302 research outputs found
Atomic structures of TDP-43 LCD segments and insights into reversible or pathogenic aggregation.
The normally soluble TAR DNA-binding protein 43 (TDP-43) is found aggregated both in reversible stress granules and in irreversible pathogenic amyloid. In TDP-43, the low-complexity domain (LCD) is believed to be involved in both types of aggregation. To uncover the structural origins of these two modes of Ξ²-sheet-rich aggregation, we have determined ten structures of segments of the LCD of human TDP-43. Six of these segments form steric zippers characteristic of the spines of pathogenic amyloid fibrils; four others form LARKS, the labile amyloid-like interactions characteristic of protein hydrogels and proteins found in membraneless organelles, including stress granules. Supporting a hypothetical pathway from reversible to irreversible amyloid aggregation, we found that familial ALS variants of TDP-43 convert LARKS to irreversible aggregates. Our structures suggest how TDP-43 adopts both reversible and irreversible Ξ²-sheet aggregates and the role of mutation in the possible transition of reversible to irreversible pathogenic aggregation
Comparative analysis of carboxysome shell proteins
Carboxysomes are metabolic modules for CO2 fixation that are found in all cyanobacteria and some chemoautotrophic bacteria. They comprise a semi-permeable proteinaceous shell that encapsulates ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase. Structural studies are revealing the integral role of the shell protein paralogs to carboxysome form and function. The shell proteins are composed of two domain classes: those with the bacterial microcompartment (BMC; Pfam00936) domain, which oligomerize to form (pseudo)hexamers, and those with the CcmL/EutN (Pfam03319) domain which form pentamers in carboxysomes. These two shell protein types are proposed to be the basis for the carboxysomeβs icosahedral geometry. The shell proteins are also thought to allow the flux of metabolites across the shell through the presence of the small pore formed by their hexameric/pentameric symmetry axes. In this review, we describe bioinformatic and structural analyses that highlight the important primary, tertiary, and quaternary structural features of these conserved shell subunits. In the future, further understanding of these molecular building blocks may provide the basis for enhancing CO2 fixation in other organisms or creating novel biological nanostructures
Self-assembled amyloid fibrils with controllable conformational heterogeneity
Amyloid fibrils are a hallmark of neurodegenerative diseases and exhibit a conformational diversity that governs their pathological functions. Despite recent findings concerning the pathological role of their conformational diversity, the way in which the heterogeneous conformations of amyloid fibrils can be formed has remained elusive. Here, we show that microwave-assisted chemistry affects the self-assembly process of amyloid fibril formation, which results in their conformational heterogeneity. In particular, microwave-assisted chemistry allows for delicate control of the thermodynamics of the self-assembly process, which enabled us to tune the molecular structure of ??-lactoglobulin amyloid fibrils. The heterogeneous conformations of amyloid fibrils, which can be tuned with microwave-assisted chemistry, are attributed to the microwave-driven thermal energy affecting the electrostatic interaction during the self-assembly process. Our study demonstrates how microwave-assisted chemistry can be used to gain insight into the origin of conformational heterogeneity of amyloid fibrils as well as the design principles showing how the molecular structures of amyloid fibrils can be controlledopen0
Amyloid-Associated Nucleic Acid Hybridisation
Nucleic acids promote amyloid formation in diseases including Alzheimer's
and Creutzfeldt-Jakob disease. However, it remains unclear whether the close
interactions between amyloid and nucleic acid allow nucleic acid secondary
structure to play a role in modulating amyloid structure and function. Here we
have used a simplified system of short basic peptides with alternating
hydrophobic and hydrophilic amino acid residues to study nucleic acid - amyloid
interactions. Employing biophysical techniques including X-ray fibre
diffraction, circular dichroism spectroscopy and electron microscopy we show
that the polymerized charges of nucleic acids concentrate and enhance the
formation of amyloid from short basic peptides, many of which would not
otherwise form fibres. In turn, the amyloid component binds nucleic acids and
promotes their hybridisation at concentrations below their solution
Kd, as shown by time-resolved FRET studies. The
self-reinforcing interactions between peptides and nucleic acids lead to the
formation of amyloid nucleic acid (ANA) fibres whose properties are distinct
from their component polymers. In addition to their importance in disease and
potential in engineering, ANA fibres formed from prebiotically-produced peptides
and nucleic acids may have played a role in early evolution, constituting the
first entities subject to Darwinian evolution
Atrial arrhythmogenesis in wild-type and Scn5a+/Ξ murine hearts modelling LQT3 syndrome
Long QT(3) (LQT3) syndrome is associated with abnormal repolarisation kinetics, prolonged action potential durations (APD) and QT intervals and may lead to life-threatening ventricular arrhythmias. However, there have been few physiological studies of its effects on atrial electrophysiology. Programmed electrical stimulation and burst pacing induced atrial arrhythmic episodes in 16 out of 16 (16/16) wild-type (WT) and 7/16 genetically modified Scn5a+/Ξ (KPQ) Langendorff-perfused murine hearts modelling LQT3 (Pβ<β0.001 for both), and in 14/16 WT and 1/16 KPQ hearts (Pβ<β0.001 for both; Fisherβs exact test), respectively. The arrhythmogenic WT hearts had significantly larger positive critical intervals (CI), given by the difference between atrial effective refractory periods (AERPs) and action potential durations at 90% recovery (APD90), compared to KPQ hearts (8.1 and 3.2Β ms, respectively, Pβ<β0.001). Flecainide prevented atrial arrhythmias in all arrhythmogenic WT (Pβ<β0.001) and KPQ hearts (Pβ<β0.05). It prolonged the AERP to a larger extent than it did the APD90 in both WT and KPQ groups, giving negative CIs. Quinidine similarly exerted anti-arrhythmic effects, prolonged AERP over corresponding APD90 in both WT and KPQ groups. These findings, thus, demonstrate, for the first time, inhibitory effects of the KPQ mutation on atrial arrhythmogenesis and its modification by flecainide and quinidine. They attribute these findings to differences in the CI between WT and mutant hearts, in the presence or absence of these drugs. Thus, prolongation of APD90 over AERP gave positive CI values and increased atrial arrhythmogenicity whereas lengthening of AERP over APD90 reduced such CI values and produced the opposite effect
An AP Endonuclease 1βDNA Polymerase Ξ² Complex: Theoretical Prediction of Interacting Surfaces
Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5β² to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-Ξ²). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-Ξ², the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-Ξ² based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-Ξ² located downstream of APEX1 (3β² to the damaged site) and three with pol-Ξ² located upstream of APEX1 (5β² to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (βΌβ10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-Ξ² at the 3β²-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-Ξ² in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-Ξ² in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3β² side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available
Conformational Proofreading: The Impact of Conformational Changes on the Specificity of Molecular Recognition
To perform recognition, molecules must locate and specifically bind their targets within a noisy biochemical environment with many look-alikes. Molecular recognition processes, especially the induced-fit mechanism, are known to involve conformational changes. This raises a basic question: Does molecular recognition gain any advantage by such conformational changes? By introducing a simple statistical-mechanics approach, we study the effect of conformation and flexibility on the quality of recognition processes. Our model relates specificity to the conformation of the participant molecules and thus suggests a possible answer: Optimal specificity is achieved when the ligand is slightly off target; that is, a conformational mismatch between the ligand and its main target improves the selectivity of the process. This indicates that deformations upon binding serve as a conformational proofreading mechanism, which may be selected for via evolution
Histidine Hydrogen-Deuterium Exchange Mass Spectrometry for Probing the Microenvironment of Histidine Residues in Dihydrofolate Reductase
Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C(2)-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK(a) values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis.Using His-HDX-MS, the pK(a) values and the half-lives (t(1/2)) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+) were determined. The results showed that the two parameters (pK(a) and t(1/2)) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK(a), t(1/2) or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK(a) and t(1/2) changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins
The Pentameric Vertex Proteins Are Necessary for the Icosahedral Carboxysome Shell to Function as a CO2 Leakage Barrier
BACKGROUND: Carboxysomes are polyhedral protein microcompartments found in many autotrophic bacteria; they encapsulate the CO(2) fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) within a thin protein shell and provide an environment that enhances the catalytic capabilities of the enzyme. Two types of shell protein constituents are common to carboxysomes and related microcompartments of heterotrophic bacteria, and the genes for these proteins are found in a large variety of bacteria. METHODOLOGY/PRINCIPAL FINDINGS: We have created a Halothiobacillus neapolitanus knockout mutant that does not produce the two paralogous CsoS4 proteins thought to occupy the vertices of the icosahedral carboxysomes and related microcompartments. Biochemical and ultrastructural analyses indicated that the mutant predominantly forms carboxysomes of normal appearance, in addition to some elongated microcompartments. Despite their normal shape, purified mutant carboxysomes are functionally impaired, although the activities of the encapsulated enzymes are not negatively affected. CONCLUSIONS/SIGNIFICANCE: In the absence of the CsoS4 proteins the carboxysome shell loses its limited permeability to CO(2) and is no longer able to provide the catalytic advantage RubisCO derives from microcompartmentalization. This study presents direct evidence that the diffusion barrier property of the carboxysome shell contributes significantly to the biological function of the carboxysome
- β¦