Alzheimer's-disease-associated conformation of intrinsically disordered tau protein studied by intrinsically disordered protein liquid-phase competitive enzyme-linked immunosorbent assay

Tau protein, the major constituent of paired helical filaments in Alzheimer's disease, belongs to the intrinsically disordered proteins (IDPs). IDPs are an emerging group in the protein kingdom characterized by the absence of a rigid three-dimensional structure. Disordered proteins usually acquire a "functional fold" upon binding to their interaction partner(s). This property of IDPs implies the need for innovative approaches to measure their binding affinity. We have mapped and measured the Alzheimer's-disease-associated epitope on intrinsically disordered tau protein with a novel two-step sandwich competitive enzyme-linked immunosorbent assay (ELISA). This approach allowed us to determine the binding affinity of disordered tau protein in liquid phase without any disturbance to the competitive equilibrium and without any need for covalent or noncovalent modification of tau protein. Furthermore, the global fitting method, used for the reconstruction of tau binding curves, significantly improved the assay readout. The proposed novel competitive ELISA allowed us to determine the changes in the standard Gibbs energy of binding, thus enabling measurement of tau protein conformation in the core of paired helical filaments. IDP competitive ELISA results showed, for the first time, that the tau protein C terminus of the Alzheimer's-disease-derived paired helical filaments core subunit adopts beta-turn type I' fold and is accessible from solution.     

Mycobacterium tuberculosis copper‐regulated protein SocB is an intrinsically disordered protein that folds upon interaction with a synthetic phospholipid bilayer

Multiple genes in Mycobacterium tuberculosis (Mtb) are regulated by copper including socAB (s mall o rf induced by c opper A and B), which is induced by copper and repressed by RicR (r egulated i n c opper r epressor). socA and socB encode hypothetical proteins of 61 and 54 amino acids, respectively. Here, we use biophysical and computational methods to evaluate the SocB structure. We find that SocB lacks evidence for secondary structure, with no thermal cooperative unfolding event, according to circular dichroism measurements. 2D NMR spectra similarly exhibit hallmarks of a disordered structural state, which is also supported by analyzing SocB diffusion. Altogether, these findings suggest that by itself SocB is intrinsically disordered. Interestingly, SocB interacts with a synthetic phospholipid bilayer and becomes helical, which suggests that it may be membrane‐associated. Proteins 2016; 84:193–200. © 2015 Wiley Periodicals, Inc.     

Membrane Tethering of SepF,a Membrane Anchor for the Mycobacterium tuberculosis Z-ring

Bacterial cell division begins with the formation of the Z-ring via polymerization of FtsZ and the localization of Z-ring beneath the inner membrane through membrane anchors. In Mycobacterium tuberculosis (Mtb), SepF is one such membrane anchor, but our understanding of the underlying mechanism is very limited. Here we used molecular dynamics simulations to characterize how SepF itself, a water-soluble protein, tethers to acidic membranes that mimic the Mtb inner membrane. In addition to an amphipathic helix (residues 1–12) at the N-terminus, membrane binding also occurs through two stretches of positively charged residues (Arg27-Arg37 and Arg95-Arg107) in the long linker preceding the FtsZ-binding core domain (residues 128–218). The additional interactions via the disordered linker stabilize the membrane tethering of SepF, and keep the core domain of SepF and hence the attached Z-ring close to the membrane. The resulting membrane proximity of the Z-ring in turn enables its interactions with and thus recruitment of two membrane proteins, FtsW and CrgA, at the late stage of cell division.     

Cryoprotective mechanism of a small intrinsically disordered dehydrin protein

Dehydration proteins (Dehydrins) are expressed during dehydration stress in plants and are thought to protect plant proteins and membranes from the loss of water during drought and at cold temperatures. Several different dehydrins have been shown to protect lactate dehydrogenase (LDH) from damage from being frozen and thawed. We show here that a 48 residue K₂ dehydrin from Vitis riparia protects LDH more effectively than bovine serum albumin, a protein with known cryoprotective function. Light scattering and 8‐anilino‐1‐naphthalene sulfonate fluorescence experiments show that dehydrins prevent aggregation and unfolding of the enzyme. The cryoprotective effects of LDH are reduced by the addition of salt, suggesting that the positively charged K‐segments are attracted to a negatively charged surface but this does not result in binding. Overall K₂ is an intrinsically disordered protein; nuclear magnetic resonance relaxation experiments indicate that the two‐terminal, Lys‐rich K‐segments show a weak propensity for α‐helicity and are flexible, and that the central, polar rich phi‐segment has no secondary structure preference and is highly flexible. We propose that the phi‐segments in dehydrins are important for maintaining the disordered structure so that the protein can act as a molecular shield to prevent partially denatured proteins from interacting with one another, whereas the K‐segments may help to localize the dehydrin near the enzyme surface.     

Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random‐coil‐like pKa values

The sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria allows the incorporation of ammonium into carbon skeletons. In the cyanobacterium Synechocystis sp. PCC 6803, the activity of GS is modulated by the interaction with proteins, which include a 65‐residue‐long intrinsically disordered protein (IDP), the inactivating factor IF7. This interaction is regulated by the presence of charged residues in both IF7 and GS. To understand how charged amino acids can affect the binding of an IDP with its target and to provide clues on electrostatic interactions in disordered states of proteins, we measured the pK_a values of all IF7 acidic groups (Glu32, Glu36, Glu38, Asp40, Asp58, and Ser65, the backbone C‐terminus) at 100 mM NaCl concentration, by using NMR spectroscopy. We also obtained solution structures of IF7 through molecular dynamics simulation, validated them on the basis of previous experiments, and used them to obtain theoretical estimates of the pK_a values. Titration values for the two Asp and three Glu residues of IF7 were similar to those reported for random‐coil models, suggesting the lack of electrostatic interactions around these residues. Furthermore, our results suggest the presence of helical structure at the N‐terminus of the protein and of conformational changes at acidic pH values. The overall experimental and in silico findings suggest that local interactions and conformational equilibria do not play a role in determining the electrostatic features of the acidic residues of IF7.     

Crystal structure of calcium dodecin (Rv0379), from Mycobacterium tuberculosis with a unique calcium-binding site

In eukaryotes, calcium-binding proteins play a pivotal role in diverse cellular processes, and recent findings suggest similar roles for bacterial proteins at different stages in their life cycle. Here, we report the crystal structure of calcium dodecin, Rv0379, from Mycobacterium tuberculosis with a dodecameric oligomeric assembly and a unique calcium-binding motif. Structure and sequence analysis were used to identify orthologs of Rv0379 with different ligand-binding specificity.     

SERCA mutant E309Q binds two Ca2+ ions but adopts a catalytically incompetent conformation

The sarco(endo)plasmic reticulum Ca²⁺‐ATPase (SERCA) couples ATP hydrolysis to transport of Ca²⁺. This directed energy transfer requires cross‐talk between the two Ca²⁺ sites and the phosphorylation site over 50 Å distance. We have addressed the mechano‐structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu³⁰⁹ contributes to Ca²⁺ coordination at site II, and a consensus has been that E309Q only binds Ca²⁺ at site I. The crystal structure of E309Q in the presence of Ca²⁺ and an ATP analogue, however, reveals two occupied Ca²⁺ sites of a non‐catalytic Ca₂E1 state. Ca²⁺ is bound with micromolar affinity by both Ca²⁺ sites in E309Q, but without cooperativity. The Ca²⁺‐bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A‐domain, requiring a shift of transmembrane segment M1 into an ‘up and kinked position’. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca²⁺ site II.     

Structural and binding properties of the PASTA domain of PonA2, a key penicillin binding protein from Mycobacterium tuberculosis

PonA2 is one of the two class A penicillin binding proteins of Mycobacterium tuberculosis, the etiologic agent of tuberculosis. It plays a complex role in mycobacterial physiology and is spotted as a promising target for inhibitors. PonA2 is involved in adaptation of M. tuberculosis to dormancy, an ability which has been attributed to the presence in its sequence of a C‐terminal PASTA domain. Since PASTA modules are typically considered as β‐lactam antibiotic binding domains, we determined the solution structure of the PASTA domain from PonA2 and analyzed its binding properties versus a plethora of potential binders, including the β‐lactam antibiotics, two typical muropeptide mimics, and polymeric peptidoglycan. We show that, despite a high structural similarity with other PASTA domains, the PASTA domain of PonA2 displays different binding properties, as it is not able to bind muropeptides, or β‐lactams, or polymeric peptidoglycan. These results indicate that the role of PASTA domains cannot be generalized, as their specific binding properties strongly depend on surface residues, which are widely variable. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 712–719, 2014.     

Crystal structure of the Mycobacterium tuberculosis phosphate binding protein PstS3

Mycobacterium tuberculosis evades host immune responses by colonizing macrophages. Intraphagosomal M. tuberculosis is exposed to environmental stresses such as reactive oxygen and nitrogen intermediates as well as acid shock and inorganic phosphate (Pi) depletion. Experimental evidence suggests that expression levels of mycobacterial protein PstS3 (Rv0928) are significantly increased when M. tuberculosis bacilli are exposed to Pi starvation. Hence, PstS3 may be important for survival of Mtb in conditions where there is limited supply of Pi. We report here the structure of PstS3 from M. tuberculosis at 2.3‐Å resolution. The protein presents a structure typical for ABC phosphate transfer receptors. Comparison with its cognate receptor PstS1 showed a different pattern distribution of surface charges in proximity to the Pi recognition site, suggesting complementary roles of the two proteins in Pi uptake. Proteins 2014; 82:2268–2274. © 2014 Wiley Periodicals, Inc.     

Mycobacterium smegmatis RecA protein is structurally similar to but functionally distinct from Mycobacterium tuberculosis RecA

In eubacteria, RecA proteins belong to a large superfamily of evolutionarily conserved, filament-forming, functional homologs of DNA strand exchange proteins. Here, we report the functional characterization of Mycobacterium smegmatis (Ms) and Mycobacterium tuberculosis (Mt) RecA proteins. Although in some respects Ms and Mt RecA proteins are structural and functional homologs of Escherichia coli (Ec) RecA, there are significant differences as well. The single-stranded DNA-binding property of RecA proteins was analyzed by electrophoretic mobility shift assays. We observed that Ms or Mt RecA proteins bound single-stranded DNA in a manner distinct from that of Ec RecA: The former two were able to form protein-DNA complexes in the presence of high salt. Further experiments indicated that Ms or Mt RecA proteins catalyzed adenosine triphosphate hydrolysis at approximately comparable rates across a wide range of pHs. Significantly, DNA strand invasion promoted by Ms or Mt RecA proteins displayed similar kinetics but distinctly different pH profiles. In contrast to MtRecA, MsRecA by itself was unable to form joint molecules across a wide range of pHs. However, regardless of the order in which SSB was added, it was able to stimulate MsRecA to form joint molecules within a narrow pH range, indicating that SSB is a required accessory factor. Together, these results provide a source of sharp contrast between EcRecA and mycobacterial RecAs on the one hand and Mt and Ms RecA proteins on the other.     

Analysis of protein structures reveals regions of rare backbone conformation at functional sites

Regions of rare conformation were located in 300 protein crystal structures representing seven major protein folds. A distance matrix algorithm was used to search rapidly for 9-residue fragments of rare backbone conformation using a comparison to a relational database of encoded fragments derived from the database of nonredundant structures. Rare fragments were found in 61% of the analyzed protein structures. Detailed analysis was performed for 78 proteins of different folds. The rare fragments were located near functional sites in 72% of the protein structures. The rare fragments often formed parts of ligand-binding sites (59%), protein-protein interfaces (8%), and domain-domain contacts (5%). Of the remaining structures, 5% had a high average B-factor or high local B-factors. Statistical analysis suggests that the association between ligands and rare regions does not occur by chance alone. The present study is likely to underestimate the number of functional sites, because not all analyzed protein structures contained a ligand. The results suggest that rapid searches for regions with rare local backbone conformations can assist in prediction of functional sites in novel proteins.     

Residual structure in disordered peptides and unfolded proteins from multivariate analysis and ab initio simulation of Raman optical activity data

Vibrational Raman optical activity (ROA), measured as a small difference in the intensity of Raman scattering from chiral molecules in right- and left-circularly polarized incident light, or as the intensity of a small circularly polarized component in the scattered light, is a powerful probe of the aqueous solution structure of proteins. The large number of structure-sensitive bands in protein ROA spectra makes multivariate analysis techniques such as nonlinear mapping (NLM) especially favorable for determining structural relationships between different proteins. We have previously used NLM to map a large dataset of peptide, protein, and virus ROA spectra into a readily visualizable two-dimensional space in which points close to or distant from each other, respectively, represent similar or dissimilar structures. As well as folded proteins, our dataset contains ROA spectra from many natively unfolded proteins, proteins containing both folded and unfolded domains, denatured partially structured molten globule and reduced protein states, together with folded proteins containing little or no alpha-helix or beta-sheet. In this article, the relative positions of these systems in the NLM plot are used to obtain information about any residual structure that they may contain. The striking differences between the structural propensities of proteins that are unfolded in their native states and those that are unfolded due to denaturation may be responsible for their often very different behavior, especially with regard to aggregation. An ab initio simulation of the Raman and ROA spectra of an alanine oligopeptide in the poly(L-proline) II-helical conformation confirms previous suggestions that this conformation is a significant structural element in disordered peptides and natively unfolded proteins. The use of ROA to identify and characterize proteins containing significant amounts of unfolded structure will, inter alia, be valuable in structural genomics/proteomics since unfolded sequences often inhibit crystallization.     

Secondary structure conversions of Mycobacterium tuberculosis ribonucleotide reductase protein R2 under varying pH and temperature conditions

The structural properties of Mycobacterium tuberculosis (Mtb) ribonucleotide reductase R2 protein were studied under varying pH and temperature conditions by circular dichroism (CD) spectroscopy as well as dynamic light scattering (DLS). Under physiological conditions this protein has a high alpha-helical content, similar to the corresponding protein from other species, e.g. mouse. Decreasing the pH induced significant structure conversions. When pH was below 6.5 an aggregated structure was observed and reached a maximum at pH 4. The aggregated state of this protein was verified by DLS and was found to be rich in beta-structure. This amyloid-like structure transformed into a molten globule state with high temperature stability (between 25 and 80 degrees C) at pH below 3. The corresponding mouse protein R2 under similar conditions showed no evidence of an aggregated state around pH 4.     

Thermal denaturation of an all β-sheet protein - identification of a stable partially structured intermediate at high temperature

The thermal unfolding of an all β-sheet protein, cardiotoxin analogue III, from the Taiwan Cobra (Naja naja atra) is studied at pH 2.0, 4.0 and 6.0. At pH 4.0, using circular dichroism and 1-anilino naphthalene-8-sulphonic acid (ANS) fluorescence binding studies, a stable partially structured intermediate is detected at 90°C     

Evidence of a conserved intrinsically disordered region in the C-terminus of the stringent response protein Rel from mycobacteria

The RelA/SpoT enzyme produces (p)ppGpp that helps the bacterium survive during stress. The domains present in it are interspersed with connecting linkers whose functions have been poorly elucidated. We rationally analyzed the sequence and structural property of the regulatory C-terminal region in the Rel family of proteins and report the presence of an intrinsically disordered region between two successive domains in this region that are separated by a defined amino acid sequence length. We show that the length and secondary structure of this linker are conserved in Rel proteins, further signifying its importance in rendering flexibility for domain movement and domain–domain interaction.