Main-chain Conformation Research Articles

Structure of the Antiviral Assembly Inhibitor CAP-1 Complex with the HIV-1 CA Protein

The CA domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein plays critical roles in both the early and late phases of viral replication and is therefore an attractive antiviral target. Compounds with antiviral activity were recently identified that bind to the N-terminal domain of CA (CA N) and inhibit capsid assembly during viral maturation. We have determined the structure of the complex between CA N and the antiviral assembly inhibitor N-(3-chloro-4-methylphenyl)- N′-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) using a combination of NMR spectroscopy and X-ray crystallography. The protein undergoes a remarkable conformational change upon CAP-1 binding, in which Phe32 is displaced from its buried position in the protein core to open a deep hydrophobic cavity that serves as the ligand binding site. The aromatic ring of CAP-1 inserts into the cavity, with the urea NH groups forming hydrogen bonds with the backbone oxygen of Val59 and the dimethylamonium group interacting with the side-chains of Glu28 and Glu29. Elements that could be exploited to improve binding affinity are apparent in the structure. The displacement of Phe32 by CAP-1 appears to be facilitated by a strained main-chain conformation, which suggests a potential role for a Phe32 conformational switch during normal capsid assembly.

Synthesis of poly( p-phenylene)s having alternating sugar and alkyl substituents by Suzuki coupling polymerization and evaluation of their main-chain conformations

This paper reports synthesis of poly( p-phenylene)s (PPPs) having alternating sugar and alkyl substituents by Suzuki coupling polymerization of a 1,4-dibromobenzene monomer having peracetylated glucose residues with a 1,4-benzene bis(boronic acid) having alkyl chains using Pd(PPh 3) 4 in a mixed solvent of THF and NaHCO 3 aq. at reflux temperature. The polymerization proceeded with the progress of frequent deacetylation, and thus, the crude product was acetylated, followed by the isolation procedures, giving the PPP having alternating peracetylated glucose and alkyl substituents. The structure of the isolated product was confirmed by the 1H and 13C NMR measurements to be the desired PPP derivative. The M n values were estimated by GPC analysis with DMF as the eluent to be 5400–12,700. The deacetylation of the polymer completely took place using sodium methoxide in methanol/THF. The conformation of the main-chain was evaluated by the CD spectrum in comparison with that of PPP only with the glucose substituents, indicating that the present PPP derivative had the flexible nature of the main-chain by introduction of the alkyl-substituted units between glucose-substituted units. The Suzuki coupling of a 1,4-dibromobenzne monomer having disaccharide substituents with the benzene bis(boronic acid) monomer was also performed under the similar conditions. The product was precipitated from the reaction mixture, which was simply isolated by filtration. The isolated polymer was purified further by reprecipitation into diethyl ether and its structure was a PPP having free disaccharide or monosaccharide residues. This indicated occurrence of complete deacetylation as well as partial degradation of the glycosidic linkages in the disaccharides during the polymerization. The main-chain of the obtained polymer had also the flexible nature. The fluorescence spectra of the obtained PPP derivatives in this study were also measured.

Ramachandran Plot on The Web (2.0)

The Ramachandran plot displays the main chain conformation angles (Phi and Psi) of the polypeptide chain of a protein molecule. The paper reports the updated version of the Ramachandran plot web server and has several improved options for displaying the conformation angles in various regions. In addition, options are provided to display the conformation angles in various secondary structural elements and regions within the user specified Phi and Psi values in the plot. The updated version is accessible at the following URL: http://dicsoft1.physics.iisc.ernet.in/rp/.

Open and Closed Structures of the UDP-glucose Pyrophosphorylase from Leishmania major

Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel beta-helix (LbetaH), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors.

Interpretation of Synchrotron Radiation Circular Dichroism Spectra of Anionic, Cationic, and Zwitterionic Dialanine Forms

Electronic absorption and synchrotron radiation circular dichroism (SRCD) spectra of the anionic, cationic, and zwitterionic forms of L-alanyl-L-alanine (AA) in aqueous solutions were measured and interpreted by molecular dynamics (MD) and ab initio computations. Time-dependent density functional theory (TD DFT) was applied to predict the electronic excited states. The modeling enabled the assessment of the role of molecular conformation, charge, and interaction with the polar environment in the formation of the spectral shapes. Particularly, inclusion of explicit solvent molecules in the computations appeared to be imperative because of the participation of water orbitals in the amide electronic structure. Implicit dielectric continuum solvent models gave inferior results for clusters, especially at low-energy transitions. Because of the dispersion of transition energies, tens of water/AA clusters had to be averaged in order to obtain reasonable spectral shapes with a more realistic inhomogeneous broadening. The modeling explained most of the observed differences, as the anionic and zwitterionic SRCD spectra were similar and significantly different from the cationic spectrum. The greatest deviation between the experimental and theoretical curves observed for the lowest-energy negative anion signal can be explained by the limited precision of the TD DFT method, but also by the complex dynamics of the amine group. The results also indicate that differences in the experimental spectral shapes do not directly correlate with the peptide main-chain conformation. Future peptide and protein conformational studies based on circular dichroic spectroscopy can be reliable only if such effects of molecular dynamics, solvent structure, and polar solvent-solute interactions are taken into account.

Prediction of side‐chain conformations on protein surfaces

An approach is described that improves the prediction of the conformations of surface side chains in crystal structures, given the main-chain conformation of a protein. A key element of the methodology involves the use of the colony energy. This phenomenological term favors conformations found in frequently sampled regions, thereby approximating entropic effects and serving to smooth the potential energy surface. Use of the colony energy significantly improves prediction accuracy for surface side chains with little additional computational cost. Prediction accuracy was quantified as the percentage of side-chain dihedral angles predicted to be within 40 degrees of the angles measured by X-ray diffraction. Use of the colony energy in predictions for single side chains improved the prediction accuracy for chi(1) and chi(1+2) from 65 and 40% to 74 and 59%, respectively. Several other factors that affect prediction of surface side-chain conformations were also analyzed, including the extent of conformational sampling, details of the rotamer library employed, and accounting for the crystallographic environment. The prediction of conformations for polar residues on the surface was generally found to be more difficult than those for hydrophobic residues, except for polar residues participating in hydrogen bonds with other protein groups. For surface residues with hydrogen-bonded side chains, the prediction accuracy of chi(1) and chi(1+2) was 79 and 63%, respectively. For surface polar residues, in general (all side-chain prediction), the accuracy of chi(1) and chi(1+2) was only 73 and 56%, respectively. The most accurate results were obtained using the colony energy and an all-atom description that includes neighboring molecules in the crystal (protein chains and hetero atoms). Here, the accuracy of chi(1) and chi(1+2) predictions for surface side chains was 82 and 73%, respectively. The root mean square deviations obtained for hydrogen-bonding surface side chains were 1.64 and 1.81 A, with and without consideration of crystal packing effects, respectively.

PSSARD (2.0): A database server for making flexible queries relating amino acid sequences to main-chain secondary structure conformations for proteins of known three-dimensional structure and certain useful applications

We have updated the Protein Sequence–Structure Analysis Relational Database (PSSARD) first published in the Int. J. Biol. Macromol. 36 (2005) 259–262 corresponding to 1573 representative protein chains selected from the Protein Data Bank (PDB). In this, the updated and revised PSSARD (Version 2.0), we have included all proteins in the Protein Data Bank available at the time of developing this database including the NMR PDB entries. The current database corresponds to 22,752 XRAY PDB entries and 3977 NMR PDB entries and is separated accordingly in order to facilitate the appropriate database search. The representative protein chains can also be separately accessed within the current database. We have made a provision to combine more than one field to query the database and the results of any search can be used to carry out further nested searches using a combination of queries. We have provided hyperlinks to the individual PDB entries obtained as the result of any search in PSSARD in order to obtain additional details relevant to the protein structure. Certain applications useful to identify domains and structural motifs are discussed.

Crystal Structures of Nonoxidative Zinc-dependent 2,6-Dihydroxybenzoate (γ-Resorcylate) Decarboxylase from Rhizobium sp. Strain MTP-10005

Reversible 2,6-dihydroxybenzoate decarboxylase from Rhizobium sp. strain MTP-10005 belongs to a nonoxidative decarboxylase family. We have determined the structures of the following three forms of the enzyme: the native form, the complex with the true substrate (2,6-dihydroxybenzoate), and the complex with 2,3-dihydroxybenzaldehyde at 1.7-, 1.9-, and 1.7-A resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of one (alphabeta)8 triose-phosphate isomerase-barrel domain with three functional linkers and one C-terminal tail. The native enzyme possesses one Zn2+ ion liganded by Glu8, His10, His164, Asp287, and a water molecule at the active site center, although the enzyme has been reported to require no cofactor for its catalysis. The substrate carboxylate takes the place of the water molecule and is coordinated to the Zn2+ ion. The 2-hydroxy group of the substrate is hydrogen-bonded to Asp287, which forms a triad together with His218 and Glu221 and is assumed to be the catalytic base. On the basis of the geometrical consideration, substrate specificity is uncovered, and the catalytic mechanism is proposed for the novel Zn2+-dependent decarboxylation.

Variation of the Network Anisotropy of Cholesteric Side Chain Elastomers

A series of cholesteric side chain elastomers with odd and even numbers of atoms along the spacer chain were prepared. Varying the content of odd and even spacer groups resulted in anisotropic main chain conformations changing from prolate to oblate. The conformation anisotropy was characterized by temperature-dependent measurements of the sample dimensions. The transmission spectra for polarized light of the elastomer films were recorded under uniaxial mechanical stress, applied perpendicular to the helical axis of the cholesteric liquid crystal. With increasing strain a blue shift of the selective reflection due to a contraction of the helix along its axis was observed. In the case of a nonvanishing conformation anisotropy in addition a distortion of the regular helical structure was indicated by changes in the transmission spectra for linearly polarized light. This distortion was found to be much weaker than predicted by theory.

Stereoelectronic Tuning of the Structure and Stability of the Trp Cage Miniprotein

Proline residues are critical structural elements in proteins, defining turns, loops, secondary structure boundaries, and polyproline helices. Control of proline conformation therefore may be used to define protein structure and stability. 4-Substituted proline derivatives may be used to control proline ring pucker, which correlates with protein main chain conformation. To examine the use of proline conformational restriction to tune globular protein stability, a series of peptides derived from the trp cage miniprotein was synthesized. Proline at residue 12 of the trp cage miniprotein, which adopts a Cgamma-exo ring pucker in the NMR structure, was replaced with 4-substituted proline derivatives, including 4R derivatives favoring a Cgamma-exo ring pucker and 4S derivatives favoring a Cgamma-endo ring pucker. Eight trp cage peptides were synthesized, five of which included residues that are not commercially available, without requiring any solution phase chemistry. Analysis of the trp cage peptides by circular dichroism and NMR indicated that the structure and stability of the trp cage miniprotein was controllable based on the conformational bias of the proline derivative. Replacement of Pro12 with 4S-substituted proline derivatives that favor the Cgamma-endo ring pucker destabilized the trp cage, while replacement of Pro12 with 4R-substituted proline derivatives that favor a Cgamma-exo ring pucker resulted in increased alpha-helicity and thermal stability of the trp cage. The most stable trp cage derivatives contained benzoates of 4R-hydroxyproline, which also exhibited the most pronounced stereoelectronic effects in TYProxN model peptides. Overall, the stability of the trp cage was tunable by over 50 degrees C depending on the identity of the proline side chain at residue 12.

The Crystal Structure of H-2Db Complexed with a Partial Peptide Epitope Suggests a Major Histocompatibility Complex Class I Assembly Intermediate

In the absence of bound peptide ligands, major histocompatibility complex (MHC) class I molecules are unstable. In an attempt to determine the minimum requirement for peptide-dependent MHC class I stabilization, we have used short synthetic peptides derived from the Sendai virus nucleoprotein epitope (residues 324-332, 1FAPGNYPAL9) to promote its folding in vitro of H-2D(b). We found that H-2D(b) can be stabilized by the pentapeptide 5NYPAL9, which is equivalent to the C-terminal portion of the optimal nonapeptide and includes both the P5 and P9 anchor residues. We have crystallized the complex of the H-2D(b) molecule with the pentamer and determined the structure to show how a quasi-stable MHC class I molecule can be formed by occupancy of a single binding pocket in the peptide-binding groove.

Structural phase transition of monoclinic crystals of hen egg-white lysozyme

Two monoclinic crystals (space group P2(1)) of hen egg-white lysozyme, a type I crystal grown at room temperature in a D2O solution with pD 4.5 containing 2%(w/v) sodium nitrate and a type II crystal grown at 313 K in a 10%(w/v) sodium chloride solution with pH 7.6, were each transformed into another monoclinic crystal with the same space group by dehydration-induced phase transition. Changes in X-ray diffraction were recorded to monitor the progress of the crystal transformation, which started with the appearance of diffuse streaks. In both crystals, the intensity of h + l odd reflections gradually weakened and finally disappeared on completion of the transformation. X-ray diffraction in the intermediate state indicated the presence of lattices of both the native and transformed crystals. In the native type I crystal, two alternate conformations were observed in the main chain of the region Gly71-Asn74. One conformer bound a sodium ion which was replaced with a water molecule in the other conformer. In the transformed crystal, the sodium ion was removed and the main-chain conformation of this region was converted to that of the water-bound form. The transformed crystal diffracted to a higher resolution than the native crystal, while the peak width of the diffraction spots increased. Analysis of the thermal motion of protein molecules using the TLS model has shown that the enhancement of the diffraction power in the transformed crystal is mainly ascribable to the suppression of rigid-body motion owing to an increase in intermolecular contacts as a result of the loss of bulk solvent.

A structurally‐defined gap function for pairwise sequence alignment of proteins in the twilight zone

Protein classification and annotation from sequence data alone require methods able to recognize remote evolutionary relationships and to produce biologically correct alignments for building three-dimensional models. Algorithms proficient at detecting remote homology tend not to align accurately enough for modeling. Good alignment algorithms lack the speed and sensitivity required for comprehensive database searching. The problem is most acute in the twilight zone of sequence identity (15–35% matched residues). Most pairwise alignment algorithms use dynamic programming with affine gap functions which impose arbitrary gap initiation and extension penalties. Yet, the insertion or deletion points and composition of the residues inserted or deleted during evolution cannot be modeled by arbitrary penalties, which ignore the structural context of main-chain conformation, solvent accessibility and hydrogen bonding. This work proposes a gap scoring function based on the propensity of gaps to open outside of helices and strands, with an extension rule adjusted for the propensity of certain amino acid residues to predominate in turn and coil contexts. Potential gap residues have been identified using STRIDE ( Frishman, & Argos, 1995 Proteins 23: 566) in high-resolution structures sharing less than 20% pairwise sequence identity, culled from the Protein Data Bank by the PISCES server ( Wang, & Dunbrack, 2003 Bioinformatics 19: 1589). Performance of the new function is assessed in JAligner, an open source implementation of the Smith-Waterman-Gotoh alignment algorithm.

Electronic and vibrational circular dichroism of aromatic amino acids by density functional theory

Structures of model compounds mimicking aromatic amino acid residues in proteins are optimized by density functional theory (DFT), assuming that the main-chain conformation was a random coil. Excitation energies and dipole and rotational strengths for the optimized structures were calculated based on time-dependent DFT (TD-DFT). The electronic circular dichroism (ECD) bands of the models were significantly affected by side-chain conformations. Hydration models of the aromatic residues were also subjected to TD-DFT calculations, and the ECD bands of these models were found to be highly perturbed by the hydration of the main-chain amide groups. In addition to calculating the random-coil conformation, we also performed TD-DFT calculations of the aromatic residue models, assuming that the main-chain conformation was an alpha-helix or beta-strand. As expected, the overall feature of the ECD bands was also perturbed by the main-chain conformations. Moreover, vibrational circular dichroism (VCD) spectra of the hydration models in a random-coil structure were simulated by DFT, which showed that the VCD spectra are more sensitive to the side-chain conformations than the ECD spectra. The present results show that analyses combining ECD and VCD spectroscopy and using DFT calculations can elucidate the main- and side-chain conformations of aromatic residues in proteins.

Accounting for loop flexibility during protein–protein docking

Although reliable docking can now be achieved for systems that do not undergo important induced conformational change upon association, the presence of flexible surface loops, which must adapt to the steric and electrostatic properties of a partner, generally presents a major obstacle. We report here the first docking method that allows large loop movements during a systematic exploration of the possible arrangements of the two partners in terms of position and rotation. Our strategy consists in taking into account an ensemble of possible loop conformations by a multi-copy representation within a reduced protein model. The docking process starts from regularly distributed positions and orientations of the ligand around the whole receptor. Each starting configuration is submitted to energy minimization during which the best-fitting loop conformation is selected based on the mean-field theory. Trials were carried out on proteins with significant differences in the main-chain conformation of the binding loop between isolated form and complexed form, which were docked to their partner considered in their bound form. The method is able to predict complexes very close to the crystal complex both in terms of relative position of the two partners and of the geometry of the flexible loop. We also show that introducing loop flexibility on the isolated protein form during systematic docking largely improves the predictions of relative position of the partners in comparison with rigid-body docking.

Crystal structures of T cell receptor β chains related to rheumatoid arthritis

The crystal structures of the Vbeta17+ beta chains of two human T cell receptors (TCRs), originally derived from the synovial fluid (SF4) and tissue (C5-1) of a patient with rheumatoid arthritis (RA), have been determined in native (SF4) and mutant (C5-1(F104-->Y/C187-->S)) forms, respectively. These TCR beta chains form homo-dimers in solution and in crystals. Structural comparison reveals that the main-chain conformations in the CDR regions of the C5-1 and SF4 Vbeta17 closely resemble those of a Vbeta17 JM22 in a bound form; however, the CDR3 region shows different conformations among these three Vbeta17 structures. At the side-chain level, conformational differences were observed at the CDR2 regions between our two ligand-free forms and the bound JM22 form. Other significant differences were observed at the Vbeta regions 8-12, 40-44, and 82-88 between C5-1/SF4 and JM22 Vbeta17, implying that there is considerable variability in the structures of very similar beta chains. Structural alignments also reveal a considerable variation in the Vbeta-Cbeta associations, and this may affect ligand recognition. The crystal structures also provide insights into the structure basis of T cell recognition of Mycoplasma arthritidis mitogen (MAM), a superantigen that may be implicated in the development of human RA. Structural comparisons of the Vbeta domains of known TCR structures indicate that there are significant similarities among Vbeta regions that are MAM-reactive, whereas there appear to be significant structural differences among those Vbeta regions that lack MAM-reactivity. It further reveals that CDR2 and framework region (FR) 3 are likely to account for the binding of TCR to MAM.

Identification of the Region Responsible for Fibril Formation in the CAD Domain of Caspase-Activated DNase

Caspase-activated DNase (CAD) has a compact domain at its N-terminus (CAD domain, 87 amino acid residues), which comprises one alpha-helix and five beta-strands forming a single sheet. The CAD domain of CAD (CAD-CD) forms amyloid fibrils containing alpha-helix at low pH in the presence of salt. To obtain insights into the mechanism of amyloid fibril formation, we identified the peptide region essential for fibril formation of CAD-CD and the region responsible for the salt requirement. We searched for these regions by constructing a series of deletion and point mutants of CAD-CD. Fibril formation by these CAD-CD mutants was examined by fluorescence analysis of thioflavin T and transmission electron microscopy. C-Terminal deletion and point mutation studies revealed that an aromatic residue near the C-terminus (Trp81) is critical for fibril formation. In addition, the main chain conformation of the beta5 strand, which forms a hydrophobic core with Trp81, was found to be important for the fibril formation by CAD-CD. The N-terminal 30 amino acid region containing two beta-strands was not essential for fibril formation. Rather, the N-terminal region was found to be responsible for the requirement of salt for fibril formation.

The CDR1 of the human λVI light chains adopts a new canonical structure

We performed a comparative analysis of the conformation of the CDR1 of the human lambdaVI variable domains JTO and WIL and the equivalent loop of the lambdaI light chains RHE and KOL, which are representative of the type I canonical structure for lambda light chains. On the basis of the differences found in the main chain conformation, as well as the identity of the residues at key positions, we showed that the L1 of some lambdaVI light chains adopts a conformation that represents a new type of canonical structure. The conformation of the L1 of those lambdaVI light chains, is primarily determined by the presence of an Arg residue at position 25. The analysis of the lambdaVI light chain sequences so far reported, showed that near 25% of those proteins have Gly at position 25 instead of Arg, which represents an allotypic variant of the lambdaVI variable locus. The presence of Gly at position 25 in the L1 of lambdaVI light chains would imply a different conformation for this loop. Additionally, the position 68 in lambdaVI light chains, which is at the top of the FR3 loop, showed such spatial orientation and variability that suggested its participation in the conformation of the antigen recognition surface in this subgroup of lambda chains.

The Orientation Stability and UV Irradiation of Poly(hexa‐2,4‐diynilene‐1,6‐dioxy)benzoates and Cinnamates Containing Disperse Red 19

The poling and orientation parameters of the four polymers para and meta benzoates and cinnamates containing Disperse Red 19 dye were studied. The para polymers were found to be orientated more efficiently, and their stabilities were much better than the meta polymers. The photobleaching takes place when irradiated at temperatures around Tg, but did not occur when irradiated under the poling conditions. After the photo cross‐linking at around Tg the orientation maintenance of the para polymers were found to be almost indefinite. The stability of the para polymers is considered to be due to their stable main chain conformation when poled.

Ab initio construction of all-atom loop conformations

In this study, a new ab initio method named CLOOP has been developed to build all-atom loop conformations. In this method, a loop main-chain conformation is generated by sampling main-chain dihedral angles from a restrained varphi/psi set, and the side-chain conformations are built randomly. The CHARMM all-atom force field was used to evaluate the loop conformations. Soft core potentials were used to treat the non-bond interactions, and a designed energy-minimization technique was used to close and optimize the loop conformations. It is shown that the two strategies improve the computational efficiency and the loop-closure rate substantially compared to normal minimization methods. CLOOP was used to construct the conformations of 4-, 8-, and 12-residue loops in Fiser's test set. The average main-chain root-mean-square deviations obtained in 1,000 trials for the 10 different loops of each size are 0.33, 1.27, and 2.77 A, respectively. CLOOP can build all-atom loop conformations with a sampling accuracy comparable with previous loop main-chain construction algorithms. [Figure: see text].