Clustering polymorphs of tau and IAPP fibrils with the CHEP algorithm.

Recent steps towards automation have improved the quality and efficiency of the entire cryo-electron microscopy workflow, from sample preparation to image processing. Most of the image processing steps are now quite automated, but there are still a few steps which need the specific intervention of researchers. One such step is the identification and separation of helical protein polymorphs at early stages of image processing. Here, we tested and evaluated our recent clustering approach on three datasets containing amyloid fibrils, demonstrating that the proposed unsupervised clustering method automatically and effectively identifies the polymorphs from cryo-EM images. As an automated polymorph separation method, it has the potential to complement automated helical picking, which typically cannot easily distinguish between polymorphs with subtle differences in morphology, and is therefore a useful tool for the image processing and structure determination of helical proteins.

Cryo-EM structure of islet amyloid polypeptide fibrils reveals similarities with amyloid-ß fibrils.

Amyloid deposits consisting of fibrillar islet amyloid polypeptide (IAPP) in pancreatic islets are associated with beta-cell loss and have been implicated in type 2 diabetes (T2D). Here, we applied cryo-EM to reconstruct densities of three dominant IAPP fibril polymorphs, formed in vitro from synthetic human IAPP. An atomic model of the main polymorph, built from a density map of 4.2-Å resolution, reveals two S-shaped, intertwined protofilaments. The segment 21-NNFGAIL-27, essential for IAPP amyloidogenicity, forms the protofilament interface together with Tyr37 and the amidated C terminus. The S-fold resembles polymorphs of Alzheimer's disease (AD)-associated amyloid-ß (Aß) fibrils, which might account for the epidemiological link between T2D and AD and reports on IAPP-Aß cross-seeding in vivo. The results structurally link the early-onset T2D IAPP genetic polymorphism (encoding Ser20Gly) with the AD Arctic mutation (Glu22Gly) of Aß and support the design of inhibitors and imaging probes for IAPP fibrils.

Integrating cryo-EM and NMR data.

Single-particle cryo-electron microscopy (cryo-EM) is increasingly used as a technique to determine the atomic structure of challenging biological systems. Recent advances in microscope engineering, electron detection, and image processing have allowed the structural determination of bigger and more flexible targets than possible with the complementary techniques X-ray crystallography and NMR spectroscopy. However, there exist many biological targets for which atomic resolution cannot be currently achieved with cryo-EM, making unambiguous determination of the protein structure impossible. Although determining the structure of large biological systems using solely NMR is often difficult, highly complementary experimental atomic-level data for each molecule can be derived from the spectra, and used in combination with cryo-EM data. We review here strategies with which both techniques can be synergistically combined, in order to reach detail and understanding unattainable by each technique acting alone; and the types of biological systems for which such an approach would be desirable.

Modeling of Specific Lipopolysaccharide Binding Sites on a Gram-Negative Porin.

Protein-lipopolysaccharide (LPS) interactions play an important role in providing a stable outer membrane to Gram-negative bacteria. However, the LPS molecules are highly viscous, and sampling LPS motions is thus challenging on a microsecond time scale in simulations. To this end, we introduce a new protocol to randomly allow the LPS molecules to self-assemble around the protein and thereby reduce the starting bias in the simulations. Here we present all-atom molecular dynamics simulations of the OmpE36 porin in an outer membrane model which sum up to a simulation time of more than 20 µs and identify the geometrical properties of the first LPS shell and the role of calcium ions in LPS binding to the protein. The simulations reproduce LPS binding to the porin observed in a recently determined crystal structure but not as compact as in the crystal structure. In addition, the influence of the outer membrane environment on the protein dynamics was analyzed. Our findings highlight the role of divalent cations in stabilizing the binding between proteins and LPS molecules in the outer membrane of Gram-negative bacteria.

Clustering cryo-EM images of helical protein polymers for helical reconstructions.

Helical protein polymers are often dynamic and complex assemblies, with many conformations and flexible domains possible within the helical assembly. During cryo-electron microscopy reconstruction, classification of the image data into homogeneous subsets is a critical step for achieving high resolution, resolving different conformations and elucidating functional mechanisms. Hence, methods aimed at improving the homogeneity of these datasets are becoming increasingly important. In this paper, we introduce a new algorithm that uses results from 2D image classification to sort 2D classes into groups of similar helical polymers. We show that our approach is able to distinguish helical polymers that differ in conformation, composition, and helical symmetry. Our results on test and experimental cases - actin filaments and amyloid fibrils - illustrate how our approach can be useful to improve the homogeneity of a data set. This method is exclusively applicable to helical polymers and other limitations are discussed.

Brownian Dynamics Approach Including Explicit Atoms for Studying Ion Permeation and Substrate Translocation across Nanopores.

A Brownian dynamics (BD) approach including explicit atoms called BRODEA is presented to model ion permeation and molecule translocation across a nanopore confinement. This approach generalizes our previous hybrid molecular dynamics-Brownian dynamics framework ( J. Chem. Theory Comput. 2016, 12, 2401) by incorporating a widespread and enhanced set of simulation schemes based on several boundary conditions and electrostatic models, as well as a temperature accelerated method for sampling free energy surfaces and determining substrate translocation pathways. As a test case, BRODEA was applied to study the ion diffusion as well as to ciprofloxacin and enrofloxacin transport through the outer membrane porin OmpC from E. coli. The equivalence between the different simulation schemes was demonstrated and their computational efficiency evaluated. The BRODEA results are able to reproduce the main features of the ion currents and free energy surfaces determined by all-atom molecular dynamics simulations and validated by experiments. Furthermore, the BRODEA results are able to determine the ciprofloxacin and enrofloxacin permeation pathways showing a remarkable agreement with the results obtained from a computational protocol that combines metadynamics and a zero-temperature string method ( J. Chem. Theory Comput. 2017, 13, 4553; J. Phys. Chem. B 2018, 122, 1417). To our knowledge, this is the first time such antibiotic permeation pathways have been characterized by a technique based on Brownian dynamics.

Simulation Study of Occk5 Functional Properties in Pseudomonas aeruginosa Outer Membranes.

The outer membrane (OM) of Gram-negative bacteria is a unique asymmetric lipid bilayer containing lipopolysaccharides (LPS) in the outer leaflet and phospholipids in the inner leaflet. Due to the lack of porins with large pores, the substrate-specific outer membrane proteins play a crucial role in the uptake of small molecules in Pseudomonas aeruginosa. In this study, we report the influences of OM compositions on the ion permeation properties of OccK5 (also known as OpdH), such as ion selectivity and diffusion constants, using all-atom molecular dynamics simulations. The simulations indicate that OccK5 shows a remarkable anion selectivity independent of the OM composition and effective cation concentration. Intriguingly, the outer core and O-antigens of LPS sterically occlude the channel entrance and decrease the diffusion constants of ions approaching the channel, which was also observed in our previous OmpF porin simulations in Escherichia coli OMs. Our results emphasize the role of native membranes in fine-tuning the functional properties of membrane channels.

Identification and characterization of smallest pore-forming protein in the cell wall of pathogenic Corynebacterium urealyticum DSM 7109.

BACKGROUND: Corynebacterium urealyticum, a pathogenic, multidrug resistant member of the mycolata, is known as causative agent of urinary tract infections although it is a bacterium of the skin flora. This pathogenic bacterium shares with the mycolata the property of having an unusual cell envelope composition and architecture, typical for the genus Corynebacterium. The cell wall of members of the mycolata contains channel-forming proteins for the uptake of solutes. RESULTS: In this study, we provide novel information on the identification and characterization of a pore-forming protein in the cell wall of C. urealyticum DSM 7109. Detergent extracts of whole C. urealyticum cultures formed in lipid bilayer membranes slightly cation-selective pores with a single-channel conductance of 1.75 nS in 1 M KCl. Experiments with different salts and non-electrolytes suggested that the cell wall pore of C. urealyticum is wide and water-filled and has a diameter of about 1.8 nm. Molecular modelling and dynamics has been performed to obtain a model of the pore. For the search of the gene coding for the cell wall pore of C. urealyticum we looked in the known genome of C. urealyticum for a similar chromosomal localization of the porin gene to known porH and porA genes of other Corynebacterium strains. Three genes are located between the genes coding for GroEL2 and polyphosphate kinase (PKK2). Two of the genes (cur_1714 and cur_1715) were expressed in different constructs in C. glutamicum ΔporAΔporH and in porin-deficient BL21 DE3 Omp8 E. coli strains. The results suggested that the gene cur_1714 codes alone for the cell wall channel. The cell wall porin of C. urealyticum termed PorACur was purified to homogeneity using different biochemical methods and had an apparent molecular mass of about 4 kDa on tricine-containing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). CONCLUSIONS: Biophysical characterization of the purified protein (PorACur) suggested indeed that cur_1714 is the gene coding for the pore-forming protein in C. urealyticum because the protein formed in lipid bilayer experiments the same pores as the detergent extract of whole cells. The study is the first report of a cell wall channel in the pathogenic C. urealyticum.

Single Residue Acts as Gate in OccK Channels.

The OccK protein subfamily located in the outer membrane of Pseudomonas aeruginosa contains dynamic channels with several conformational states that range from open to closed forms. The molecular determinants of the OccK channels that contribute to the diverse gating have, however, remained elusive so far. Performing molecular dynamics (MD) simulations on OccK5 (OpdH) as an example, local fluctuations of loop L7 mediated by a single residue were identified that effectively gate the channel. The features of this gate residue were studied by single-channel electrophysiology and site-directed mutagenesis demonstrating that this gate residue indeed confers unique gating properties to the OccK channels. In support of these functional measurements, MD simulations highlight the correlations between the size of the side-chain belonging to the gate residue on one side and the pore size as well as the L7 flexibility on the other side.

Structural basis for nutrient acquisition by dominant members of the human gut microbiota.

The human large intestine is populated by a high density of microorganisms, collectively termed the colonic microbiota, which has an important role in human health and nutrition. The survival of microbiota members from the dominant Gram-negative phylum Bacteroidetes depends on their ability to degrade dietary glycans that cannot be metabolized by the host. The genes encoding proteins involved in the degradation of specific glycans are organized into co-regulated polysaccharide utilization loci, with the archetypal locus sus (for starch utilisation system) encoding seven proteins, SusA-SusG. Glycan degradation mainly occurs intracellularly and depends on the import of oligosaccharides by an outer membrane protein complex composed of an extracellular SusD-like lipoprotein and an integral membrane SusC-like TonB-dependent transporter. The presence of the partner SusD-like lipoprotein is the major feature that distinguishes SusC-like proteins from previously characterized TonB-dependent transporters. Many sequenced gut Bacteroides spp. encode over 100 SusCD pairs, of which the majority have unknown functions and substrate specificities. The mechanism by which extracellular substrate binding by SusD proteins is coupled to outer membrane passage through their cognate SusC transporter is unknown. Here we present X-ray crystal structures of two functionally distinct SusCD complexes purified from Bacteroides thetaiotaomicron and derive a general model for substrate translocation. The SusC transporters form homodimers, with each ß-barrel protomer tightly capped by SusD. Ligands are bound at the SusC-SusD interface in a large solvent-excluded cavity. Molecular dynamics simulations and single-channel electrophysiology reveal a 'pedal bin' mechanism, in which SusD moves away from SusC in a hinge-like fashion in the absence of ligand to expose the substrate-binding site to the extracellular milieu. These data provide mechanistic insights into outer membrane nutrient import by members of the microbiota, an area of major importance for understanding human-microbiota symbiosis.

BROMOCEA Code: An Improved Grand Canonical Monte Carlo/Brownian Dynamics Algorithm Including Explicit Atoms.

All-atom molecular dynamics simulations have a long history of applications studying ion and substrate permeation across biological and artificial pores. While offering unprecedented insights into the underpinning transport processes, MD simulations are limited in time-scales and ability to simulate physiological membrane potentials or asymmetric salt solutions and require substantial computational power. While several approaches to circumvent all of these limitations were developed, Brownian dynamics simulations remain an attractive option to the field. The main limitation, however, is an apparent lack of protein flexibility important for the accurate description of permeation events. In the present contribution, we report an extension of the Brownian dynamics scheme which includes conformational dynamics. To achieve this goal, the dynamics of amino-acid residues was incorporated into the many-body potential of mean force and into the Langevin equations of motion. The developed software solution, called BROMOCEA, was applied to ion transport through OmpC as a test case. Compared to fully atomistic simulations, the results show a clear improvement in the ratio of permeating anions and cations. The present tests strongly indicate that pore flexibility can enhance permeation properties which will become even more important in future applications to substrate translocation.

Simulations of outer membrane channels and their permeability.

Channels in the outer membrane of Gram-negative bacteria provide essential pathways for the controlled and unidirectional transport of ions, nutrients and metabolites into the cell. At the same time the outer membrane serves as a physical barrier for the penetration of noxious substances such as antibiotics into the bacteria. Most antibiotics have to pass through these membrane channels to either reach cytoplasmic bound targets or to further cross the hydrophobic inner membrane. Considering the pharmaceutical significance of antibiotics, understanding the functional role and mechanism of these channels is of fundamental importance in developing strategies to design new drugs with enhanced permeation abilities. Due to the biological complexity of membrane channels and experimental limitations, computer simulations have proven to be a powerful tool to investigate the structure, dynamics and interactions of membrane channels. Considerable progress has been made in computer simulations of membrane channels during the last decade. The goal of this review is to provide an overview of the computational techniques and their roles in modeling the transport across outer membrane channels. A special emphasis is put on all-atom molecular dynamics simulations employed to better understand the transport of molecules. Moreover, recent molecular simulations of ion, substrate and antibiotics translocation through membrane pores are briefly summarized. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.