Solvent Exposure Of Hydrophobic Residues Research Articles

Initial Structural Models of the Aβ42 Dimer from Replica Exchange Molecular Dynamics Simulations.

Experimental characterization of the molecular structure of small amyloid (A)β oligomers that are currently considered as toxic agents in Alzheimer’s disease is a formidably difficult task due to their transient nature and tendency to aggregate. Such structural information is of importance because it can help in developing diagnostics and an effective therapy for the disease. In this study, molecular simulations and protein–protein docking are employed to explore a possible connection between the structure of Aβ monomers and the properties of the intermonomer interface in the Aβ42 dimer. A structurally diverse ensemble of conformations of the monomer was sampled in microsecond timescale implicit solvent replica exchange molecular dynamics simulations. Representative structures with different solvent exposure of hydrophobic residues and secondary structure content were selected to build structural models of the dimer. Analysis of these models reveals that formation of an intramonomer salt bridge (SB) between Asp23 and Lys28 residues can prevent the building of a hydrophobic interface between the central hydrophobic clusters (CHCs) of monomers upon dimerization. This structural feature of the Aβ42 dimer is related to the difference in packing of hydrophobic residues in monomers with the Asp23–Lys28 SB in on and off states, in particular, to a lower propensity to form hydrophobic contacts between the CHC domain and C-terminal residues in monomers with a formed SB. These findings could have important implications for understanding the difference between aggregation pathways of Aβ monomers leading to neurotoxic oligomers or inert fibrillar structures.

Insights into Formation and Structure of Aβ Oligomers Cross-Linked via Tyrosines.

Alzheimer's disease (AD) pathology is hypothesized to be triggered by amyloid β-protein (Aβ) assembly into oligomers. Oligomer size distributions of both predominant Aβ alloforms, Aβ40 and Aβ42, can be determined in vitro using cross-linking followed by gel electrophoresis. Cross-linking, which can occur in vivo in the presence of copper and hydrogen peroxide, was recently shown to stabilize Aβ oligomers by inhibiting their conversion into fibrils. Whereas several studies showed that cross-linking is facilitated by dityrosine bond formation, the molecular-level mechanism of cross-linking remains unclear. Here, we use efficient discrete molecular dynamics with DMD4B-HYDRA force field to examine the effect of cross-linking via tyrosines on Aβ oligomer formation. Our results show that cross-linking via tyrosines promotes Aβ self-assembly, in particular that of Aβ40, but does not account for cross-linked oligomers larger than Aβ40 trimers and Aβ42 tetramers. Cross-linking via tyrosines profoundly alters Aβ40 and Aβ42 oligomer conformations by increasing the solvent exposure of hydrophobic residues, resulting in elongated oligomeric morphologies that differ from globular structures of noncross-linked oligomers. When compared to available experimental data, our findings imply that amino acids other than tyrosines are involved in Aβ cross-linking, a proposition that is currently under investigation.

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations.

Living systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 μm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules.

Comparison of the structural characteristics of Cu2+-bound and unbound α-syn12 peptide obtained in simulations using different force fields

The effects of Cu(2+) binding and the utilization of different force fields when modeling the structural characteristics of α-syn12 peptide were investigated. To this end, we performed extensive temperature replica exchange molecular dynamics (T-REMD) simulations on Cu(2+)-bound and unbound α-syn12 peptide using the GROMOS 43A1, OPLS-AA, and AMBER03 force fields. Each replica was run for 300 ns. The structural characteristics of α-syn12 peptide were studied based on backbone dihedral angle distributions, free-energy surfaces obtained with different reaction coordinates, favored conformations, the formation of different Turn structures, and the solvent exposure of the hydrophobic residues. The findings show that AMBER03 prefers to sample helical structures for the unbound α-syn12 peptide and does not sample any β-hairpin structure for the Cu(2+)-bound α-syn12 peptide. In contrast, the central structure of the major conformational clusters for the Cu(2+)-bound and unbound α-syn12 peptide according to simulations performed using the GROMOS 43A1 and OPLS-AA force fields is a β-hairpin with Turn(9-6). Cu(2+) can also promote the formation of the β-hairpin and increase the solvent exposure of hydrophobic residues, which promotes the aggregation of α-syn12 peptide. This study can help us to understand the mechanisms through which Cu(2+) participates in the fibrillation of α-syn12 peptide at the atomic level, which in turn represents a step towards elucidating the nosogenesis of Parkinson's disease.

Aggregation of γ-crystallins associated with human cataracts via domain swapping at the C-terminal β-strands.

The prevalent eye disease age-onset cataract is associated with aggregation of human γD-crystallins, one of the longest-lived proteins. Identification of the γ-crystallin precursors to aggregates is crucial for developing strategies to prevent and reverse cataract. Our microseconds of atomistic molecular dynamics simulations uncover the molecular structure of the experimentally detected aggregation-prone folding intermediate species of monomeric native γD-crystallin with a largely folded C-terminal domain and a mostly unfolded N-terminal domain. About 30 residues including a, b, and c strands from the Greek Key motif 4 of the C-terminal domain experience strong solvent exposure of hydrophobic residues as well as partial unstructuring upon N-terminal domain unfolding. Those strands comprise the domain-domain interface crucial for unusually high stability of γD-crystallin. We further simulate the intermolecular linkage of these monomeric aggregation precursors, which reveals domain-swapped dimeric structures. In the simulated dimeric structures, the N-terminal domain of one monomer is frequently found in contact with residues 135-164 encompassing the a, b, and c strands of the Greek Key motif 4 of the second molecule. The present results suggest that γD-crystallin may polymerize through successive domain swapping of those three C-terminal β-strands leading to age-onset cataract, as an evolutionary cost of its very high stability. Alanine substitutions of the hydrophobic residues in those aggregation-prone β-strands, such as L145 and M147, hinder domain swapping as a pathway toward dimerization. These findings thus provide critical molecular insights onto the initial stages of age-onset cataract, which is important for understanding protein aggregation diseases.

Tropomyosin: Long Range Perturbations In The Hydrophobic Interface

Skeletal tropomyosin, (Tm), is an α-helical coiled-coil which binds to actin, and with troponin, regulates muscle contraction. We previously demonstrated that a conserved Asp 137 in the hydrophobic interface produces a dynamic region in the middle of Tm, and that this region is involved in the myosin dependent activation of the thin filament at high Ca2+, (Sumida et. al. JBC 283 2008). The current work characterizes a long-range interaction between positions 137 and 190. The thermodynamic properties of wild type (WT) Tm and two single mutants, C190A and D137L, are compared with those of the double mutant, D137L/C190A, using differential scanning calorimetry, (DSC), and circular dichroism, (CD). CD measurements show that Ala 190 increases the fraction of helix unfolding in the 40°C pre-transition, before the main transition. DSC measurements support this finding, indicating a large enthalpic pre-transition, (ΔH=150kcal/mol), for the C190A mutant relative to D137L, D137L/C190A, or WT, (average ΔH=20kcal/mol). Additionally, Ala 190 increases the ΔCp, (heat capacity), of Tm ∼5 fold, reflecting an increase in solvent exposure of hydrophobic residues in the pre-transition during unfolding. Since the D137L/C190A and D137L mutants do not exhibit the large enthalpic pre-transition observed for C190A, the Leu mutation at 137 must stabilize the alanine effect observed for C190A mutation 77Å away. This demonstrates how a locally dynamic region near 137 is able to produce global effects along the thin filament, and in this manner provide the proper regulation of the myosin dependent activation of the thin filament. This observation may also contribute to our understanding about the manner in which single point mutations significantly affect function in cardio-myopathies such as FHC and DCM.

Six Amino Acid Residues in a 1200 Å2 Interface Mediate Binding of Factor VIII to An IgG4 Inhibitory Antibody.

Approximately one quarter of severe hemophilia A patients who receive Factor VIII (FVIII) injections develop antibodies, clinically referred to as “inhibitors”, which interfere with FVIII procoagulant activity. The effects of these antibody inhibitors can be difficult and quite expensive to manage. Inhibitors are associated with high morbidity and mortality and impaired quality of life; therefore, there is a compelling need to develop new therapeutic options. One approach is to design recombinant versions of FVIII that are less immunogenic (less likely to stimulate T cells) or less antigenic (containing fewer B-cell epitopes, i.e. surfaces that bind to anti-FVIII IgG). Proteins with reduced antigenicity will by definition bind to inhibitory IgG with lower affinity and therefore could be useful in attempting to achieve hemostasis in patients with an established inhibitor response. To design such FVIII proteins, common inhibitor epitopes must be characterized by determining which amino acid residues are essential to form high-affinity antigen-antibody complexes. A crystal structure of the FVIII C2 domain bound to an Fab fragment from a patient-derived inhibitory IgG4 antibody, BO2C11, provides the most detailed characterization to date of a human inhibitor epitope (Spiegel et al., Blood 38, 13–19, 2001). Although this structure clearly shows which FVIII residues interact with the antibody surface, the contributions of particular residues to the overall affinity must be determined experimentally. In this study, we systematically modified each of the C2 side chains at the C2-Fab interface, which buries 1200 Å2 of each protein surface, then used surface plasmon resonance (SPR) to measure the contributions of individual residues to the kon and koff rates and to the overall affinity. The experiments were carried out on a Biacore T100 instrument, which allowed us to analyze several samples in parallel and to carry out SPR runs at different temperatures. Substitutions at only six sites decreased the affinity significantly relative to that of wild-type C2. R2220A and R2220Q completely abrogated binding to BO2C11, while F2196A, N2198A, M2199A, L2200A and R2215A displayed markedly higher off-rate kinetic constants compared to wild-type C2 but retained some binding affinity. SPR runs were carried out for the latter five proteins using a temperature gradient (10–40°C), and thermodynamic values derived from van't Hoff analysis were used to roughly quantitate the energetic consequences of these mutations compared to wild-type C2 binding. Although a relative order of energetic contributions was established (F2200 > F2196 = R2215 > N2198 > M2199) the ΔΔGº values were similar (approx. 11 ± 5 kJ/mol). Furthermore, the data suggest that the loss of binding energy was mostly an entropic, not enthalpic, effect, as the ΔH values were remarkably stable for the set of C2 mutants. In other words, the mutations increased the ordering of the system consisting of BO2C11 bound to C2 plus solvent, or else they increased the disorder of the uncomplexed system, e.g. by allowing greater flexibility of protein side chains or backbone, or by changing the solvent exposure of hydrophobic residues, thereby affecting ordering of water molecules. Interestingly, only one of two beta-hairpin turns that comprise part of this epitope contributes appreciably to the binding of the C2 domain to BO2C11. Substitutions at L2251 and L2252 in the second hairpin turn had surprisingly little effect on the off-rate and overall affinity, despite their extensive contact with the antibody that shielded this hydrophobic region from solvent. IgG4 antibodies are common in anti-FVIII immune responses, as is inhibition of FVIII binding to activated membranes and von Willebrand factor. BO2C11 is a human-derived IgG4 that inhibits these binding interactions. Our results for this prototypical inhibitor suggest that a limited number of amino acid substitutions could produce modified FVIII proteins capable of eluding inhibitors that bind to similar epitopes, even in the case of antibodies that form an extensive antigen-antibody interface.

Structural Basis of Lytic Cycle Activation by the Epstein-Barr Virus ZEBRA Protein

Epstein-Barr virus (EBV) causes infectious mononucleosis and is linked to several human malignancies. EBV has a biphasic infection cycle consisting of a latent and a lytic, replicative phase. The switch from latent to lytic infection is triggered by the EBV immediate-early transcription factor ZEBRA (BZLF1, Zta, Z, EB1). We present the crystal structure of ZEBRA's DNA binding domain bound to an EBV lytic gene promoter element. ZEBRA exhibits a variant of the basic-region leucine zipper (bZIP) fold in which a C-terminal moiety stabilizes the coiled coil involved in dimer formation. The structure provides insights into ZEBRA's broad target site specificity, preferential activation of specific EBV promoters in their methylated state, ability to dimerize despite lacking a leucine zipper motif, and failure to heterodimerize with cellular bZIP proteins. The structure will allow for the design of new therapeutic agents that block activation of the EBV lytic cycle.

Solution Structure of the N-terminal RNP Domain of U1A Protein: The Role of C-terminal Residues in Structure Stability and RNA Binding

The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an α-helix that lies across the β-sheet, with residues Ile93, Ile94 and Met97 making contacts with Leu44, Phe56 and Ile58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the β-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135° to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from Ile93 and Ile94 to Ile58, Leu 41, Val62 and His10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface.