Molecular dynamics model structures for the molten globule state of {alpha}-lactalbumin: aromatic residue clusters I and II

To model the molten globule structure of <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-lactalbumin, moleculardynamics (MD) simulations were carried out for the protein inexplicit water at high temperature. In these simulations, long-rangeCoulomb interactions were evaluated explicitly with an originalmethod (particle–particle and particle–cell: PPPC)to avoid artifacts caused by the cut-off. The MD simulationswere started from two initial conditions to verify that similarresults would be obtained. From the last 150 ps trajectoriesof the two MD simulations, two partially unfolded average structureswere obtained. These structures had the following common structuralfeatures which are characteristic of the molten globule state.The radii of gyration for these conformations were 7.4 and 9.6%larger than that of the native state. These values were almostthe same as the experimental value (9.6%) observed recentlyby small-angle X-ray scattering (Kataoka,M., Kuwajima,K., Tokunaga,F.and Goto,Y., 1997, Protein Sci., 6, 422–430). Furthermore,aromatic residues of clusters I and II in these structures werefar apart from each other except for Try103–Trp104. Thisresult is in good agreement with NMR experimental results forthe acid-denatured molten globule state (Alexandrescu et al.,1992, 1993); that is, NOE signals between the aromatic residueswere not observed, except for that of Try103–Trp104 inthe molten globule state. Other structural features of thesemodels for the molten globule state are discussed with referenceto native state structures.     

A holistic approach to protein structure alignment

A method of protein structure comparison developed previouslyis extended to incorporate other aspects of protein structurein addition to the inter-atomic vectors on which it was originallybased. Each additional aspect, which included hydrogen bonding,solvent exposure, torsional angles and sequence, was introducedseparately and evaluated for its ability to improve alignmentquality. The components were then combined, suitably weighted,to produce a more holistic comparison method. The method wastested on a group of remotely related ß/<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0"> type proteinsthat share a common feature in their overall chain fold. Theresults indicated that while the original inter-atomic vectorcomponent was sufficient to give the correct alignment of mostpairs of topologically equivalent proteins, the inclusion ofhydrogen bonds, torsion angles and a measure of solvent exposureled to improvements in the more difficult comparisons. Considerationof amino acid properties, including hydrophobicity, had no beneficialeffect. The failure of the latter component was not unexpectedconsidering the almost total lack of sequence similarity amongthe proteins considered.     

Calcium binding mode of {gamma}-carboxyglutamic acids in conantokins

Conantokin-T (con-T) and conantokin-G (con-G) are two highlyhomologous peptide toxins found in Conus venom. The former isa 21-residue peptide with four <IMG SRC="http://peds.oxfordjournals.org/math/gamma.gif" ALT="{gamma}" BORDER="0">-carboxyglutamic acid (Gla) residues(at positions 3, 4, 10 and 14), while the latter is a 17-residuepeptide with five <IMG SRC="http://peds.oxfordjournals.org/math/gamma.gif" ALT="{gamma}" BORDER="0">-carboxyglutamic acid residues (at positions3, 4, 7, 10 and 14). Despite the apparent similarity in numberand relative positions of the <IMG SRC="http://peds.oxfordjournals.org/math/gamma.gif" ALT="{gamma}" BORDER="0">-carboxyglutamic acid residues,¹¹³Cd-NMR studies indicated a distinct metal binding behaviorfor con-G and con-T. There appears to be four binding sitesin con-G in contrast to one metal binding site in con-T. Toelucidate the mode of calcium binding by the <IMG SRC="http://peds.oxfordjournals.org/math/gamma.gif" ALT="{gamma}" BORDER="0">-carboxyglutamicacid residues in these conantokins, we designed various analogouspeptides with their <IMG SRC="http://peds.oxfordjournals.org/math/gamma.gif" ALT="{gamma}" BORDER="0">-carboxyglutamic acid replaced by otheramino acid residues. ¹¹³Cd-NMR experiments on conantokin analoguesreveal that the major difference in the number of metal bindingsites between con-G and con-T is due to the residue at position7. We also performed molecular simulations to calculate therelative binding free energies of several potential bindingsites. Based on our theoretical and experimental results, wepropose a `four-site' binding model for conantokin-G and a `single-site'binding model for conantokin-T.     

Molecular modelling of Staphylococcal {delta}-toxin ion channels by restrained molecular dynamics

<IMG SRC="http://peds.oxfordjournals.org/math/delta.gif" ALT="{delta}" BORDER="0">-Toxin is a 26-residue channel-forming peptide from Staphylococcusaureus which forms an amphipathic <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helix in a membrane environment.Channel formation in planar bilayers suggests that an averageof six <IMG SRC="http://peds.oxfordjournals.org/math/delta.gif" ALT="{delta}" BORDER="0">-toxin helices self-assemble to form transbilayer pores.Molecular models for channels formed by <IMG SRC="http://peds.oxfordjournals.org/math/delta.gif" ALT="{delta}" BORDER="0">-toxin and by a syntheticanalogue have been generated using a simulated annealing protocolapplied via restrained molecular dynamics. These models areanalysed in terms of the predicted geometric and energetic propertiesof the transbilayer pores. Pore radius calculations of the modelsdemonstrate that rings of channel-lining residues contributea series of constrictions along the pore. Electrostatic propertiesof the pores are determined both by pore-lining charged sidechains and by the aligned helix dipoles of the parallel helixbundle. Molecular dynamics simulations (100 ps) of <IMG SRC="http://peds.oxfordjournals.org/math/delta.gif" ALT="{delta}" BORDER="0">-toxin modelscontaining intra-pore water were performed. Analysis of theresultant dynamics trajectories further supports the proposalthat alternative conformations of pore-constricting side chainsmay be responsible for the observed conductance heterogeneityof <IMG SRC="http://peds.oxfordjournals.org/math/delta.gif" ALT="{delta}" BORDER="0">-toxin ion channels.     

Computer simulations of signal transduction mechanism in {alpha}1B-adrenergic and m3-muscarinic receptors

Molecular dynamices simulations of the hamster <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">_1Badrenergicand the rat m3-muscarinic seven-helix bundle receptor modelshave been carried out. The free, agonist-bound and antagonist-boundforms have been considered. Moreover, three mutant forms ofthe m3-muscarinic recep-tor (N507<IMG SRC="http://peds.oxfordjournals.org/math/larr.gif" ALT="<-" BORDER="0">A, N507<IMG SRC="http://peds.oxfordjournals.org/math/larr.gif" ALT="<-" BORDER="0">D and N507<IMG SRC="http://peds.oxfordjournals.org/math/larr.gif" ALT="<-" BORDER="0">S) have alsobeen simulated; among these, the N507<IMG SRC="http://peds.oxfordjournals.org/math/larr.gif" ALT="<-" BORDER="0">S mutant shows a constitutiveactivity. A comparative structural/dynamics analysis has beenperformed to elucidate (i) the perturbations induced by thefunctionally different ligands upon binding to their targetreceptor, (ii) the features of the three single-point mutantswith respect to the receptor wild type and (iii) the propertiesshared by the agonist-boundforms of the <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">_1B-adrenergic receptorand the m3-muscarinic receptor and by the constitutively activemutant N507<IMG SRC="http://peds.oxfordjournals.org/math/larr.gif" ALT="<-" BORDER="0">S. The consistency obtained between the structuralrearrangement of the transmembrane seven-helix bundle modelsconsidered, and the experimental pharmacological efficaciesof the ligands and of the mutants, constitute an important validationof the 3-D models obtained and allow the inference of the mechanismof ligand- or mutation-induced receptor activation at the molecularlevel.     

Functionally essential, invariant glutamate near the C-terminus of strand {beta}5 in various ({alpha}/{beta})8-barrel enzymes as a possible indicator of their evolutionary relatedness

Twelve different (<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">/ß)₈-barrel enzymes belonging tothree structurally distinct families were found to contain,near the C-terminus of their strand ß5, a conservedinvariant glutamic acid residue that plays an important functionalrole in each of these enzymes. The search was based on the ideathat a conserved sequence region of an (<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">/ß)₈-barrelenzyme should be more or less conserved also in the equivalentpart of the structure of the other enzymes with this foldingmotif owing to their mutual evolutionary relatedness. For thispurpose, the sequence region around the well conserved fifthß-strand of a-amylase containing catalytic glutamate(Glu230, Aspergillus oryzae <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-amylase numbering), was used asthe sequence-structural template. The isolated sequence stretchesof the 12 (<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">/ß)₈-barrels are discussed from both thesequence-structural and the evolutionary point of view, theinvariant glutamate residue being proposed to be a joining featureof the studied group of enzymes remaining from their ancestral(<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">/ß)₈-barrel     

Solution structure and dynamics of a serpin reactive site loop using interleukin 1 as a presentation scaffold

Human interleukin-1ß (IL1ß) was used as a presentationscaffold for the characterization of the reactive site loop(RSL) of the serpin <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">1-antitrypsin (A1AT), the physiologicalinhibitor of leukocyte elastase. A chimeric protein was generatedby replacement of residues 50–53 of IL1ß, correspondingto an exposed reverse turn in IL1ß, with the 10-residueP5-P5' sequence EAIPMSIPPE from A1AT. The chimera (antitrypsin-interleukin,AT-IL) inhibits elastase specifically and also binds the IL1ßreceptor. Multinuclear NMR characterization of AT-IL establishedthat, with the exception of the inserted sequence, the structureof the IL1ß scaffold is preserved in the chimera. Thestructure of the inserted RSL was analyzed relative to thatof the isolated 10-residue RSL peptide, which was shown to beessentially disordered in solution. The chimeric RSL was alsofound to be solvent exposed and conformationally mobile in comparisonwith the IL1ß scaffold, and there was no evidence of persistinginteractions with the scaffold outside of the N- and C-terminallinkages. However, AT-IL exhibits sigificant differences inchemical shift and NOE patterns relative to the isolated RSLthat are consistent with local features of non-random structure.The proximity of these features to the P1-P1' residues suggeststhat they may be responsible for the inhibitory activity ofthe chimera.