The C-terminal head domain of Burkholderia pseudomallei BpaC has a striking hydrophilic core with an extensive solvent network.

Gram-negative pathogens like Burkholderia pseudomallei use trimeric autotransporter adhesins such as BpaC as key molecules in their pathogenicity. Our 1.4 Å crystal structure of the membrane-proximal part of the BpaC head domain shows that the domain is exclusively made of left-handed parallel ß-roll repeats. This, the largest such structure solved, has two unique features. First, the core, rather than being composed of the canonical hydrophobic Ile and Val, is made up primarily of the hydrophilic Thr and Asn, with two different solvent channels. Second, comparing BpaC to all other left-handed parallel ß-roll structures showed that the position of the head domain in the protein correlates with the number and type of charged residues. In BpaC, only negatively charged residues face the solvent-in stark contrast to the primarily positive surface charge of the left-handed parallel ß-roll "type" protein, YadA. We propose extending the definitions of these head domains to include the BpaC-like head domain as a separate subtype, based on its unusual sequence, position, and charge. We speculate that the function of left-handed parallel ß-roll structures may differ depending on their position in the structure.

Do Sideline Tests of Vestibular and Oculomotor Function Accurately Diagnose Sports-Related Concussion in Adults? A Systematic Review and Meta-analysis.

BACKGROUND: Sports-related concussion (SRC) assessment tools are primarily based on subjective assessments of somatic, cognitive, and psychosocial/emotional symptoms. SRC symptoms remain underreported, and objective measures of SRC impairments would be valuable to assist diagnosis. Measurable impairments to vestibular and oculomotor processing have been shown to occur after SRC and may provide valid objective assessments. PURPOSE: Determine the diagnostic accuracy of sideline tests of vestibular and oculomotor dysfunction to identify SRC in adults. STUDY DESIGN: Systematic review; Level of evidence, 4. METHODS: Electronic databases and gray literature were searched from inception until February 12, 2020. Physically active individuals (>16 years of age) who participated in sports were included. The reference standard for SRC was a combination of clinical signs and symptoms (eg, the Sport Concussion Assessment Tool [SCAT]), and index tests included any oculomotor assessment tool. The QUADAS tool was used to assess risk of bias, with the credibility of the evidence being rated according to GRADE. RESULTS: A total of 8 studies were included in this review. All included studies used the King-Devick test, with no other measures being identified. Meta-analysis was performed on 4 studies with a summary sensitivity and specificity of 0.77 and 0.82, respectively. The overall credibility of the evidence was rated as very low. CONCLUSION: Caution must be taken when interpreting these results given the very low credibility of the evidence, and the true summary sensitivity and specificity may substantially differ from the values calculated within this systematic review. Therefore, we recommend that clinicians using the King-Devick test to diagnose SRC in adults do so in conjunction with other tools such as the SCAT. PROSPERO REGISTRATION: CRD42018106632.

Modelling the active SARS-CoV-2 helicase complex as a basis for structure-based inhibitor design.

The RNA helicase (non-structural protein 13, NSP13) of SARS-CoV-2 is essential for viral replication, and it is highly conserved among the coronaviridae family, thus a prominent drug target to treat COVID-19. We present here structural models and dynamics of the helicase in complex with its native substrates based on thorough analysis of homologous sequences and existing experimental structures. We performed and analysed microseconds of molecular dynamics (MD) simulations, and our model provides valuable insights to the binding of the ATP and ssRNA at the atomic level. We identify the principal motions characterising the enzyme and highlight the effect of the natural substrates on this dynamics. Furthermore, allosteric binding sites are suggested by our pocket analysis. Our obtained structural and dynamical insights are important for subsequent studies of the catalytic function and for the development of specific inhibitors at our characterised binding pockets for this promising COVID-19 drug target.

Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides.

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

Spectrum of pathogenic variants and founder effects in amelogenesis imperfecta associated with MMP20.

Amelogenesis imperfecta (AI) describes a heterogeneous group of developmental enamel defects that typically have Mendelian inheritance. Exome sequencing of 10 families with recessive hypomaturation AI revealed four novel and one known variants in the matrix metallopeptidase 20 (MMP20) gene that were predicted to be pathogenic. MMP20 encodes a protease that cleaves the developing extracellular enamel matrix and is necessary for normal enamel crystal growth during amelogenesis. New homozygous missense changes were shared between four families of Pakistani heritage (c.625G>C; p.(Glu209Gln)) and two of Omani origin (c.710C>A; p.(Ser237Tyr)). In two families of UK origin and one from Costa Rica, affected individuals were homozygous for the previously reported c.954-2A>T; p.(Ile319Phefs*19) variant. For each of these variants, microsatellite haplotypes appeared to exclude a recent founder effect, but elements of haplotype were conserved, suggesting more distant founding ancestors. New compound heterozygous changes were identified in one family of the European heritage: c.809_811+12delinsCCAG; p.(?) and c.1122A>C; p.(Gln374His). This report further elucidates the mutation spectrum of MMP20 and the probable impact on protein function, confirms a consistent hypomaturation phenotype and shows that mutations in MMP20 are a common cause of autosomal recessive AI in some communities.

Sequence-dependent structural properties of B-DNA: what have we learned in 40 years?

The structure of B-DNA, the physiological form of the DNA molecule, has been a central topic in biology, chemistry and physics. Far from uniform and rigid, the double helix was revealed as a flexible and structurally polymorphic molecule. Conformational changes that lead to local and global changes in the helix geometry are mediated by a complex choreography of base and backbone rearrangements affecting the ability of the B-DNA to recognize ligands and consequently on its functionality. In this sense, the knowledge obtained from the sequence-dependent structural properties of B-DNA has always been thought crucial to rationalize how ligands and, most notably, proteins recognize B-DNA and modulate its activity, i.e. the structural basis of gene regulation. Honouring the anniversary of the first high-resolution X-ray structure of a B-DNA molecule, in this contribution, we present the most important discoveries of the last 40 years on the sequence-dependent structural and dynamical properties of B-DNA, from the early beginnings to the current frontiers in the field.

New variants and in silico analyses in GRK1 associated Oguchi disease.

Biallelic mutations in G-Protein coupled receptor kinase 1 (GRK1) cause Oguchi disease, a rare subtype of congenital stationary night blindness (CSNB). The purpose of this study was to identify disease causing GRK1 variants and use in-depth bioinformatic analyses to evaluate how their impact on protein structure could lead to pathogenicity. Patients' genomic DNA was sequenced by whole genome, whole exome or focused exome sequencing. Disease associated variants, published and novel, were compared to nondisease associated missense variants. The impact of GRK1 missense variants at the protein level were then predicted using a series of computational tools. We identified twelve previously unpublished cases with biallelic disease associated GRK1 variants, including eight novel variants, and reviewed all GRK1 disease associated variants. Further structure-based scoring revealed a hotspot for missense variants in the kinase domain. In addition, to aid future clinical interpretation, we identified the bioinformatics tools best able to differentiate disease associated from nondisease associated variants. We identified GRK1 variants in Oguchi disease patients and investigated how disease-causing variants may impede protein function in-silico.

Continuum mechanical parameterisation of cytoplasmic dynein from atomistic simulation.

Cytoplasmic dynein is responsible for intra-cellular transport in eukaryotic cells. Using Fluctuating Finite Element Analysis (FFEA), a novel algorithm that represents proteins as continuum viscoelastic solids subject to thermal noise, we are building computational tools to study the mechanics of these molecular machines. Here we present a methodology for obtaining the material parameters required to represent the flexibility of cytoplasmic dynein within FFEA from atomistic molecular dynamics (MD) simulations, and show that this continuum representation is sufficient to capture the principal dynamic properties of the motor.

Combined Force-Torque Spectroscopy of Proteins by Means of Multiscale Molecular Simulation.

Assessing the structural properties of large proteins is important to gain an understanding of their function in, e.g., biological systems or biomedical applications. We propose a method to examine the mechanical properties of proteins subject to applied forces by means of multiscale simulation. Both stretching and torsional forces are considered, and these may be applied independently of each other. As a proof of principle, we apply torsional forces to a coarse-grained continuum model of the antibody protein immunoglobulin G using fluctuating finite element analysis and use it to identify the area of strongest deformation. This region is essential to the torsional properties of the molecule as a whole because it represents the softest, most deformable domain. Zooming in, this part of the molecule is subjected to torques and stretching forces using molecular dynamics simulations on an atomistically resolved level to investigate its torsional properties. We calculate the torsional resistance as a function of the rotation of the domain while subjecting it to various stretching forces. From this, we assess how the measured twist-torque profiles develop with increasing stretching force and show that they exhibit torsion stiffening, in qualitative agreement with experimental findings. We argue that combining the twist-torque profiles for various stretching forces effectively results in a combined force-torque spectroscopy analysis, which may serve as a mechanical signature for a biological macromolecule.

Exploring the dynamics of flagellar dynein within the axoneme with Fluctuating Finite Element Analysis.

Flagellar dyneins are the molecular motors responsible for producing the propagating bending motions of cilia and flagella. They are located within a densely packed and highly organised super-macromolecular cytoskeletal structure known as the axoneme. Using the mesoscale simulation technique Fluctuating Finite Element Analysis (FFEA), which represents proteins as viscoelastic continuum objects subject to explicit thermal noise, we have quantified the constraints on the range of molecular conformations that can be explored by dynein-c within the crowded architecture of the axoneme. We subsequently assess the influence of crowding on the 3D exploration of microtubule-binding sites, and specifically on the axial step length. Our calculations combine experimental information on the shape, flexibility and environment of dynein-c from three distinct sources; negative stain electron microscopy, cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET). Our FFEA simulations show that the super-macromolecular organisation of multiple protein complexes into higher-order structures can have a significant influence on the effective flexibility of the individual molecular components, and may, therefore, play an important role in the physical mechanisms underlying their biological function.