Modeling the intact form of the {alpha}1-proteinase inhibitor

The structure of the intact form of the serpin <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁-proteinaseinhibitor has been modeled based on the assumption that thecentral strand s4A of the six-stranded ß-sheet A ofthe cleaved inhibitor is not incorporated into the sheet ofintact <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">₁-proteinase inhibitor. This strand was removed fromits position in the center of the sheet by suitable rotationsabout the backbone dihedrals of Lys343 using molecular graphics.The resulting structure was then annealed using molecular dynamics(MD) while applying progressive distance restraints to the reactivepeptide bond (Met358-Ser359) for 50 ps. During this time, thedisrupted ß-sheet reformed to create a five-strandedß-sheet with strands 3 and 5 in a parallel arrangement.This change and accompanying structural rearrangements are largelyconfirmed by the X-ray structure of plakalbumin, whose structurereflects the overall structure of intact serpins. The successfulmodeling experiment demonstrates the utility of MD for makinggross structural predictions based on related structures. Thebinding loop of the intact form is modeled to allow dockingwith serine proteinases, in particular thrombin, which mosthighly constrains the possible conformations of the bindingloop.     

Replacing the ({beta}{alpha})-unit 8 of E.coli TIM with its chicken homologue leads to a stable and active hybrid enzyme

In order to investigate how structural modifications interferewith protein stability, we modified a (ß<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">)-unit inE.coli triosephosphate isomerase (TIM), a typical (ß<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">)-barrelprotein, assuming that the pseudosymmetrical ß-barrelcan be divided into eight successive loop/ß-strand/loop/<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helixmotifs. We replaced the eighth (ß<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">)-unit of E.coliTIM with the corresponding chicken (ß<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">)-unit. The substitution,involving the replacement of 10 of the 23 residues of this (ß<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">)-unit, was evaluated first by modelling, then experimentally.Modelling by bomology suggests how the amino add replacementsmight be accommodated in the hybrid E.coli/chicken TIM (ETCM8CHI).Both natural and hybrid recombinant TIMs, overproduced in E.coli,were purified to homogeneity and characterized as to their stabilityand kinetics. Our kinetic studies show that the modificationperformed here leads to an active enzyme. The stability studiesindicate that the stability of ETIM8CHI is comparable to thatof the wild type TIM.     

A sequence motif in the transmembrane region of growth factor receptors with tyrosine kinase activity mediates dimerization

A mechanism by which ligand binding to the extracellular domainof a growth factor receptor causes activation of its cytoplasmictyrosine kinase domain is that binding promotes receptor dimerization.Recently we proposed a model in which dimerization of the transmembrane<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helices in one member of this family, rat neu, is mediatedby the presence of three specific residues. This paper showsthat a similar sequence motif is observed in 18 of the 20 transmembrane<IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helices of the tyrosine kinase family of growth factor receptors.The motif encompasses a five residue segment in which position0 (P0) requires a small side chain (Gly, Ala, Ser, Thr or Pro),P3 an aliphatic side chain (Ala, Val, Leu or Ile) and P4 onlythe smallest side chains (Gly or Ala). In addition other featuresof the transmembrane sequences are reported. It is concludedthat the dimerization of transmembrane <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helices may be a generalmechanism of tyrosine kinase activation in this family of growthfactor receptors.     

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.     

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.     

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.     

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     

The relative positions of alanine residues in the hydrophobic core control the formation of two-stranded or four-stranded {alpha}-helical coiled-coils

The objective of this study was to investigate the positionaleffect of hydrophobic interactions in the <IMG SRC="http://peds.oxfordjournals.org/math/alpha.gif" ALT="{alpha}" BORDER="0">-helical interfacein controlling the formation of two-stranded and four-strandedcoiled-coils. Two disulfide-bridged antiparallel coiled-coilswere designed which differ only in the position of a singleAla residue in the middle heptad: in peptide 2H the Ala residuesare in register (in the same rung), while in peptide 4H theyare not. Data from size-exclusion chromatography and sedimentationequilibrium experiments showed that under benign conditionspeptides 2H and 4H were two-stranded and four-stranded coiled-coilsrespectively. These results, in conjunction with molecular modelingstudies, suggest that when four Ala residues are in the sameplane of a potential four-stranded coiled-coil, the small sidechains of Ala would create a large cavity in the hydrophobicinterface of the potential four-stranded structure which isdestabilizing and favors the two-stranded, disulfide-bridgedcoiled-coil. In contrast, an alternating Leu-Ala hydrophobicpacking in the two planes distributes the potential cavity overa larger region, which may be partially filled by minor adjustmentsof the neighboring Leu side chains. As a result, there is stillsufficient hydrophobic contact to maintain the four-strandedstructure.     

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.