Markov State Model of Lassa Virus Nucleoprotein Reveals Large Structural Changes during the Trimer to Monomer Transition.
Ontology highlight
ABSTRACT:
SUBMITTER: Pattis JG
PROVIDER: S-EPMC7205590 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Protein fibril self-assembly is a universal transition implicated in neurodegenerative diseases. Although fibril structure/growth are well characterized, fibril nucleation is poorly understood. Here, we use a computational-experimental approach to resolve fibril nucleation. We show that monomer hairpin content quantified from molecular dynamics simulations is predictive of experimental fibril formation kinetics across a tau motif mutant library. Hairpin trimers are predicted to be fibril transition states; one hairpin spontaneously converts into the cross-beta conformation, templating subsequent fibril growth. We designed a disulfide-linked dimer mimicking the transition state that catalyzes fibril formation, measured by ThT fluorescence and TEM, of wild-type motif - which does not normally fibrillize. A dimer compatible with extended conformations but not the transition-state fails to nucleate fibril at any concentration. Tau repeat domain simulations show how long-range interactions sequester this motif in a mutation-dependent manner. This work implies that different fibril morphologies could arise from disease-dependent hairpin seeding from different loci.
| S-EPMC10980705 | biostudies-literature
Project description:We describe methods to express the nucleoprotein (NP) of Lassa fever virus (LASV) in E. coli, to purify and crystallize it using the sitting-drop vapor diffusion method. The crystals were screened using Rigaku micro-007 X-ray generator and a dataset was collected at a resolution of 2.36 Å. The crystals belong to space group P3, with the unit cell parameters a = b = 176.35 Å, c = 56.40 Å, α = β = 90°, and γ = 120°. Using the X-ray diffraction method, we constructed a three-dimensional structure of the LASV NP that should aid in the development of novel therapeutic strategies against this virus, for which vaccine and effective treatment modalities are currently unavailable.
| S-EPMC6351422 | biostudies-literature
Project description:Electrostatic channeling is a naturally occurring approach to control the flux of charged intermediates in catalytic cascades. Computational techniques have enabled quantitative understanding of such mechanisms, augmenting experimental approaches by modeling molecular interactions in atomic detail. In this work, we report the first utilization of a Markov-state model (MSM) to describe the surface diffusion of a reaction intermediate, glucose 6-phosphate, on an artificially modified cascade where hexokinase and glucose-6-phosphate dehydrogenase are covalently conjugated by a cationic oligopeptide bridge. Conformation space networks are used to represent intermediate transport on enzyme surfaces, along with committor probabilities that assess the desorption probability of the intermediate on each segment of the channeling pathway. For the region between the peptide bridge and downstream active site, the ionic strength dependence of desorption probability by MSM agreed well with that by transition state theory. A kinetic Monte Carlo model integrates parameters from different computational methods to evaluate the contribution of desorption during each step. The approach is validated by calculation of kinetic lag time, which agrees well with experimental results. These results further demonstrate the applicability of molecular simulations and advanced sampling techniques to the design of chemical networks.
| S-EPMC6602406 | biostudies-literature
Project description:EhCaBP1 is a well-characterized calcium binding protein from Entamoeba histolytica with four canonical EF-hand motifs. The crystal structure of EhCaBP1 reveals the trimeric organization of N-terminal domain. The solution structure obtained at pH 6.0 indicated its monomeric nature, similar to that of calmodulin. Recent domain-wise studies showed clearly that the N-terminal domain of EhCaBP1 is capable of performing most of the functions of the full-length protein. Additionally, the mode of target binding in the trimer is similar to that found in calmodulin. To study the dynamic nature of this protein and further validate the trimerization of N-terminal domain at physiological conditions, the crystal structure of N-terminal domain was determined at 2.5 A resolution. The final structure consists of EF-1 and EF-2 motifs separated by a long straight helix as seen in the full-length protein. The spectroscopic and stability studies, like far and near-ultraviolet circular dichroism spectra, intrinsic and extrinsic fluorescence spectra, acrylamide quenching, thermal denaturation, and dynamic light scattering, provided clear evidence for a conversion from trimeric state to monomeric state. As the pH was lowered from the physiological pH, a dynamic trimer-monomer transition was observed. The trimeric state and monomeric state observed in spectroscopic studies may represent the x-ray and NMR structures of the EhCaBP1. At pH 6.0, the endogenous kinase activation function was almost lost, indicating that the monomeric state of the protein, where EF-hand motifs are far apart, is not a functional state.
| S-EPMC2884261 | biostudies-literature
Project description:Vault is the largest nonicosahedral cytosolic nucleoprotein particle ever described. The widespread presence and evolutionary conservation of vaults suggest important biologic roles, although their functions have not been fully elucidated. X-ray structure of vault from rat liver was determined at 3.5 Å resolution. It exhibits an ovoid shape with a size of 40 × 40 × 67 nm(3). The cage structure of vault consists of a dimer of half-vaults, with each half-vault comprising 39 identical major vault protein (MVP) chains. Each MVP monomer folds into 12 domains: nine structural repeat domains, a shoulder domain, a cap-helix domain and a cap-ring domain. Interactions between the 42-turn-long cap-helix domains are key to stabilizing the particle. The other components of vaults, telomerase-associated proteins, poly(ADP-ribose) polymerases and small RNAs, are in location in the vault particle by electron microscopy.
| S-EPMC3491081 | biostudies-literature
Project description:The question of whether, during the evolution of an enzyme, the transition state of the catalysed reaction is largely unchanged, or whether transition state and protein change together, was examined using the egb beta-galactosidases of Escherichia coli. Charge development at the first chemical state was assumed [Konstantinidis and Sinnott (1991) Biochem. J. 279, 587-593] to be proportional to delta delta G++, the ratio of second-order rate constants for the hydrolysis of beta-D-galactopyranosyl fluoride and 1-fluoro-D-galactopyranosyl fluoride, expressed as a free-energy difference. delta delta G++ (kJ.mol-1) falls from 10.4 for wild-type enzyme to 6.8 and 7.2 as a consequence of two different single amino-acid changes (which arise from single evolutionary events), to 6.3 as a consequence of the two amino-acid changes together, and then increases slightly to 7.3 as a consequence of a third single evolutionary change involving three further amino-acid changes.
| S-EPMC1132473 | biostudies-other
Project description:The conversion of prion protein from normal to scrapie followed by the aggregation and deposition of this scrapie form leads to various neurodegenerative diseases. A few studies carried out by researchers suggest that E219K prion mutant (glutamate to lysine mutation at residue position 219) is more stable than wild type protein. However a similar point mutation E200K (glutamate to lysine mutation at residue position 200) is pathogenic. In this study we have carried out detailed atomistic simulation of the wild type, pathogenic mutant E200K and E219K mutant which provides more stability. The aim of the study was to detect the early structural changes present in all the three variants which might be responsible for the stability or for their conversion from PrPC to PrPSc. MSM based analyses have been carried out to find out the differences between WT, E200K and E219K systems. Markov state model (MSM) analysis was able to predict the intermediate states which helped to understand the effect of same mutation at two different locations. The MSM analysis was able to show that the extra stability of E219K mutant may be a result of the increase in number of native contacts, strong salt bridges and less random motions. While pathogenicity of E200K mutant can be attributed to loss of some crucial salt-bridge interactions, increased random motions between helix 2 and helix 3.
| S-EPMC9064127 | biostudies-literature
Project description:Lassa virus, the causative agent of Lassa fever, causes thousands of deaths annually and is a biological threat agent, for which there is no vaccine and limited therapy. The nucleoprotein (NP) of Lassa virus has essential roles in viral RNA synthesis and immune suppression, the molecular mechanisms of which are poorly understood. Here we report the crystal structure of Lassa virus NP at 1.80 Å resolution, which reveals amino (N)- and carboxy (C)-terminal domains with structures unlike any of the reported viral NPs. The N domain folds into a novel structure with a deep cavity for binding the m7GpppN cap structure that is required for viral RNA transcription, whereas the C domain contains 3'-5' exoribonuclease activity involved in suppressing interferon induction. To our knowledge this is the first X-ray crystal structure solved for an arenaviral NP, which reveals its unexpected functions and indicates unique mechanisms in cap binding and immune evasion. These findings provide great potential for vaccine and drug development.
| S-EPMC3057469 | biostudies-literature
Project description:Diagnostic systems for Lassa fever (LF), a viral hemorrhagic fever caused by Lassa virus (LASV), such as enzyme immunoassays for the detection of LASV antibodies and LASV antigens, were developed using the recombinant nucleoprotein (rNP) of LASV (LASV-rNP). The LASV-rNP was expressed in a recombinant baculovirus system. LASV-rNP was used as an antigen in the detection of LASV-antibodies and as an immunogen for the production of monoclonal antibodies. The LASV-rNP was also expressed in HeLa cells by transfection with the expression vector encoding cDNA of the LASV-NP gene. An immunoglobulin G enzyme-linked immunosorbent assay (ELISA) using LASV-rNP and an indirect immunofluorescence assay using LASV-rNP-expressing HeLa cells were confirmed to have high sensitivity and specificity in the detection of LASV-antibodies. A novel monoclonal antibody to LASV-rNP, monoclonal antibody 4A5, was established. A sandwich antigen capture (Ag-capture) ELISA using the monoclonal antibody and an anti-LASV-rNP rabbit serum as capture and detection antibodies, respectively, was then developed. Authentic LASV nucleoprotein in serum samples collected from hamsters experimentally infected with LASV was detected by the Ag-capture ELISA. The Ag-capture ELISA specifically detected LASV-rNP but not the rNPs of lymphocytic choriomeningitis virus or Junin virus. The sensitivity of the Ag-capture ELISA in detecting LASV antigens was comparable to that of reverse transcription-PCR in detecting LASV RNA. These LASV rNP-based diagnostics were confirmed to be useful in the diagnosis of LF even in institutes without a high containment laboratory, since the antigens can be prepared without manipulation of the infectious viruses.
| S-EPMC2043324 | biostudies-literature
Project description:A central problem in understanding enzyme regulation is to define the conformational states that account for allosteric changes in catalytic activity. For Escherichia coli aspartate transcarbamoylase (ATCase; EC) the active, relaxed (R state) holoenzyme is generally assumed to be represented by the crystal structure of the complex of the holoenzyme with the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA). It is unclear, however, which conformational differences between the unliganded, inactive, taut (T state) holoenzyme and the PALA complex are attributable to localized effects of inhibitor binding as contrasted to the allosteric transition. To define the conformational changes in the isolated, nonallosteric C trimer resulting from the binding of PALA, we determined the 1.95-A resolution crystal structure of the C trimer-PALA complex. In contrast to the free C trimer, the PALA-bound trimer exhibits approximate threefold symmetry. Conformational changes in the C trimer upon PALA binding include ordering of two active site loops and closure of the hinge relating the N- and C-terminal domains. The C trimer-PALA structure closely resembles the liganded C subunits in the PALA-bound holoenzyme. This similarity suggests that the pronounced hinge closure and other changes promoted by PALA binding to the holoenzyme are stabilized by ligand binding. Consequently, the conformational changes attributable to the allosteric transition of the holoenzyme remain to be defined.
| S-EPMC25784 | biostudies-literature