated eukaryotic mechanism of lipid saturation sensing, however, remains elusive. Here we show that Mga2, a transcription factor conserved among fungi, acts as a lipid-packing sensor in the ER membrane to control the production of unsaturated fatty acids. Systematic mutagenesis, molecular dynamics simulations, and electron paramagnetic resonance spectroscopy identify a pivotal role of the oligomeric transmembrane helix (TMH) of Mga2 for intra-membrane sensing, and they show that the lipid environment controls the proteolytic activation of Mga2 by stabilizing alternative rotational orientations of the TMH region. This work establishes a eukaryotic strategy of lipid saturation sensing that differs significantly from the analogous bacterial mechanism relying on hydrophobic thickness. [ABSTRACT FROM AUTHOR]

The T cell receptor (TCR) expressed by T lymphocytes initiates protective immune responses to pathogens and tumors. To explore the structural basis of how TCR signaling is initiated when the receptor binds to peptide-loaded major histocompatibility complex (pMHC) molecules, we used cryogenic electron microscopy to determine the structure of a tumor-reactive TCRαβ/CD3δγε 2 ζ 2 complex bound to a melanoma-specific human class I pMHC at 3.08 Å resolution. The antigen-bound complex comprises 11 subunits stabilized by multivalent interactions across three structural layers, with clustered membrane-proximal cystines stabilizing the CD3-εδ and CD3-εγ heterodimers. Extra density sandwiched between transmembrane helices reveals the involvement of sterol lipids in TCR assembly. The geometry of the pMHC/TCR complex suggests that efficient TCR scanning of pMHC requires accurate pre-positioning of T cell and antigen-presenting cell membranes. Comparisons of the ligand-bound and unliganded receptors, along with molecular dynamics simulations, indicate that TCRs can be triggered in the absence of spontaneous structural rearrangements. [Display omitted] • Connecting peptides create rigid links to stabilize membrane-bound TCR subunits • Sterol lipid contributes to transmembrane assembly of the TCR complex • A pMHC/TCR structure highlights the cooperative nature of antigen recognition • TCR signaling may be triggered without spontaneous structural rearrangements Cryo-EM structural analysis of a T cell receptor (TCR) complex bound to a tumor-specific human class I pMHC indicates the functional impact of connecting peptides and sterol lipid for complex assembly and suggests that TCR signaling may be triggered without spontaneous structural rearrangements. [ABSTRACT FROM AUTHOR]

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation-induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high-affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family. [Display omitted] • CK1 enzymes autophosphorylate a conserved threonine in the kinase domain, T220 • Phospho-T220 increases the structural plasticity of the substrate binding cleft • Phospho-T220 alters the substrate specificity of CK1 Cullati et al. discovered that CK1 enzymes autophosphorylate a conserved threonine in their kinase domains. Phosphorylation at this site alters the conformation of the substrate binding cleft, affecting substrate specificity. CK1 targets substrates in many different signaling pathways, and this mechanism may regulate which are phosphorylated under different conditions. [ABSTRACT FROM AUTHOR]

Summary: Protein kinase C (PKC) isozymes are the paradigmatic effectors of lipid signaling. PKCs translocate to cell membranes and are allosterically activated upon binding of the lipid diacylglycerol to their C1A and C1B domains. The crystal structure of full-length protein kinase C βII was determined at 4.0 Å, revealing the conformation of an unexpected intermediate in the activation pathway. Here, the kinase active site is accessible to substrate, yet the conformation of the active site corresponds to a low-activity state because the ATP-binding side chain of Phe629 of the conserved NFD motif is displaced. The C1B domain clamps the NFD helix in a low-activity conformation, which is reversed upon membrane binding. A low-resolution solution structure of the closed conformation of PKCβII was derived from small-angle X-ray scattering. Together, these results show how PKCβII is allosterically regulated in two steps, with the second step defining a novel protein kinase regulatory mechanism. [Copyright &y& Elsevier]

The family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella -infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella -containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection. • Phosphoribosyl serine ubiquitination can be reverted by DupA and DupB • DUP specifically binds to and cleaves PR-ubiquitin from serine on substrates • Catalytically inactive DupA mutants can capture PR-ubiquitinated proteins • PR ubiquitination on multiple ER structural proteins causes ER fragmentation Shin et al. show that phosphoribosyl serine ubiquitination can be reversed by two deubiquitinases (DupA and DupB) from Legionella. Two DUPs specifically cleave PR-ubiquitin from serine on substrates. Catalytically inactive DupA-based proteomics approach reveals PR-ubiquitinated proteins and their roles in endoplasmic reticulum (ER) remodeling. [ABSTRACT FROM AUTHOR]

The plasma membrane (PM) is composed of a complex lipid mixture that forms heterogeneous membrane environments. Yet, how small-scale lipid organization controls physiological events at the PM remains largely unknown. Here, we show that ORP-related Osh lipid exchange proteins are critical for the synthesis of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P 2 ], a key regulator of dynamic events at the PM. In real-time assays, we find that unsaturated phosphatidylserine (PS) and sterols, both Osh protein ligands, synergistically stimulate phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity. Biophysical FRET analyses suggest an unconventional co-distribution of unsaturated PS and phosphatidylinositol 4-phosphate (PI4P) species in sterol-containing membrane bilayers. Moreover, using in vivo imaging approaches and molecular dynamics simulations, we show that Osh protein-mediated unsaturated PI4P and PS membrane lipid organization is sensed by the PIP5K specificity loop. Thus, ORP family members create a nanoscale membrane lipid environment that drives PIP5K activity and PI(4,5)P 2 synthesis that ultimately controls global PM organization and dynamics. • The Osh lipid exchange proteins are required to maintain PI(4,5)P 2 levels in the PM • Unsaturated PS and sterols synergistically stimulate PIP5K activity • The specificity loop conserved in PIP5Ks serves as a lipid sensor • A simulation model of the PIP5K specificity loop embedded in a lipid bilayer Nishimura et al. show that the Osh lipid exchange proteins are required for PI(4,5)P 2 synthesis, a key regulator of global plasma membrane organization and function. The Osh proteins form a unique membrane lipid environment with unsaturated phosphatidylserine and sterols that specifies PIP 5-kinase activity at the plasma membrane. [ABSTRACT FROM AUTHOR]

Summary Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. The interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first β sheet of the BECN1 BARA domain and the UVRAG BARA2 domain by hydrogen-deuterium exchange and cryo-EM. These data suggest that the BARA β sheet 1 unfolds to directly engage the membrane. This mechanism was confirmed using protein engineering, giant unilamellar vesicle assays, and molecular simulations. Using this mechanism, a BECN1 β sheet-1 derived peptide activates both PI3KC3 complexes I and II, while HIV-1 Nef inhibits complex II. These data reveal how BECN1 switches on and off PI3KC3 binding to membranes. The observations explain how PI3KC3 inhibition by Rubicon, activation by autophagy-inducing BECN1 peptides, and inhibition by HIV-1 Nef are mediated by the switchable ability of the BECN1 BARA domain to partially unfold and insert into membranes. Graphical Abstract Highlights • Structural mapping of Rubicon binding to PI3KC3-C2 • Rubicon stabilizes BECN1 BARA β sheet-1 and blocks membrane docking • BECN1 autophagy-activating peptide promotes BARA membrane binding • HIV-1 Nef modulates PI3KC3-C2 by a similar mechanism as Rubicon A structural, biochemical, and biophysical study of PI 3-kinase regulation by Rubicon, HIV-1 Nef, and an autophagy-activating peptide shows how BECN1 BARA domain is switched on and off in both physiology and infection, and how it can be targeted for therapeutic induction of autophagy. [ABSTRACT FROM AUTHOR]

Summary The unfolded protein response (UPR) is a conserved homeostatic program that is activated by misfolded proteins in the lumen of the endoplasmic reticulum (ER). Recently, it became evident that aberrant lipid compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent in activating the UPR. The underlying molecular mechanism, however, remained unclear. We show that the most conserved transducer of ER stress, Ire1, uses an amphipathic helix (AH) to sense membrane aberrancies and control UPR activity. In vivo and in vitro experiments, together with molecular dynamics (MD) simulations, identify the physicochemical properties of the membrane environment that control Ire1 oligomerization. This work establishes the molecular mechanism of UPR activation by lipid bilayer stress. [ABSTRACT FROM AUTHOR]

PglK is a lipid-linked oligosaccharide (LLO) flippase essential for asparagine-linked protein glycosylation in Campylobacter jejuni. Previously we have proposed a non-alternating-access LLO translocation mechanism, where postulated outward-facing states play a primary role. To investigate this unusual mechanistic proposal, we have determined a high-resolution structure of PglK that displays an outward semi-occluded state with the two nucleotide binding domains forming an asymmetric closed dimer with two bound ATPγS molecules. Based on this structure, we performed extensive molecular dynamics simulations to investigate LLO recognition and flipping. Our results suggest that PglK may employ a "substrate-hunting" mechanism to locally increase the LLO concentration and facilitate its jump into the translocation pathway, for which sugars from the LLO head group are essential. We further conclude that the release of LLO to the outside occurs before ATP hydrolysis and is followed by the closing of the periplasmic cavity of PglK., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Eukaryotic cells face the challenge of maintaining the complex composition of several coexisting organelles. The molecular mechanisms underlying the homeostasis of subcellular membranes and their adaptation during stress are only now starting to emerge. Here, we discuss three membrane property sensors of the endoplasmic reticulum (ER), namely OPI1, MGA2, and IRE1, each controlling a large cellular program impacting the lipid metabolic network. OPI1 coordinates the production of membrane and storage lipids, MGA2 regulates the production of unsaturated fatty acids required for membrane biogenesis, and IRE1 controls the unfolded protein response (UPR) to adjust ER size, protein folding, and the secretory capacity of the cell. Although these proteins use remarkably distinct sensing mechanisms, they are functionally connected via the ER membrane and cooperate to maintain membrane homeostasis. As a rationalization of the recently described mechanism of UPR activation by lipid bilayer stress, we propose that IRE1 can sense the protein-to-lipid ratio in the ER membrane to ensure a balanced production of membrane proteins and lipids., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ductile materials can absorb spikes in mechanical force, whereas brittle ones fail catastrophically. Here we develop a theory to quantify the kinetic ductility of single molecules from force spectroscopy experiments, relating force-spike resistance to the differential responses of the intact protein and the unfolding transition state to an applied mechanical force. We introduce a class of unistable one-dimensional potential surfaces that encompass previous models as special cases and continuously cover the entire range from ductile to brittle. Compact analytic expressions for force-dependent rates and rupture-force distributions allow us to analyze force-clamp and force-ramp pulling experiments. We find that the force-transmitting protein domains of filamin and titin are kinetically ductile when pulled from their two termini, making them resistant to force spikes. For the mechanostable muscle protein titin, a highly ductile model reconciles data over 10 orders of magnitude in force loading rate from experiment and simulation., (Copyright © 2016 Biophysical Society. All rights reserved.)

Structures of biomolecular systems are increasingly computed by integrative modeling that relies on varied types of experimental data and theoretical information. We describe here the proceedings and conclusions from the first wwPDB Hybrid/Integrative Methods Task Force Workshop held at the European Bioinformatics Institute in Hinxton, UK, on October 6 and 7, 2014. At the workshop, experts in various experimental fields of structural biology, experts in integrative modeling and visualization, and experts in data archiving addressed a series of questions central to the future of structural biology. How should integrative models be represented? How should the data and integrative models be validated? What data should be archived? How should the data and models be archived? What information should accompany the publication of integrative models?, (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The biogenesis of autophagosomes commences at the phagophore assembly site (PAS), a protein-vesicle ultrastructure that is organized by the Atg1 complex. The Atg1 complex consists of the Atg1 protein kinase, the intrinsically disordered region-rich Atg13, and the dimeric double crescent-shaped Atg17-Atg31-Atg29 subcomplex. We show that the PAS contains a relatively uniform ∼28 copies of Atg17, and upon autophagy induction, similar numbers of Atg1 and Atg13 molecules. We then apply ensemble refinement of small-angle X-ray scattering to determine the solution structures of the Atg1-Atg13 and Atg17-Atg31-Atg29 subcomplexes and the Atg1 complex, using a trimmed minipentamer tractable to biophysical studies. We observe tetramers of Atg1 pentamers that assemble via Atg17-Atg31-Atg29. This leads to a model for the higher organization of the Atg1 complex in PAS scaffolding., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The theoretical prediction of water drying transitions near nonpolar surfaces has stimulated an intensive search for biological processes exploiting this extreme form of hydrophobicity. Here we quantitatively demonstrate that drying of a hydrophobic constriction is the major determinant of ion conductance in the GLIC pentameric ion channel. Molecular-dynamics simulations show that in the closed state, the channel conductance is ∼12 orders-of-magnitude lower than in the open state. This large drop in conductance is remarkable because even in the functionally closed conformation the pore constriction remains wide enough for the passage of sodium ions, aided by a continuous bridge of ∼12 water molecules. However, we find that the free energy cost of hydrating the hydrophobic gate is large, accounting almost entirely for the energetic barrier blocking ion passage. The free energies of transferring a sodium ion into a prehydrated gate in functionally closed and open states differ by only 1.2 kcal/mol, compared to an 11 kcal/mol difference in the costs of hydrating the hydrophobic gate. Conversely, ion desolvation effects play only minor roles in GLIC ion channel gating. Our simulations help rationalize experiments probing the gating kinetics of the nicotinic acetylcholine receptor in response to mutations of pore-lining residues. The molecular character and phase behavior of water should thus be included in quantitative descriptions of ion channel gating., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The ESCRT-I and ESCRT-II supercomplex induces membrane buds that invaginate into the lumen of endosomes, a process central to the lysosomal degradation of ubiquitinated membrane proteins. The solution conformation of the membrane-budding ESCRT-I-II supercomplex from yeast was refined against small-angle X-ray scattering (SAXS), single-molecule Förster resonance energy transfer (smFRET), and double electron-electron resonance (DEER) spectra. These refinements yielded an ensemble of 18 ESCRT-I-II supercomplex structures that range from compact to highly extended. The crescent shapes of the ESCRT-I-II supercomplex structures provide the basis for a detailed mechanistic model, in which ESCRT-I-II stabilizes membrane buds and coordinates cargo sorting by lining the pore of the nascent bud necks. The hybrid refinement used here is general and should be applicable to other dynamic multiprotein assmeblies., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We developed and implemented an ensemble-refinement method to study dynamic biomolecular assemblies with intrinsically disordered segments. Data from small angle X-ray scattering (SAXS) experiments and from coarse-grained molecular simulations were combined by using a maximum-entropy approach. The method was applied to CHMP3 of ESCRT-III, a protein with multiple helical domains separated by flexible linkers. Based on recent SAXS data by Lata et al. (J. Mol. Biol. 378, 818, 2008), we constructed ensembles of CHMP3 at low- and high-salt concentration to characterize its closed autoinhibited state and open active state. At low salt, helix α(5) is bound to the tip of helices α(1) and α(2), in excellent agreement with a recent crystal structure. Helix α(6) remains free in solution and does not appear to be part of the autoinhibitory complex. The simulation-based ensemble refinement is general and effectively increases the resolution of SAXS beyond shape information to atomically detailed structures., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Pentameric ligand-gated ion channels are an important family of membrane proteins and play key roles in physiological processes, including signal transduction at chemical synapses. Here, we study the conformational changes associated with the opening and closing of the channel pore. Based on recent crystal structures of two prokaryotic members of the family in open and closed states, respectively, mixed elastic network models are constructed for the transmembrane domain. To explore the conformational changes in the gating transition, a coarse-grained transition path is computed that smoothly connects the closed and open conformations of the channel. We find that the conformational transition involves no major rotations of the transmembrane helices, and is instead characterized by a concerted tilting of helices M2 and M3. In addition, helix M2 changes its bending state, which results in an early closure of the pore during the open-to-closed transition.

The human Hrs and STAM proteins comprise the ESCRT-0 complex, which sorts ubiquitinated cell surface receptors to lysosomes for degradation. Here we report a model for the complete ESCRT-0 complex based on the crystal structure of the Hrs-STAM core complex, previously solved domain structures, hydrodynamic measurements, and Monte Carlo simulations. ESCRT-0 expressed in insect cells has a hydrodynamic radius of RH = 7.9 nm and is a 1:1 heterodimer. The 2.3 Angstroms crystal structure of the ESCRT-0 core complex reveals two domain-swapped GAT domains and an antiparallel two-stranded coiled-coil, similar to yeast ESCRT-0. ESCRT-0 typifies a class of biomolecular assemblies that combine structured and unstructured elements, and have dynamic and open conformations to ensure versatility in target recognition. Coarse-grained Monte Carlo simulations constrained by experimental RH values for ESCRT-0 reveal a dynamic ensemble of conformations well suited for diverse functions.

Accurate force fields are essential for the success of molecular dynamics simulations. In apparent contrast to the conformational preferences of most force fields, recent NMR experiments suggest that short polyalanine peptides in water populate the polyproline II structure almost exclusively. To investigate this apparent contradiction, with its ramifications for the assessment of molecular force fields and the structure of unfolded proteins, we performed extensive simulations of Ala(5) in water ( approximately 5 micros total time), using twelve different force fields and three different peptide terminal groups. Using either empirical or density-functional-based Karplus relations for the J-couplings, we find that most current force fields do overpopulate the alpha-region, with quantitative results depending on the choice of Karplus relation and on the peptide termini. Even after reweighting to match experiment, we find that Ala(5) retains significant alpha- and beta-populations. In fact, several force fields match the experimental data well before reweighting and have a significant helical population. We conclude that radical changes to the best current force fields are not necessary, based on the NMR data. Nevertheless, experiments on short peptides open the way toward the systematic improvement of current simulation models.

Single-molecule force experiments provide powerful new tools to explore biomolecular interactions. Here, we describe a systematic procedure for extracting kinetic information from force-spectroscopy experiments, and apply it to nanopore unzipping of individual DNA hairpins. Two types of measurements are considered: unzipping at constant voltage, and unzipping at constant voltage-ramp speeds. We perform a global maximum-likelihood analysis of the experimental data at low-to-intermediate ramp speeds. To validate the theoretical models, we compare their predictions with two independent sets of data, collected at high ramp speeds and at constant voltage, by using a quantitative relation between the two types of measurements. Microscopic approaches based on Kramers theory of diffusive barrier crossing allow us to estimate not only intrinsic rates and transition state locations, as in the widely used phenomenological approach based on Bell's formula, but also free energies of activation. The problem of extracting unique and accurate kinetic parameters of a molecular transition is discussed in light of the apparent success of the microscopic theories in reproducing the experimental data.

The yeast Vps27/Hse1 complex and the homologous mammalian Hrs/STAM complex deliver ubiquitinated transmembrane proteins to the ESCRT endosomal-sorting pathway. The Vps27/Hse1 complex directly binds to ubiquitinated transmembrane proteins and recruits both ubiquitin ligases and deubiquitinating enzymes. We have solved the crystal structure of the core responsible for the assembly of the Vps27/Hse1 complex at 3.0 A resolution. The structure consists of two intertwined GAT domains, each consisting of two helices from one subunit and one from the other. The two GAT domains are connected by an antiparallel coiled coil, forming a 90 A-long barbell-like structure. This structure places the domains of Vps27 and Hse1 that recruit ubiquitinated cargo and deubiquitinating enzymes close to each other. Coarse-grained Monte Carlo simulations of the Vps27/Hse1 complex on a membrane show how the complex binds cooperatively to lipids and ubiquitinated membrane proteins and acts as a scaffold for ubiquitination reactions.

We explore the relative contributions of different structural elements to the stability of Abeta fibrils by molecular-dynamics simulations performed over a broad range of temperatures (298 K to 398 K). Our fibril structures are based on solid-state nuclear magnetic resonance experiments of Abeta(1-40) peptides, with sheets of parallel beta-strands connected by loops and stabilized by interior salt bridges. We consider models with different interpeptide interfaces, and different staggering of the N- and C-terminal beta-strands along the fibril axis. Multiple 10-20 ns molecular-dynamics simulations show that fibril segments with 12 peptides are stable at ambient temperature. The different models converge toward an interdigitated side-chain packing, and present water channels solvating the interior D23/K28 salt bridges. At elevated temperatures, we observe the early phases of fibril dissociation as a loss of order in the hydrophilic loops connecting the two beta-strands, and in the solvent-exposed N-terminal beta-sheets. As the most dramatic structural change, we observe collective sliding of the N- and C-terminal beta-sheets on top of each other. The interior C-terminal beta-sheets in the hydrophobic core remain largely intact, indicating that their formation and stability is crucial to the dissociation/elongation and stability of Abeta fibrils.

Conformational transitions underlie the function of many biomolecular systems. Resolving intermediate structural changes, however, is challenging for both experiments and all-atom simulations because the duration of transitions is short relative to the lifetime of the stable species. Simplified descriptions based on a single experimental structure, such as elastic network models or Gō models, are not immediately applicable. Here, we develop a general method that combines multiple coarse-grained models to capture slow conformational transitions. Individually, each model describes one of the experimental structures; together, they approximate the complete energy surface. We demonstrate the method for the helix-to-sheet transition in Arc repressor N11L. We find that the transition involves the partial unfolding of the switch region, and rapid refolding into the alternate structure. Transient local unfolding is consistent with the low hydrogen exchange protection factors of the switch region. Also in agreement with experiment, the isomerization occurs independently of the global folding/dimerization transition.

Narrow hydrophobic regions are a common feature of biological channels, with possible roles in ion-channel gating. We study the principles that govern ion transport through narrow hydrophobic membrane pores by molecular dynamics simulation of model membranes formed of hexagonally packed carbon nanotubes. We focus on the factors that determine the energetics of ion translocation through such nonpolar nanopores and compare the resulting free-energy barriers for pores with different diameters corresponding to the gating regions in closed and open forms of potassium channels. Our model system also allows us to compare the results from molecular dynamics simulations directly to continuum electrostatics calculations. Both simulations and continuum calculations show that subnanometer wide pores pose a huge free-energy barrier for ions, but a small increase in the pore diameter to approximately 1 nm nearly eliminates that barrier. We also find that in those wider channels the ion mobility is comparable to that in the bulk phase. By calculating local electrostatic potentials, we show that the long range Coulomb interactions of ions are strongly screened in the wide water-filled channels. Whereas continuum calculations capture the overall energetics reasonably well, the local water structure, which is not accounted for in this model, leads to interesting effects such as the preference of hydrated ions to move along the pore wall rather than through the center of the pore.

Alignment of macromolecules in nearly neutral aqueous lyotropic liquid-crystalline media such as bicelles, commonly used in macromolecular NMR studies, can be predicted accurately by a steric obstruction model (Zweckstetter and Bax, 2000). A simple extension of this model is described that results in improved predictions for both the alignment orientation and magnitude of protein and DNA solutes in charged nematic media, such as the widely used medium of filamentous phage Pf1. The extended model approximates the electrostatic interaction between a solute and an ordered phage particle as that between the solute's surface charges and the electric field of the phage. The model is evaluated for four different proteins and a DNA oligomer. Results indicate that alignment in charged nematic media is a function not only of the solute's shape, but also of its electric multipole moments of net charge, dipole, and quadrupole. The relative importance of these terms varies greatly from one macromolecule to another, and evaluation of the experimental data indicates that these terms scale differently with ionic strength. For several of the proteins, the calculated alignment is sensitive to the precise position of the charged groups on the protein surface. This suggests that NMR alignment measurements can potentially be used to probe protein electrostatics. Inclusion of electrostatic interactions in addition to steric effects makes the extended model applicable to all liquid crystals used in biological NMR to date.

Hydrodynamic properties of small single-stranded RNA homopolymers with three and six nucleotides in free solution are determined from molecular dynamics simulations in explicit solvent. We find that the electrophoretic mobility increases with increasing RNA length, consistent with experiment. Diffusion coefficients of RNA, corrected for finite-size effects and solvent viscosity, agree well with those estimated from experiments and hydrodynamic calculations. The diffusion coefficients and electrophoretic mobilities satisfy a Nernst-Einstein relation in which the effective charge of RNA is reduced by the charge of transiently bound counterions. Fluctuations in the counterion atmosphere are shown to enhance the diffusive spread of RNA molecules drifting along the direction of the external electric field. As a consequence, apparent diffusion coefficients measured by capillary zone electrophoresis can be significantly larger than the actual values at certain experimental conditions.

Mechanical forces exerted by laser tweezers or atomic force microscopes can be used to drive rare transitions in single molecules, such as unfolding of a protein or dissociation of a ligand. The phenomenological description of pulling experiments based on Bell's expression for the force-induced rupture rate is found to be inadequate when tested against computer simulations of a simple microscopic model of the dynamics. We introduce a new approach of comparable complexity to extract more accurate kinetic information about the molecular events from pulling experiments. Our procedure is based on the analysis of a simple stochastic model of pulling with a harmonic spring and encompasses the phenomenological approach, reducing to it in the appropriate limit. Our approach is tested against computer simulations of a multimodule titin model with anharmonic linkers and then an illustrative application is made to the forced unfolding of I27 subunits of the protein titin. Our procedure to extract kinetic information from pulling experiments is simple to implement and should prove useful in the analysis of experiments on a variety of systems.