Folding landscapes of the Alzheimer amyloid-beta(12-28) peptide

The energy landscape for folding of the 12-28 fragment of the Alzheimer amyloid beta (Abeta) peptide is characterized using replica-exchange molecular dynamics simulations with an all-atom peptide model and explicit solvent. At physiological temperatures, the peptide exists mostly as a collapsed random coil, populating a small fraction (less than 10%) of hairpins with a beta-turn at position V18F19, with another 10% of hairpin-like conformations possessing a bend rather than a turn in the central VFFA positions. A small fraction of the populated states, approximately 14%, adopt polyproline II (PPII) conformations. Folding of the structured hairpin states proceeds through the assembly of two locally stable segments, VFFAE and EDVGS. The interactions stabilizing these locally folded structural motifs are in conflict with those stabilizing the global fold of A12-28, a signature of underlying residual frustration in this peptide. At increased temperature, the population of both beta-strand and PPII conformations diminishes in favor of beta-turn and random-coil states. On the basis of the conformational preferences of Abeta 12-28 monomers, two models for the molecular structure of amyloid fibrils formed by this peptide are proposed.     

Substrate-induced conformational changes in sarcoplasmic reticulum Ca(2+)-ATPase probed by surface modification using diethylpyrocarbonate with mass spectrometry

We have identified 15 residues from the surface of sarcoplasmic reticulum Ca²⁺-pump ATPase, by mass spectrometry using diethylpyrocarbonate modification. The reactivity of 9 residues remained high under all the conditions. The reactivity of Lys-515 at the nucleotide site was severely inhibited by ATP, whereas that of Lys-158 in the A-domain decreased by one-half and increased by five-fold in the presence of Ca²⁺ and MgF₄, respectively. These are well explained by solvent accessibility, pK_a and nearby hydrophobicity of the reactive atom on the basis of the atomic structure. However, the reactivity of 4 residues near the interface among A-, N- and P-domain suggested larger conformational changes of these domains in membrane upon binding of Ca²⁺ (Lys-436), ATP (Lys-158) and MgF₄ (His-5, -190, Lys-436).     

The Osmolyte TMAO Stabilizes Native RNA Tertiary Structures in the Absence of Mg: Evidence for a Large Barrier to Folding from Phosphate Dehydration

The stabilization of RNA tertiary structures by ions is well known, but the neutral osmolyte trimethylamine oxide (TMAO) can also effectively stabilize RNA tertiary structure. To begin to understand the physical basis for the effects of TMAO on RNA, we have quantitated the TMAO-induced stabilization of five RNAs with known structures. So-called m values, the increment in unfolding free energy per molal of osmolyte at constant KCl activity, are ∼ 0 for a hairpin secondary structure and between 0.70 and 1.85 kcal mol^− 1m^− 1 for four RNA tertiary structures (30-86 nt). Further analysis of two RNAs by small-angle X-ray scattering and hydroxyl radical probing shows that TMAO reduces the radius of gyration of the unfolded ensemble to the same endpoint as seen in titration with Mg²⁺ and that the structures stabilized by TMAO and Mg²⁺ are indistinguishable. Remarkably, TMAO induces the native conformation of a Mg²⁺ ion chelation site formed in part by a buried phosphate, even though Mg²⁺ is absent. TMAO interacts weakly, if at all, with KCl, ruling out the possibility that TMAO stabilizes RNA indirectly by increasing salt activity. TMAO is, however, strongly excluded from the vicinity of dimethylphosphate (unfavorable interaction free energy, + 211 cal mol^− 1m^− 1 for the potassium salt), an ion that mimics the RNA backbone phosphate. We suggest that formation of RNA tertiary structure is accompanied by substantial phosphate dehydration (loss of 66-173 water molecules in the RNA structures studied) and that TMAO works principally by reducing the energetic penalty associated with this dehydration. The strong parallels we find between the effects of TMAO and Mg²⁺ suggest that RNA sequence is more important than specific ion interactions in specifying the native structure.     

Dynamics of a benzo[a]pyrene-derived guanine DNA lesion in TGT and CGC sequence contexts: enhanced mobility in TGT explains conformational heterogeneity, flexible bending, and greater susceptibility to nucleotide excision repair

The nucleotide excision repair (NER) machinery excises a variety of bulky DNA lesions, but with varying efficiencies. The structural features of the DNA lesions that govern these differences are not well understood. An intriguing model system for studying structure-function relationships in NER is the major adduct derived from the reaction of the highly tumorigenic metabolite of benzo[a]pyrene, (+)-anti-benzo[a]pyrene diol epoxide, with the exocyclic amino group of guanine ((+)-trans-anti-[BP]-N²-dG, or G*). The rates of incision of the stereochemically identical lesions catalyzed by the prokaryotic UvrABC system was shown to be greater by a factor of 2.3 ± 0.3 in the TG*T than in the CG*C sequence context [Biochemistry 46 (2007) 7006-7015]. Here we employ molecular dynamics simulations to elucidate the origin of the greater excision efficiency in the TG*T case and, more broadly, to delineate structural parameters that enhance NER. Our results show that the BP aromatic ring system is 5′-directed along the modified strand in the B-DNA minor groove in both sequence contexts. However, the TG*T modified duplex is much more dynamically flexible, featuring more perturbed and mobile Watson-Crick hydrogen bonding adjacent to the lesion, a greater impairment in stacking interactions, more dynamic local roll/bending, and more minor groove flexibility. These characteristics explain a number of experimental observations concerning the (+)-trans-anti-[BP]-N²-dG adduct in double-stranded DNA with the TG*T sequence context: its conformational heterogeneity in NMR solution studies, its highly flexible bend, and its lower thermal stability. By contrast, the CG*C modified duplex is characterized by a single BP conformation and a rigid bend. While current recognition models of bulky lesions by NER factors have stressed the importance of impaired Watson-Crick pairing/stacking and bending, our results highlight the likelihood of an important role for the local dynamics in the vicinity of the lesion.     

Structural Basis for Aβ1-42 Toxicity Inhibition by Aβ C-Terminal Fragments: Discrete Molecular Dynamics Study

Amyloid β-protein (Aβ) is central to the pathology of Alzheimer's disease. Of the two predominant Aβ alloforms, Aβ_1-40 and Aβ_1-42, the latter forms more toxic oligomers. C-terminal fragments (CTFs) of Aβ were recently shown to inhibit Aβ_1-42 toxicity in vitro. Here, we studied Aβ_1-42 assembly in the presence of three effective CTF inhibitors and an ineffective fragment, Aβ_21-30. Using a discrete molecular dynamics approach that recently was shown to capture key differences between Aβ_1-40 and Aβ_1-42 oligomerization, we compared Aβ_1-42 oligomer formation in the absence and presence of CTFs or Aβ_21-30 and identified structural elements of Aβ_1-42 that correlated with Aβ_1-42 toxicity. CTFs co-assembled with Aβ_1-42 into large heterooligomers containing multiple Aβ_1-42 and inhibitor fragments. In contrast, Aβ_21-30 co-assembled with Aβ_1-42 into heterooligomers containing mostly a single Aβ_1-42 and multiple Aβ_21-30 fragments. The CTFs, but not Aβ_21-30, decreased the β-strand propensity of Aβ_1-42 in a concentration-dependent manner. CTFs and Aβ_21-30 had a high binding propensity to the hydrophobic regions of Aβ_1-42, but only CTFs were found to bind the Aβ_1-42 region A2-F4. Consequently, only CTFs but not Aβ_21-30 reduced the solvent accessibility of Aβ_1-42 in region D1-R5. The reduced solvent accessibility of Aβ_1-42 in the presence of CTFs was comparable to the solvent accessibility of Aβ_1-40 oligomers formed in the absence of Aβ fragments. These findings suggest that region D1-R5, which was more exposed to the solvent in Aβ_1-42 than in Aβ_1-40 oligomers, is involved in mediating Aβ_1-42 oligomer neurotoxicity.     

Unfolding pathways of goat alpha-lactalbumin as revealed in multiple alignment of molecular dynamics trajectories

Molecular dynamics simulations of protein unfolding were performed at an elevated temperature for the authentic and recombinant forms of goat alpha-lactalbumin. Despite very similar three-dimensional structures, the two forms have significantly different unfolding rates due to an extra N-terminal methionine in the recombinant protein. To identify subtle differences between the two forms in the highly stochastic kinetics of unfolding, we classified the unfolding trajectories using the multiple alignment method based on the analogy between the biological sequences and the molecular dynamics trajectories. A dendrogram derived from the multiple trajectory alignment revealed a clear difference in the unfolding pathways of the authentic and recombinant proteins, i.e. the former reached the transition state in an all-or-none manner while the latter unfolded less cooperatively. It was also found in the classification that the two forms of the protein shared a common transition state structure, which was in excellent agreement with the transition state structure observed experimentally in the Phi-value analysis.     

Coarse-grained models for simulations of multiprotein complexes: application to ubiquitin binding

We develop coarse-grained models and effective energy functions for simulating thermodynamic and structural properties of multiprotein complexes with relatively low binding affinity (K_d > 1 μM) and apply them to binding of Vps27 to membrane-tethered ubiquitin. Folded protein domains are represented as rigid bodies. The interactions between the domains are treated at the residue level with amino-acid-dependent pair potentials and Debye-Hückel-type electrostatic interactions. Flexible linker peptides connecting rigid protein domains are represented as amino acid beads on a polymer with appropriate stretching, bending, and torsion-angle potentials. In simulations of membrane-attached protein complexes, interactions between amino acids and the membrane are described by residue-dependent short-range potentials and long-range electrostatics. We parameterize the energy functions by fitting the osmotic second virial coefficient of lysozyme and the binding affinity of the ubiquitin-CUE complex. For validation, extensive replica-exchange Monte Carlo simulations are performed of various protein complexes. Binding affinities for these complexes are in good agreement with the experimental data. The simulated structures are clustered on the basis of distance matrices between two proteins and ranked according to cluster population. In ∼ 70% of the complexes, the distance root-mean-square is less than 5 Å from the experimental structures. In ∼ 90% of the complexes, the binding interfaces on both proteins are predicted correctly, and in all other cases at least one interface is correct. Transient and nonspecifically bound structures are also observed. With the validated model, we simulate the interaction between the Vps27 multiprotein complex and a membrane-tethered ubiquitin. Ubiquitin is found to bind preferentially to the two UIM domains of Vps27, but transient interactions between ubiquitin and the VHS and FYVE domains are observed as well. These specific and nonspecific interactions are found to be positively cooperative, resulting in a substantial enhancement of the overall binding affinity beyond the ∼ 300 μM of the specific domains. We also find that the interactions between ubiquitin and Vps27 are highly dynamic, with conformational rearrangements enabling binding of Vps27 to diverse targets as part of the multivesicular-body protein-sorting pathway.     

Computational investigation of pathogenic nsSNPs in CEP63 protein

Centrosomes are central regulators of mitosis that are often amplified in cancer cells. Centrosomal protein of 63kDa (CEP63) is a centrosomal protein that has an effective role in mitotic spindle assembly and cell cycle regulation. Genetic alterations in CEP63 coding gene has been widely studied for inducing aneuploidy and solid tumors in humans. The nonsynonymous single nucleotide polymorphisms (nsSNPs) are a genetic variant resulting in amino acid substitution and are reported in a wide range of human diseases. Here we report one new SNP (rs112926188) in a CEP63 coding region that can potentially disrupt the structure and basic functionality of a CEP63 protein. We used extensive functional and structural level analyses of an available SNP in a CEP63 coding gene. Furthermore the disease-association analysis was carried out to examine the possible pathogenic variant among the available dataset. To understand atomic arrangement in 3D space, native and pathogenic mutant structures were modeled. Molecular dynamics simulations were performed to understand structural consequences of prioritized deleterious mutation. Our analysis showed that rs112926188 allele substituting proline at the 61st residue position (L61P) produced more flexibility in 3D space. Moreover the flexible nature of mutant L61P was validated by a hydrogen bond network. This nature of mutant L61P CEP63 may restrict the recruitment of essential centrosomal proteins to their respective location and may play an active role in inducing aneuploidy.     

Energetics of MazG Unfolding in Correlation with Its Structural Features

MazG is a homodimeric α-helical protein that belongs to the superfamily of all-α NTP pyrophosphatases. Its function has been connected to the regulation of the toxin-antitoxin module mazEF, implicated in programmed growth arrest/cell death of Escherichia coli cells under conditions of amino acid starvation. The goal of the first detailed biophysical study of a member of the all-α NTP pyrophosphatase superfamily, presented here, is to improve molecular understanding of the unfolding of this type of proteins. Thermal unfolding of MazG monitored by differential scanning calorimetry, circular dichroism spectroscopy, and fluorimetry at neutral pH in the presence of a reducing agent (dithiothreitol) can be successfully described as a reversible four-state transition between a dimeric native state, two dimeric intermediate states, and a monomeric denatured state. The first intermediate state appears to have a structure similar to that of the native state while the final thermally denatured monomeric state is not fully unfolded and contains a significant fraction of residual α-helical structure. In the absence of dithiothreitol, disulfide cross-linking causes misfolding of MazG that appears to be responsible for the formation of multimeric aggregates. MazG is most stable at pH 7-8, while at pH < 6, it exists in a molten-globule-like state. The thermodynamic parameters characterizing each step of MazG denaturation transition obtained by global fitting of the four-state model to differential scanning calorimetry, circular dichroism, and fluorimetry temperature profiles are in agreement with the observed structural characteristics of the MazG conformational states and their assumed functional role.     

Carbonylation of mitochondrial aconitase with 4-hydroxy-2-(E)-nonenal: Localization and relative reactivity of addition sites

Mass spectrometry was used to investigate the effects of exposing mitochondrial aconitase (ACO2) to the membrane lipid peroxidation product, 4-hydroxy-2-(E)-nonenal (HNE). ACO2 was selected for this study because (1) it is known to be inactivated by HNE, (2) elevated concentrations of HNE-adducted ACO2 have been associated with disease states, (3) extensive structural information is available, and (4) the iron–sulfur cluster in ACO2 offers a critical target for HNE adduction. The aim of this study was to relate the inactivation of ACO2 by HNE to structural features. Initially, Western blotting and an enzyme activity assay were used to assess aggregate effects and then gel electrophoresis, in-gel digestion, and tandem mass spectrometry (MS/MS) were used to identify HNE addition sites. HNE addition reaction rates were determined for the most significant sites using the iTRAQ approach. The most reactive sites were Cys³⁵⁸, Cys⁴²¹, and Cys⁴²⁴, the three iron–sulfur cluster-coordinating cysteines, Cys⁹⁹, the closest non-ligated cysteine to the cluster, and Cys⁵⁶⁵, which is located in the cleft leading to the active site. Interestingly, both enzyme activity assay and iTRAQ relative abundance plots appeared to be trending toward horizontal asymptotes, rather than completion.