Side-chain Hydrogen Bonds Research Articles

Vibrational spectral signatures of peptide secondary structures:N-methylation and side chain hydrogen bond in cyclosporin A

Molecular dynamics simulations are performed to explore important conformations of cyclosporin A, an immunosuppressive cyclic undecapeptide drug, in different media including gas-phase, chloroform, and acetonitrile. Density functional theory calculations are used to refine the low-lying conformers and to predict their infrared and vibrational circular dichroism spectra. Vibrational spectral signatures in the important amide II, I, and A regions are identified for typical peptide secondary structures including β-turn (type II' or I), antiparallel β-sheet (flat or twisted), inverse γ-turn, N-methylated peptide bond, and side chain H-bond. New insights into the spectral signatures of secondary structures especially with N-methylation and side chain hydrogen bond are provided, which can be very useful for further peptide conformation analysis in general.

Structure of a TCR with high affinity for self-antigen reveals basis for escape from negative selection

The failure to eliminate self-reactive T cells during negative selection is a prerequisite for autoimmunity. To escape deletion, autoreactive T-cell receptors (TCRs) may form unstable complexes with self-peptide-MHC by adopting suboptimal binding topologies compared with anti-microbial TCRs. Alternatively, escape can occur by weak binding between self-peptides and MHC. We determined the structure of a human autoimmune TCR (MS2-3C8) bound to a self-peptide from myelin basic protein (MBP) and the multiple sclerosis-associated MHC molecule HLA-DR4. MBP is loosely accommodated in the HLA-DR4-binding groove, accounting for its low affinity. Conversely, MS2-3C8 binds MBP-DR4 as tightly as the most avid anti-microbial TCRs. MS2-3C8 engages self-antigen via a docking mode that resembles the optimal topology of anti-foreign TCRs, but is distinct from that of other autoreactive TCRs. Combined with a unique CDR3β conformation, this docking mode compensates for the weak binding of MBP to HLA-DR4 by maximizing interactions between MS2-3C8 and MBP. Thus, the MS2-3C8-MBP-DR4 complex reveals the basis for an alternative strategy whereby autoreactive T cells escape negative selection, yet retain the ability to initiate autoimmunity.

Homophilic Adhesion Mechanism of Neurofascin, a Member of the L1 Family of Neural Cell Adhesion Molecules

The L1 family neural cell adhesion molecules play key roles in specifying the formation and remodeling of the neural network, but their homophilic interaction that mediates adhesion is not well understood. We report two crystal structures of a dimeric form of the headpiece of neurofascin, an L1 family member. The four N-terminal Ig-like domains of neurofascin form a horseshoe shape, akin to several other immunoglobulin superfamily cell adhesion molecules such as hemolin, axonin, and Dscam. The neurofascin dimer, captured in two crystal forms with independent packing patterns, reveals a pair of horseshoes in trans-synaptic adhesion mode. The adhesion interaction is mediated mostly by the second Ig-like domain, which features an intermolecular β-sheet formed by the joining of two individual GFC β-sheets and a large but loosely packed hydrophobic cluster. Mutagenesis combined with gel filtration assays suggested that the side chain hydrogen bonds at the intermolecular β-sheet are essential for the homophilic interaction and that the residues at the hydrophobic cluster play supplementary roles. Our structures reveal a conserved homophilic adhesion mode for the L1 family and also shed light on how the pathological mutations of L1 affect its structure and function.

Amyloid Formation from an α‐Helix Peptide Bundle Is Seeded by 310‐Helix Aggregates

Transformation of proteins and peptides to fibrillar aggregates rich in β sheets underlies many diseases, but mechanistic details of these structural transitions are poorly understood. To simulate aggregation, four equivalents of a water-soluble, α-helical (65 %) amphipathic peptide (AEQLLQEAEQLLQEL) were assembled in parallel on an oxazole-containing macrocyclic scaffold. The resulting 4α-helix bundle is monomeric and even more α helical (85 %), but it is also unstable at pH 4 and undergoes concentration-dependent conversion to β-sheet aggregates and amyloid fibrils. Fibrils twist and grow with time, remaining flexible like rope (>1 μm long, 5-50 nm wide) with multiple strings (2 nm), before ageing to matted fibers. At pH 7 the fibrils revert back to soluble monomeric 4α-helix bundles. During α→β folding we were able to detect soluble 3(10) helices in solution by using 2D-NMR, CD and FTIR spectroscopy. This intermediate satisfies the need for peptide elongation, from the compressed α helix to the fully extended β strand/sheet, and is driven here by 3(10) -helix aggregation triggered in this case by template-promoted helical bundling and by hydrogen-bonding glutamic acid side chains. A mechanism involving α⇌α(4) ⇌(3(10) )(4) ⇌(3(10) )(n) ⇌(β)(n) ⇋m(β)(n) equilibria is plausible for this peptide and also for peptides lacking hydrogen-bonding side chains, with unfavourable equilibria slowing the α→β conversion.

Poly(N‐vinylimidazole) based hydrogen bonded side chain liquid crystalline polymer

AbstractA new poly(N‐vinylimidazole) (PVI) based side chain liquid crystalline polymer (HB‐PLC) was prepared from PVI as a hydrogen bond acceptor polymer and 11‐(4‐cyanobiphenyl‐4′‐oxy)undekan‐1‐ol (LC11) by molecular self‐assembly processes via hydrogen bond formation between nitrogen of PVI and hydroxyl group of the LC11. The formation of H‐bond was confirmed by using FTIR spectroscopy. The liquid crystalline behavior of the HB‐PLC was investigated by differential scanning calorimeter (DSC) and polarized optical microscopy. The thermal behaviors of the H‐bonded liquid crystalline polymer were investigated by DSC measurements under various heating rates. The HB‐PLC shows a transition temperature of 97°C with an relatively high enthalpy change of 140.6 J/g corresponding to the transition from liquid crystalline nematic phase to isotropic phase. The activation energy required for liquid crystal phase transformation was found to be 551.75 kJ/mol. Copyright © 2010 John Wiley & Sons, Ltd.

In Silico Study of Human Gap Junction Beta-2 Protein by Homology Modeling

Abstract Asp66his, Asp54Lys, and Asp50Asn are mutations in connexin 26 that are observed in the clinic and give rise to autosomal dominant syndromes. They are the result of point mutations in the human gap junction β-2 gene. In order to investigate the structural mechanism of Bart-Pumphrey Syndrome, Keratitis-Ichthyosis-Deafness Syndrome, and Vohwinkel Syndrome, homology model-ing was carried out. Asp66 has direct contact with Asn62 by two hydrogen bonds in the wild-type protein, and in Asp66His, the biggest change observed is a tre-mendous energy increase caused by hydrogen bond breakage to Asn62. Shifts in the side chain and new hy-drogen bond formation are observed for Lys54 com-pared to the wild-type protein (Asn54) and result in clos-er contact to Val84. Asp50Asn causes a significant de-crease in bond energy, and residual charge reversal re-pels the ion and metabolites and, hence, inhibits their transportation. Such perturbations are likely to be a fac-tor contributing to abnormal functioning of ion channels, resulting cell death and disease.Keywords: Bart-Pumphrey syndrome, connexin 26, gap junction, β-2 protein, hearing impairment, Keratitis-Ich-thyosis-Deafness syndrome, knuckle pads, leukonychia, vohwinkel syndrome

MPlot--a server to analyze and visualize tertiary structure contacts and geometrical features of helical membrane proteins

MPlot is a webserver that provides a quick and easy way for structural biologists to analyze, visualize and plot tertiary structure contacts of helical membrane proteins. As input, experimentally determined or computationally modeled protein structures in PDB format are required. The automatic analysis concatenates in house tools to calculate cut-off dependent van der Waals contacts or crossing angles of transmembrane helices with third party tools to compute main chain or side chain hydrogen bonds or membrane planes. Moreover, MPlot allows new features and tools to be added on a regular basis. For that purpose, MPlot was embedded in a framework that facilitates advanced users to compose new workflows from existing tools, or to substitute intermediate results with results from their (own) tools. The outputs can be viewed online in a Jmol based protein viewer, or via automatically generated scripts in PyMOL. For further illustration, the results can be downloaded as a 2D graph, representing the spatial arrangement of transmembrane helices true to scale. For analysis and statistics, all results can be downloaded as text files that may serve as inputs for or as standard data to validate the output of knowledge based tertiary structure prediction tools.URL: http://proteinformatics.charite.de/mplot/.

Comparative Characterization of Short Monomeric Polyglutamine Peptides by Replica Exchange Molecular Dynamics Simulation

Polyglutamine (polyQ) diseases are caused by an abnormal expansion of CAG repeats. While their detailed structure remains unclear, polyQ peptides assume beta-sheet structures when they aggregate. To investigate the conformational ensemble of short, monomeric polyQ peptides, which consist of 15 glutamine residues (Q(15)), we performed replica exchange molecular dynamics (REMD) simulations. We found that Q(15) can assume multiple configurations due to all of the residues affecting the formation of side-chain hydrogen bonds. Analysis of the free energy landscape reveals that Q(15) has a basin for random-coil structures and another for alpha-helix or beta-turn structures. To investigate properties of aggregated polyQ peptides, we performed multiple molecular dynamics (MMD) simulations for monomeric and oligomeric Q(15). MMD revealed that the formation of oligomers stabilizes the beta-turn structure by increasing the number of hydrogen bonds between the main chains.

Natural polyphenols as inhibitors of amyloid aggregation. Molecular dynamics study of GNNQQNY heptapeptide decamer

Amyloid-like fibrils had been associated with many fatal diseases, and the rational design of the fibrillization inhibitors holds the great promise of finding the prevention and treatment options. The understanding of the mechanisms by which the small molecules can inhibit the aggregation plays the key role in such design. Here we present the results of MD simulations that provide the atomistic details of the process, by which the small molecules may destabilize the ordered amyloid oligomers formed by the model hexapeptide. We select a heptapeptide fragment (GNNQQNY) from Sup-35 yeast prion protein, which is capable to form both amyloid fibrils and microcrystals. Atomic-resolution structures of its crystals were reported by Eisenberg et al. (Nature 447:453, 2007). We analyze several MD trajectories describing the evolution of the decamer fragment taken from this crystal structure, both by itself and in the presence of myricetin and kaempferol (two naturally occurring polyphenols, found to be strong and weak aggregation inhibitors). While the decamer of GNNQQNY demonstrates remarkable stability of its structure after 2 ns simulation, myricretin disturbs the aggregation. The simulations show myricetin interacts with the β-sheet due to polar interactions with side chains of the peptide weakening the interstrand hydrogen bonds, wrapping the β-sheet and disaggregating the outer layer. Both backbone to backbone and side chain to side chain hydrogen bonds are lost, and the β-sheets are moving away from each other. This leads to the loss of backbone H-bonding and eventual separation of one β-strands from the outer layer. We also test several AMBER force fields and implicit solvent models for their ability to keep the decamer of GNNQQNY aggregated. The RMSDs of decamer of GNNQQNY with force field 99SB and implicit solvent models of igb2 and igb5, were maintained at less than 4 Å.

Intermonomer Hydrogen Bonds Enhance GxxxG-Driven Dimerization of the BNIP3 Transmembrane Domain: Roles for Sequence Context in Helix–Helix Association in Membranes

We determined the sequence dependence of human BNIP3 transmembrane domain dimerization using the biological assay TOXCAT. Mutants in which intermonomer hydrogen bonds between Ser172 and His173 are abolished show moderate interaction, indicating that side-chain hydrogen bonds contribute to dimer stability but are not essential to dimerization. Mutants in which a GxxxG motif composed of Gly180 and Gly184 has been abolished show little or no interaction, demonstrating the critical nature of the GxxxG motif to BNIP3 dimerization. These findings show that side-chain hydrogen bonds can enhance the intrinsic dimerization of a GxxxG motif and that sequence context can control how hydrogen bonds influence helix–helix interactions in membranes. The dimer interface mapped by TOXCAT mutagenesis agrees closely with the interfaces observed in the NMR structure and inferred from mutational analysis of dimerization on SDS-PAGE, showing that the native dimer structure is retained in detergents. We show that TOXCAT and SDS-PAGE give complementary and consistent information about BNIP3 transmembrane domain dimerization: TOXCAT is insensitive to mutations that have modest effects on self-association in detergents but readily discriminates among mutations that completely disrupt detergent-resistant dimerization. The close agreement between conclusions reached from TOXCAT and SDS-PAGE data for BNIP3 suggests that accurate estimates of the relative effects of mutations on native-state protein–protein interactions can be obtained even when the detergent environment is strongly disruptive.

Hydration mechanism on a poly(methacrylic acid) film studied by in situ attenuated total reflection infrared spectroscopy

A drastic structure change during a hydration process of a poly(methacrylic acid) (PMAA) film was observed by time-resolved in situ attenuated total reflection infrared (ATR-IR) spectroscopy. Inter- or intra-hydrogen-bonds via side-chain carboxyl groups are formed as cyclic dimers, side-on dimers and linear open chain oligomers including open dimers in a dried PMAA film. By contacting water, the side-chain hydrogen-bonds in PMAA are dissociated instantly, and then the subsequent carboxyl groups which have no hydrogen-bond newly hydrate with water molecules in a side-on form. Quantum chemical calculations (QCCs) using a model monomer of propionic acid (PA) also support the hydrogen-bonded and hydrated structures explored by the ATR-IR spectroscopy. It has been concluded from the present study that the dissociation of hydrogen-bonded and newly created hydrated structures via the carboxyl groups play an important role for the swelling of PMAA in water.

Model Transient Networks from Strongly Hydrogen-Bonded Polymers

Random copolymers consisting of n-butyl acrylate backbones with quadruple hydrogen-bonding side chains based on 2-ureido-4[1H]-pyrimidinone (UPy) have been synthesized via controlled radical polymerization and postpolymerization functionalization. Through this synthetic strategy high UPy monomer content (15 mol %) can be reached while maintaining low polydispersity and excellent control over molecular weight, providing model reversible networks with well-defined molecular architecture. Despite low Tgs and a lack of entanglements or crystallinity, these materials behave as thermoplastic elastomers through the strong but reversible association of UPy groups. Bulk properties such as the plateau modulus, tensile modulus, and relaxation time scale are primarily determined by the average distance between UPy’s along the chain. Starting from a difunctional initiator, triblock copolymers can also be synthesized containing a homopolymer midblock and random copolymer end blocks, effectively concentrating the hydrogen-bonding groups near the chain ends. By controlling both the average composition and distribution of UPy’s along the polymer chain, macroscopic material properties such as stiffness and resistance to creep can be independently tuned.

The Effect of Side Chain Length and Hydrogen Bonding on the Viscoelastic Property of Isobutene/Maleimide Copolymers

AbstractThe viscoelastic properties of the alternating copolymers of N‐substituted maleimides with n‐alkyl and ω‐carboxy‐n‐alkyl groups as the N‐substituent with isobutene were investigated by dynamic mechanical analysis. The transition temperatures in the α‐relaxation region depended on the alkyl chain length and the presence or absence of a terminal carboxy group in the side chain. β‐Relaxation due to side chain dynamics was also observed in a lower temperature region without merging the α‐ and β‐relaxation processes for all the copolymers. The molecular motions of the semiflexible main chain and the flexible side alkyl groups were investigated as well as the intermolecular interaction by hydrogen bonding of the carboxyl groups.magnified image

Structural Basis of the Catalytic Mechanism Operating in Open-Closed Conformers of Lipocalin Type Prostaglandin D Synthase

Lipocalin type prostaglandin D synthase (L-PGDS) is a multifunctional protein acting as a somnogen (PGD2)-producing enzyme, an extracellular transporter of various lipophilic ligands, and an amyloid-β chaperone in human cerebrospinal fluid. In this study, we determined the crystal structures of two different conformers of mouse L-PGDS, one with an open cavity of the β-barrel and the other with a closed cavity due to the movement of the flexible E-F loop. The upper compartment of the central large cavity contains the catalytically essential Cys65 residue and its network of hydrogen bonds with the polar residues Ser45, Thr67, and Ser81, whereas the lower compartment is composed of hydrophobic amino acid residues that are highly conserved among other lipocalins. SH titration analysis combined with site-directed mutagenesis revealed that the Cys65 residue is activated by its interaction with Ser45 and Thr67 and that the S45A/T67A/S81A mutant showed less than 10% of the L-PGDS activity. The conformational change between the open and closed states of the cavity indicates that the mobile calyx contributes to the multiligand binding ability of L-PGDS.

The zinc fingers of the SR-like protein ZRANB2 are single-stranded RNA-binding domains that recognize 5′ splice site-like sequences

The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA(N(x))AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5' splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine "ladder." A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.

The other 90% of the protein: Assessment beyond the Cαs for CASP8 template‐based and high‐accuracy models

For template-based modeling in the CASP8 Critical Assessment of Techniques for Protein Structure Prediction, this work develops and applies six new full-model metrics. They are designed to complement and add value to the traditional template-based assessment by the global distance test (GDT) and related scores (based on multiple superpositions of Calpha atoms between target structure and predictions labeled "Model 1"). The new metrics evaluate each predictor group on each target, using all atoms of their best model with above-average GDT. Two metrics evaluate how "protein-like" the predicted model is: the MolProbity score used for validating experimental structures, and a mainchain reality score using all-atom steric clashes, bond length and angle outliers, and backbone dihedrals. Four other new metrics evaluate match of model to target for mainchain and sidechain hydrogen bonds, sidechain end positioning, and sidechain rotamers. Group-average Z-score across the six full-model measures is averaged with group-average GDT Z-score to produce the overall ranking for full-model, high-accuracy performance. Separate assessments are reported for specific aspects of predictor-group performance, such as robustness of approximately correct template or fold identification, and self-scoring ability at identifying the best of their models. Fold identification is distinct from but correlated with group-average GDT Z-score if target difficulty is taken into account, whereas self-scoring is done best by servers and is uncorrelated with GDT performance. Outstanding individual models on specific targets are identified and discussed. Predictor groups excelled at different aspects, highlighting the diversity of current methodologies. However, good full-model scores correlate robustly with high Calpha accuracy.

Typical aromatic noncovalent interactions in proteins: A theoretical study using phenylalanine

A systematic study of CH...pi, OH...pi, NH...pi, and cation...pi interactions has been done using complexes of phenylalanine in its cationic, anionic, neutral, and zwitterionic forms with CH(4), H(2)O, NH(3), and NH(4) (+) at B3LYP, MP2, MPWB1K, and M06-2X levels of theory. All noncovalent interactions are identified by the presence of bond critical points (bcps) of electron density (rho(r)) and the values of rho(r) showed linear relationship to the binding energies (E(total)). The estimated E(total) from supermolecule, fragmentation, and rho(r) approaches suggest that cation...pi interactions are in the range of 36 to 46 kcal/mol, whereas OH...pi, and NH...pi interactions have comparable strengths of 6 to 27 kcal/mol and CH...pi interactions are the weakest (0.62-2.55 kcal/mol). Among different forms of phenylalanine, cationic form generally showed the highest noncovalent interactions at all levels of theory. Cooperativity of multiple interactions is analyzed on the basis of rho(r) at bcps which suggests that OH...pi and NH...pi interactions show positive, whereas CH...pi and cation...pi interactions exhibit negative cooperativity with respect to the side chain hydrogen bond interactions. In general, side chain interactions are strengthened as a result of aromatic interaction. Solvation has no significant effect on the overall geometry of the complex though slight weakening of noncovalent interactions by 1-2 kcal/mol is observed. An assessment of the four levels of theory studied herein suggests that both MPWB1K and M06-2X give better performance for noncovalent interactions. The results also support the fact that B3LYP is inadequate for the study of weak interactions.

Implementing the LIM code: the structural basis for cell type-specific assembly of LIM-homeodomain complexes

LIM-homeodomain (LIM-HD) transcription factors form a combinatorial 'LIM code' that contributes to the specification of cell types. In the ventral spinal cord, the binary LIM homeobox protein 3 (Lhx3)/LIM domain-binding protein 1 (Ldb1) complex specifies the formation of V2 interneurons. The additional expression of islet-1 (Isl1) in adjacent cells instead specifies the formation of motor neurons through assembly of a ternary complex in which Isl1 contacts both Lhx3 and Ldb1, displacing Lhx3 as the binding partner of Ldb1. However, little is known about how this molecular switch occurs. Here, we have identified the 30-residue Lhx3-binding domain on Isl1 (Isl1(LBD)). Although the LIM interaction domain of Ldb1 (Ldb1(LID)) and Isl1(LBD) share low levels of sequence homology, X-ray and NMR structures reveal that they bind Lhx3 in an identical manner, that is, Isl1(LBD) mimics Ldb1(LID). These data provide a structural basis for the formation of cell type-specific protein-protein interactions in which unstructured linear motifs with diverse sequences compete to bind protein partners. The resulting alternate protein complexes can target different genes to regulate key biological events.

Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme

CCA-adding enzyme builds the 3'-end CCA of tRNA without a nucleic acid template. The mechanism for the maintenance of fidelity during the CCA-adding reaction remains elusive. Here, we present almost a dozen complex structures of the class I CCA-adding enzyme and tRNA mini-helices (mini-D(73)N(74), mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75); D(73) is a discriminator nucleotide and N is either A, G, or U). The mini-D(73)N(74) complexes adopt catalytically inactive open forms, and CTP shifts the enzymes to the active closed forms and allows N(74) to flip for CMP incorporation. In contrast, unlike the catalytically active closed form of the mini-D(73)C(74)C(75) complex, the mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75) complexes adopt inactive open forms. Only the mini-D(73)C(74)U(75) accepts AMP to a similar extent as mini-D(73)C(74)C(75), and ATP shifts the enzyme to a closed, active form and allows U(75) to flip for AMP incorporation. These findings suggest that the 3'-region of RNA is proofread, after two nucleotide additions, in the closed, active form of the complex at the AMP incorporation stage. This proofreading is a prerequisite for the maintenance of fidelity for complete CCA synthesis.

Structural Basis for the Recognition of Histone H4 by the Histone-Chaperone RbAp46

SummaryRbAp46 and RbAp48 (pRB-associated proteins p46 and p48, also known as RBBP7 and RBBP4, respectively) are highly homologous histone chaperones that play key roles in establishing and maintaining chromatin structure. We report here the crystal structure of human RbAp46 bound to histone H4. RbAp46 folds into a seven-bladed β propeller structure and binds histone H4 in a groove formed between an N-terminal α helix and an extended loop inserted into blade six. Surprisingly, histone H4 adopts a different conformation when interacting with RbAp46 than it does in either the nucleosome or in the complex with ASF1, another histone chaperone. Our structural and biochemical results suggest that when a histone H3/H4 dimer (or tetramer) binds to RbAp46 or RbAp48, helix 1 of histone H4 unfolds to interact with the histone chaperone. We discuss the implications of our findings for the assembly and function of RbAp46 and RbAp48 complexes.