Burial Of Hydrophobic Residues Research Articles

Scrutiny of the mechanism of small molecule inhibitor preventing conformational transition of amyloid-β42 monomer: insights from molecular dynamics simulations

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is characterized by loss of intellectual functioning of brain and memory loss. According to amyloid cascade hypothesis, aggregation of amyloid-β42 (Aβ42) peptide can generate toxic oligomers and their accumulation in the brain is responsible for the onset of AD. In spite of carrying out a large number of experimental studies on inhibition of Aβ42 aggregation by small molecules, the detailed inhibitory mechanism remains elusive. In the present study, comparable molecular dynamics (MD) simulations were performed to elucidate the inhibitory mechanism of a sulfonamide inhibitor C1 (2,5-dichloro-N-(4-piperidinophenyl)-3-thiophenesulfonamide), reported for its in vitro and in vivo anti-aggregation activity against Aβ42. MD simulations reveal that C1 stabilizes native α-helix conformation of Aβ42 by interacting with key residues in the central helix region (13–26) with hydrogen bonds and π–π interactions. C1 lowers the solvent-accessible surface area of the central hydrophobic core (CHC), KLVFF (16–20), that confirms burial of hydrophobic residues leading to the dominance of helical conformation in the CHC region. The binding free energy analysis with MM–PBSA demonstrates that Ala2, Phe4, Tyr10, Gln15, Lys16, Leu17, Val18, Phe19, Phe20, Glu22, and Met35 contribute maximum to binding free energy (−43.1 kcal/mol) between C1 and Aβ42 monomer. Overall, MD simulations reveal that C1 inhibits Aβ42 aggregation by stabilizing native helical conformation and inhibiting the formation of aggregation-prone β-sheet conformation. The present results will shed light on the underlying inhibitory mechanism of small molecules that show potential in vitro anti-aggregation activity against Aβ42.

Self-Assembly of Catenanes from Lasso Peptides.

Lasso peptides exist naturally in a threaded state as [1]rotaxanes, and we reasoned that lasso peptides cleaved in their loop region could serve as building blocks for catenanes. Mutagenesis of the lasso peptide microcin J25 (MccJ25) with two cysteine residues followed by cleavage of the peptide with trypsin led to a [2]rotaxane structure that self-assembled into a [3]catenane and [4]catenanes at room temperature in aqueous solution. The [3]catenane represents the smallest ring size of a catenane composed solely of polypeptide segments. The NMR structure of the [3]catenane was determined, suggesting that burial of hydrophobic residues may be a driving force for assembly of the catenane structure.

High-resolution structures and orientations of antimicrobial peptides piscidin 1 and piscidin 3 in fluid bilayers reveal tilting, kinking, and bilayer immersion.

While antimicrobial peptides (AMPs) have been widely investigated as potential therapeutics, high-resolution structures obtained under biologically relevant conditions are lacking. Here, the high-resolution structures of the homologous 22-residue long AMPs piscidin 1 (p1) and piscidin 3 (p3) are determined in fluid-phase 3:1 phosphatidylcholine/phosphatidylglycerol (PC/PG) and 1:1 phosphatidylethanolamine/phosphatidylglycerol (PE/PG) bilayers to identify molecular features important for membrane destabilization in bacterial cell membrane mimics. Structural refinement of 1H–15N dipolar couplings and 15N chemical shifts measured by oriented sample solid-state NMR and all-atom molecular dynamics (MD) simulations provide structural and orientational information of high precision and accuracy about these interfacially bound α-helical peptides. The tilt of the helical axis, τ, is between 83° and 93° with respect to the bilayer normal for all systems and analysis methods. The average azimuthal rotation, ρ, is 235°, which results in burial of hydrophobic residues in the bilayer. The refined NMR and MD structures reveal a slight kink at G13 that delineates two helical segments characterized by a small difference in their τ angles (<10°) and significant difference in their ρ angles (∼25°). Remarkably, the kink, at the end of a G(X)4G motif highly conserved among members of the piscidin family, allows p1 and p3 to adopt ρ angles that maximize their hydrophobic moments. Two structural features differentiate the more potent p1 from p3: p1 has a larger ρ angle and less N-terminal fraying. The peptides have comparable depths of insertion in PC/PG, but p3 is 1.2 Å more deeply inserted than p1 in PE/PG. In contrast to the ideal α-helical structures typically assumed in mechanistic models of AMPs, p1 and p3 adopt disrupted α-helical backbones that correct for differences in the amphipathicity of their N- and C-ends, and their centers of mass lie ∼1.2–3.6 Å below the plane defined by the C2 atoms of the lipid acyl chains.

A Molecular Dynamics Study of the Flexibility and Protein-Binding of the Trigger Factor Chaperone in Solution

Trigger factor (TF) is a chaperone, found in bacterial cells (and chloroplasts), that interacts with nascent polypeptide chains to guide protein folding, and prevent spontaneous misfolding and aggregation. While its crystal structure has been resolved, the solution structure and dynamics is largely unknown. using all-atom and coarse-grained molecular dynamics (MD) simulations in combination with Markov State Modeling (MSM) on the extended crystal structure of TF in solution, we show that the collective motions of its tertiary domains, hinged about inter-domain linkers with minimal or no loss in secondary structure, leads to a substantially different conformation in solution, with a relatively stable compact collapsed state and several intermediate metastable states. These states are the results of domain-pair formations that are triggered by burial of hydrophobic residues and stabilized by hydrophilic contacts. A hydrophobic probes method, which uses methane-like Lennard-Jones particles to identify hydrophobic patches, is employed to characterize these contacts as well as the surface hydrophobicity of TF. The flexibility and promiscuity of TF facilitate its chaperone action on a variety of substrate proteins; however, its direct influence, or that of chaperones in general, on protein-folding pathways remains poorly understood. We employ steered MD to complement AA-MD simulations to understand the relatively unknown binding mechanism of maltose binding protein (MBP) to TF. We show that, in solution, TF, by conserving secondary structural elements in the folding intermediates of MBP, stabilizes native contacts in MBP and prevents the protein aggregation. Contrary to the current viewpoint, our results also illustrate a crucial role for N-terminal of TF in its binding with protein substrates through hydrophilic interactions.

Environment Polarity in Proteins Mapped Noninvasively by FTIR Spectroscopy

The polarity pattern of a macromolecule is of utmost importance to its structure and function. For example, one of the main driving forces for protein folding is the burial of hydrophobic residues. Yet polarity remains a difficult property to measure experimentally, due in part to its non-uniformity in the protein interior. Herein, we show that FTIR linewidth analysis of noninvasive 1-(13)C=(18)O labels can be used to obtain a reliable measure of the local polarity, even in a highly multi-phasic system, such as a membrane protein. We show that in the Influenza M2 H(+) channel, residues that line the pore are located in an environment that is as polar as fully solvated residues, while residues that face the lipid acyl chains are located in an apolar environment. Taken together, FTIR linewidth analysis is a powerful, yet chemically non-perturbing approach to examine one of the most important properties in proteins - polarity.

Influence of the Helical Backbone in the Behavior of a Simple Model for Dimeric Coiled-Coil Proteins

Using computer simulations as a tool for thought experiments, we investigate the influence of the helical backbone geometry in the association process and the final structures of simple model which mimics parallel, two-stranded coiled-coil proteins. We define three types of helices: two of them have straight helical axes and 3.5 or 3.6 residues per helical turn; the third type presents a coiled helical axis, according to the canonical scheme defined by Crick. By using a Monte Carlo simulation algorithm, we observe that the three models exhibit different transition temperatures for the formation of the dimeric structure from two independent peptides, and a different behavior concerning the appearance of out-of-register structures. The energy minimized dimer structures present strong deviations from the correct association for straight helices with 3.6 residues/ turn, especially for long peptides, deviations which are absent for the other two types when only the burial of hydrophobic residues is considered. A careful analysis of the energies for the out-of-register configurations and the contact maps reveals also differences between dimers resulting from the model with Crick parameterization and with 3.5 residues/ turn, The results presented in this paper may be relevant for the design of simple models which use rigid α-helices built from predicted elements of secondary structure.

Sodium Dodecyl Sulfate Stability of HLA-DR1 Complexes Correlates with Burial of Hydrophobic Residues in Pocket 1

Certain class II MHC-peptide complexes are resistant to SDS-induced dissociation. This property, which has been used as an in vivo as well as an in vitro peptide binding assay, is not understood at the molecular level. Here we have investigated the mechanistic basis of SDS stability of HLA-DR1 complexes by using a biosensor-based assay and SDS-PAGE with a combination of wild-type and mutant HLA-DR1 and variants of hemagglutinin peptide HA306-318. Experiments with wild-type DR1 along with previously published results establish that the SDS-stable complexes are formed only when the hydrophobic pocket 1 (P1) is occupied by a bulky aromatic (Trp, Phe, Tyr) or an aliphatic residue (Met, Ile, Val, Leu). To further explore whether the SDS sensitivity is primarily due to the exposed hydrophobic regions, we mutated residue beta Gly86 at the bottom of P1 to tyrosine, presumably reducing the depth of the pocket and the exposure of hydrophobic residues and increasing the contacts between subunits. In direct contrast to wild-type DR1, the peptide-free mutant DR1 exists as an alpha/beta heterodimer in SDS. Moreover, the presence of a smaller hydrophobic residue, such as alanine, as P1 anchor with no contribution from any other anchor is sufficient to enhance the SDS stability of the mutant complexes, demonstrating that the basis of SDS resistance may be localized to P1 interactions. The good correlation between SDS sensitivity and the exposure of hydrophobic residues provides a biochemical rationale for the use of this assay to investigate the maturation of class II molecules and the longevity of the complexes.

Improved recognition of native-like protein structures using a combination of sequence-dependent and sequence-independent features of proteins.

We describe the development of a scoring function based on the decomposition P(structure/sequence) proportional to P(sequence/structure) *P(structure), which outperforms previous scoring functions in correctly identifying native-like protein structures in large ensembles of compact decoys. The first term captures sequence-dependent features of protein structures, such as the burial of hydrophobic residues in the core, the second term, universal sequence-independent features, such as the assembly of beta-strands into beta-sheets. The efficacies of a wide variety of sequence-dependent and sequence-independent features of protein structures for recognizing native-like structures were systematically evaluated using ensembles of approximately 30,000 compact conformations with fixed secondary structure for each of 17 small protein domains. The best results were obtained using a core scoring function with P(sequence/structure) parameterized similarly to our previous work (Simons et al., J Mol Biol 1997;268:209-225] and P(structure) focused on secondary structure packing preferences; while several additional features had some discriminatory power on their own, they did not provide any additional discriminatory power when combined with the core scoring function. Our results, on both the training set and the independent decoy set of Park and Levitt (J Mol Biol 1996;258:367-392), suggest that this scoring function should contribute to the prediction of tertiary structure from knowledge of sequence and secondary structure.

High-resolution solution structure of the beta chemokine hMIP-1 beta by multidimensional NMR.

The three-dimensional structure of a member of the beta subfamily of chemokines, human macrophage inflammatory protein-1 beta (hMIP-1 beta), has been determined with the use of solution multidimensional heteronuclear magnetic resonance spectroscopy. Human MIP-1 beta is a symmetric homodimer with a relative molecular mass of approximately 16 kilodaltons. The structure of the hMIP-1 beta monomer is similar to that of the related alpha chemokine interleukin-8 (IL-8). However, the quaternary structures of the two proteins are entirely distinct, and the dimer interface is formed by a completely different set of residues. Whereas the IL-8 dimer is globular, the hMIP-1 beta dimer is elongated and cylindrical. This provides a rational explanation for the absence of cross-binding and reactivity between the alpha and beta chemokine subfamilies. Calculation of the solvation free energies of dimerization suggests that the formation and stabilization of the two different types of dimers arise from the burial of hydrophobic residues.

Unusual clustering of carboxyl side chains in the core of iron-free ribonucleotide reductase.

The principal driving forces of protein folding are the burial of hydrophobic residues in the interior of proteins and the exposure of charged residues at the surface. Charged residues are only occasionally found in the interior, where they form hydrogen bonds to oppositely charged residues or main-chain atoms. Ribonucleotide reductase, a key enzyme in DNA synthesis, catalyses the de novo production of deoxyribonucleotide precursors. It is composed of two different dimeric proteins R1 and R2 (refs 3-5). R2 subunits contain buried iron-centres with each centre formed by two ferric ions coordinated by four carboxylates and two histidine ligands. Iron-free R2, apoR2, is a precursor of active R2 and folds into a stable protein which is transformed into active R2 by ferrous ions and molecular oxygen. Here we show that the iron-free protein does not undergo any major structural changes compared with the iron-containing R2. The effect of this is a clustering of four carboxyl side chains in the interior of the subunit, in contrast to the normal distribution of charged residues in proteins.

Modeling the anti-CEA antibody combining site by homology and conformational search.

A model for an antibody specific for the carcinoembryonic antigen (CEA) has been constructed using a method which combines the concept of canonical structures with conformational search. A conformational search technique is introduced which couples random generation of backbone loop conformations to a simulated annealing method for assigning side chain conformations. This technique was used both to verify conformations selected from the set of known canonical structures and to explore conformations available to the H3 loop in CEA ab initio. Canonical structures are not available for H3 due to its variability in length, sequence, and observed conformation in known antibody structures. Analysis of the results of conformational search resulted in three equally probable conformations for H3 loop in CEA. Force field energies, solvation free energies, exposure of charged residues and burial of hydrophobic residues, and packing of hydrophobic residues at the base of the loop were used as selection criteria. The existence of three equally plausible structures may reflect the high degree of flexibility expected for an exposed loop of this length. The nature of the combining site and features which could be important to interaction with antigen are discussed.