3D modeling, ligand binding and activation studies of the cloned mouse {delta}, {micro} and {kappa} opioid receptors

Refined 3D models of the transmembrane domains of the cloned, µ and opioid receptors belonging to the superfamilyof G-protein coupled receptors (GPCRs) were constructed froma multiple sequence alignment using the alpha carbon templateof rhodopsin recently reported. Other key steps in the procedurewere relaxation of the 3D helix bundle by unconstrained energyoptimization and assessment of the stability of the structureby performing unconstrained molecular dynamics simulations ofthe energy optimized structure. The results were stable ligand-freemodels of the TM domains of the three opioid receptors. Theligand-free receptor was then used to develop a systematicand reliable procedure to identify and assess putative bindingsites that would be suitable for similar investigation of theother two receptors and GPCRs in general. To this end, a non-selective,`universal' antagonist, naltrexone, and agonist, etorphine,were used as probes. These ligands were first docked in allsites of the model opioid receptor which were sterically accessibleand to which the protonated amine of the ligands could be anchoredto a complementary proton-accepting residue. Using these criteria,nine ligand–receptor complexes with different bindingpockets were identified and refined by energy minimization.The properties of all these possible ligand–substratecomplexes were then examined for consistency with known experimentalresults of mutations in both opioid and other GPCRs. Using thisprocedure, the lowest energy agonist–receptor and antagonist–receptorcomplexes consistent with these experimental results were identified.These complexes were then used to probe the mechanism of receptoractivation by identifying differences in receptor conformationbetween the agonist and the antagonist complex during unconstraineddynamics simulation. The results lent support to a possibleactivation mechanism of the mouse opioid receptor similar tothat recently proposed for several other GPCRs. They also allowedthe selection of candidate sites for future mutagenesis experiments.     

An EGF-pseudomonas exotoxin A recombinant protein with a deletion in toxin binding domain specifically kills EGF receptor bearing cells

We constructed two chimeric toxins; one composed of epidermalgrowth factor (EGF) and pseudomonas exotoxin A (PE), designatedEGF-PE and the other composed of EGF and PE with a deletionof the Ia domain (cell-binding domain), designated EGF-PE (Ia).Both chimeric toxins reacted with anti-EGF and anti-PE antibodies.The cell-killing experiments showed that EGF-PE, but not EGF-PE(Ia),was cytotoxic to the murine fibroblast cell line NR6, whichcarried the PE receptor, but not the EGF receptor. However,after NR6 was transfected with DNA for the expression of humanEGF receptor, the transfected cell line, designated NRHER5,overexpressed human EGF receptors and became sensitive to EGF-PE(IA).The cytotoxicity of EGF-PE(Ia), but not EGF-PE, to NRHER5 canbe completely blocked by an excess amount of EGF. To completelyreverse the cytotoxicity of EGF-PE on NRHER5, both the EGF receptorpathway and the PE receptor pathway need to be blocked. Theseresults suggest that EGF-PE exhibits both EGF and PE bindingactivities, while EGF-PE(IA) possesses only EGF binding activity.Thus, EGF-PE(Ia) may be a better chimeric toxin than EGF-PEin terms of target specificity to EGF receptor bearing cells.We, therefore, examined the cytotoxicity of EGF-PE(Ia) to varioushuman cancer cell lines. We find that human cancer cells containingmore EGF receptors are more sensitive to EGF-PE(Ia).     

Family of G protein {alpha} chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulationof enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase.Each G protein contains an subunit that binds and hydrolyzesguanine nucleotides and interacts with ß subunitsand specific receptor and effector proteins. Amphipathic andsecondary structure analysis of the primary sequences of fivedifferent chains (bovine _s, _t1 and _t2, mouse _i, and rat _o)predicted the secondary structure of a composite chain (_avg).The chains contain four short regions of sequence homologousto regions in the GDP binding domain of bacterial elongationfactor Tu (EF-Tu). Similarities between the predicted secondarystructures of these regions in _avg and the known secondary structureof EF-Tu allowed us to construct a three-dimensional model ofthe GDP binding domain of _avg. Identification of the GDP bindingdomain of _avg defined three additional domains in the compositepolypeptide. The first includes the amino terminal 41 residuesof _avg, with a predicted am phipathic helical structure; thisdomain may control binding of the chains to the ßcomplex. The second domain, containing predicted ßstrands and helices, several of which are strongly amphipathic,probably contains sequences responsible for interaction of chains with effector enzymes. The predicted structure of thethird domain, containing the carhoxy terminal 100 amino acids,is predominantly ß sheet with an amphipathic helixat the carboxy terminus. We propose that this domain is reponsiblefor receptor binding. Our model should help direct further experimentsinto the structure and function of the G protein chain.     

Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase

Aspergillus awamori glucoamylase (GA) contains globular catalyticand starch-binding domains (residues 1–471 and 509–616,respectively). A heavily O-glycosylated sequence comprises twoparts. The first (residues 441–471) in the crystal structurewraps around an /-barrel formed by residues 1–440. Thesecond (residues 472–508) is an extended, semi-rigid linkerbetween the two domains. To investigate the functional roleof this linker, we made internal deletions to remove residues466–512 (GA1), 485–512 (GA2) and 466–483 (GA3).GA2 and GA3 were expressed in Saccharomyces cerevisiae culturesupernatants at 60 and 20% the wild-type level, respectively,while GA1 was almost undetectable. Western blots comparing extracellularand intracellular fractions indicated that the region deletedin GA3 was critical for secretion, while the region deletedin GA2 contributed to the production of a stable enzyme structure.The activities of purified GA2 and GA3 on soluble and insolublestarch were similar to those of wild-type GA, indicating thatfor soluble starch their deletions did not affect the catalyticdomain and for insoluble starch the linker does not coordinatethe activities of the catalytic and starch-binding domains.The deletions had a significant negative effect on GA2 and GA3thermos tabilities.     

Folding and stability of the active N-terminal domain of tissue inhibitor of metalloproteinases-1 and -2

The truncated forms of tissue inhibitor of metalloproteinase-1and -2 (TIMP-1 and -2), comprising the N-terminal active domain,are ideal molecules for structural analysis by intrinsic fluorescenceas each contains a single conserved tryptophan residue. In thispaper we describe studies on their conformational stability,unfolding/refolding kinetics and the environment of the uniquetryptophan as judged by its fluorescence properties in the nativestate and exposure to an external quencher, acrylamide. Twoforms of TIMP-2 were studied: TIMP-2 T21 derived from the full-lengthcDNA clone isolated from a mixed-tumour library, and TIMP-2A21 containing the highly conserved V18IRAK22 sequence. In allthree TIMP proteins the tryptophan environments in the nativestate appeared to be similar, but substantial differences wereseen in their conformational stabilities and refolding kinetics.TIMP-1 was approximately twice as stable as TIMP-2 T21 and 1.4-foldmore stable than TIMP-2 A21. This stability difference betweenTIMP-1 and TIMP-2 was shown to be independent of N-linked glycosylation.TTMP-1 and TIMP-2 A21 both showed simple two-state refoldingkinetics, whereas TIMP-2 T21 refolding was more complex andbiphasic in character. These differences between TIMP-2 T21and A21 suggest that residue 21 is a structurally importantsite in the TIMP protein.All three truncated molecules can beconsidered as stable independent folding domains ideally suitedfor further structural analysis     

Inhibition of {beta}S-chain dependent polymerization by synergistic complementation of contact site perturbations of {alpha}-chain: application of semisynthetic chimeric {alpha}-chains

Mouse _1–30-horse _31–141 chimeric -chain, a semisyntheticsuper-inhibitory -chain, inhibits ß^S-chain dependent polymerizationbetter than both parent -chains. Although contact site sequencedifferences are absent in the _1–30 region of the chimericchain, the four sequence differences of the region _17-22 couldinduce perturbations of the side chains at ₁₆, ₂₀ and ₂₃, thethree contact sites of the region. A synergistic complementationof such contact site perturbation with that of horse _31–141probably results in the super-inhibitory activity of the chimeric-chain. The inhibitory contact site sequence differences, bythemselves, could also exhibit similar synergistic complementation.Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin(LM) mutation [His20()Gln], a contact site sequence difference,engineered into human–horse chimeric -chain has been investigatedto map such a synergistic complementation. Gln20() has littleeffect on the O₂ affinity of HbS, but in human–horse chimeric-chain it reduces the O₂ affinity slightly. In the chimeric-chain, Gln20() increased sensitivity of the ßßcleft for the DPG influence, reflecting a cross-talk betweenthe ₁ß₁ interface and ßß cleft in this semisyntheticchimeric HbS. In the human -chain frame, the polymerizationinhibitory activity of Gln20() is higher compared with horse_1–30, but lower than mouse _1–30. Gln20() synergisticallycomplements the inhibitory propensity of horse _31–141.However, the inhibitory activity of LM–horse chimeric-chain is still lower than that of mouse–horse chimeric-chain. Therefore, perturbation of multiple contact sites inthe _1–30 region of the mouse–horse chimeric -chainand its linkage with the inhibitory propensity of horse _31–141has been now invoked to explain the super-inhibitory activityof the chimeric -chain. The `linkage-map' of contact sites canserve as a blueprint for designing synergistic complementationof multiple contact sites into -chains as a strategy for generatingsuper-inhibitory antisickling hemoglobins for gene therapy ofsickle cell disease.     

Expression of recombinant {alpha}Ains-crystallin and not {alpha}A-crystallin inhibits bacterial growth

A-Crystallin and A^ins-crystallin are derived from the A-crystallingene via alternative splicing. They are identical except forthe presence of a polypeptide, 23 amino acids long, encodedby the ‘insert’ exon. Evolutionary logic would suggestthat the insertion of a 23 amino acid peptide in the middleof A-crystallin, a protein evolving more slowly than eitherhistone H1, cytochrome c or hemoglobin, would lead to appreciablestructural and functional changes. However, based on physico-chemicalstudies, it is presently believed that A-crystallin and A^ins-crystallinare functionally equivalent and that the presence of the ‘insert’peptide in A^Ins-crystallin is inconsequential. We report herethat the independent expression of recombinant A^Ins-crystallin,and not A-crystallin, inhibits growth of the bacterial host.These observations were confirmed in co-expression experiments,wherein both the proteins were expressed in the same cell. Interestingly,growth inhibition is reversible. Importantly, the data demonstratethat it is catalytic amounts and not the gross accumulationof A^Ins-crystalline which causes growth inhibition. Given theprior knowledge that A-crystallin and A^Ins-crystallin differby a peptide of 23 amino acids, these data suggest that the‘insert peptide’ in A^Ins-crystallin imparts propertieson this protein that are different from A-crystallin.     

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

-Toxin is a 26-residue channel-forming peptide from Staphylococcusaureus which forms an amphipathic -helix in a membrane environment.Channel formation in planar bilayers suggests that an averageof six -toxin helices self-assemble to form transbilayer pores.Molecular models for channels formed by -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 -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 -toxin ion channels.     

Production and characterization of anti-human interferon {gamma} receptor antibody fragments that inhibit cytokine binding to the receptor

Three single-chain antibody fragments that recognize the extracellularhuman interferon receptor -chain (IFNR), and inhibit the bindingof human IFN, have been produced in Escherichia coli. Thesefragments are derived from murine anti-receptor monoclonal antibodies,and comprise the variable heavy (V_H) domain linked to the variablelight (V_L) chain through a 15 amino acid linker [(GGGGS)₃].Using surface plasmon resonance technology (BIAcore), the solubleproteins were shown to retain a high affinity for recombinantIFNR, and by radioimmunoassay to possess high inhibitory activitytowards IFN-binding to human Raji cells. The antibody fragmentsmost likely recognize epitopes that overlap the cytokine bindingsite on the receptor surface. Attempts to dissect further theantibodies to isolated V_H- and V_L-chains and to synthetic linearand cyclic peptides derived from the individual complementaritydetermining regions failed to afford fragments with significantIFNR binding affinity. Nevertheless, these native-like variableregion fragments and petidomimetics derived from them are ofinterest in the design of novel IFNR antagonists.     

Structure-function analysis of human IL-1{alpha}: identification of residues required for binding to the human type I IL-1 receptor

Using oligonucleotide-directed mutagenesis, the binding siteon human interleukin-1 (IL-1) for the human type I IL-1 receptor(IL-1R) has been analyzed. Substitution of seven amino acids(Arg12, Ile14, Asp60, Asp61, Ile64, Lys96 and Trp109) resultedin a significant loss of binding to the receptor. Based on crystallographicinformation, the side chains of these residues are clusteredin one region of IL-1 and exposed on the surface of the protein.Five of the residues in the IL-1 binding site align with thebinding residues previously determined in human IL-1ß,demonstrating that the type I IL-1R recognizes homologous regionsin both ligands. Unexpectedly, only three of the aligned residuesare identical between IL-1 and IL-1ß. These observationssuggest that the composition of contact residues in the bindingsite is unique for each ligand–receptor complex in theIL-1 system.     

Structural and functional domains in human tumour necrosis factors

Human tumour necrosis factors (hTNFs) and ß are relatedpleiotropic cytokines which share many activities and competewith each other for binding to two receptor components on manycell types. Although structural and biological data indicatethat the active form of hTNF- may be a symmetrical trimer, themanner in which hTNFs interact with their receptors to triggera myriad of cell type-dependent responses is not clear. A combinationof chemical modification, epitope mapping and site-directedmutagenesis approaches suggest that at least four distinct peptidesequences are Important for the biological activity of hTNF-.In particular, certain peptide sequences between amino acidpositions 11 and 35 in hTNF- appear to be critical for receptorbinding and triggering biological responses. The recent cloningof the two hTNF-/ß receptors opens the way for precisemapping of the functional domains in hTNFs     

Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha

To determine the region of human tumor necrosis factor-alpha(TNF-), essential for cytotoxic activity against mouse L-M cells,single amino-acid-substituted TNF- mutant proteins (muteins)were produced in Escherichia coli by protein engineering techniques.An expression plasmid for TNF- was mutagenized by passage throughan E.coli mutD5 mutator strain and by oligonucleotide-directedmutagenesis. Approximately 100 single amino-acid-substitutedTNF- muteins were produced and assayed for cytotoxic activity.The cytotoxic activities of purified TNF- muteins, e.g. TNF-31T,-32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were < 1%of that of parent TNF-. These results indicate that the integrityof at least four distinct regions of the TNF- molecule is requiredfor full biological activity. These regions are designated asfollows: region I, from position 30 to 32; region II, from position82 to 89; region III, from position 115 to 117; region FV, fromposition 141 to 146. In addition, TNF-141Y could not completelycompete with parent TNF- for binding to the receptor. This demonstratesthat region IV, and at least aspartk acid at position 141, mustbe involved in the TNF receptor binding site.     

A point mutation that decreases the thermal stability of human interferon {gamma}

We have identified a mutation of human gamma-interferon (IFN)causing a temperature-sensitive phenotype. We used a randomizedoligonucleotide to mutagenize a synthetic human IFN gene, thenscreened the resulting mutants produced in Escherichia colifor proteins with altered biological activity. One mutant proteinselected for detailed characterization exhibited < 0.3% ofthe specific biological activity of native IFN in an antiviralactivity assay performed at 37°C. However, the protein boundthe human IFN receptor with native efficiency at 4°C. Sequencingthe plasmid DNA encoding this protein snowed that the mutationchanged the lysine residue at amino acid 43 to glutamic acid(IFN/K43E). Site-specific mutagenesis at amino acid 43 showedthat this protein's phenotype resulted from positioning a negativecharge at position 43. Structural characterization of IFN/K43Eusing CD demonstrated that the protein had native conformationat 25°C, but assumed an altered conformation at 37°C.IFN/K43E in this altered conformation bound poorly to the IFNreceptor at 37°C, providing a rationale for the mutant'sdecreased antiviral activity.     

Comparative stability of dihydrofolate reductase mutants in vitro and in vivo

Dihydrofolate reductase mutants with amino acid replacementsin the active center (Thr35 Asp mutant, Arg57 His mutant andthe mutant with triple replacement Thr35 Asp, Asn37 Ser, Arg57 His) were obtained by site-directed mutagenesis. The stabilizationeffect of trimethoprim and NADP·H on the protein tertiarystructure in vitro has been investigated. In the case of mutantswith a ‘weak’ tertiary structure (Thr35 Asp35 andthe triple mutant) the separate addition of ligands does notaffect their stability. The simultaneous addition of these ligandsto Thr35 Asp35 and the triple mutant leads to the large increasein their stability. A distinct correlation was found betweenthe in vitro studied stability of the mutant proteins to theurea- or heat-induced denaturation and the level of proteolyticdegradation of these mutants previously observed in vivo.     

Site-directed mutagenesis of glutathione synthetase from Escherichia coli B: mapping of the {gamma}-L-glutamyl-L-cysteine-binding site

Lysl8, Arg86, Asn283, Ser286, Thr288 and Glu292 of glutathionesynthetase from Escherichia coli B are presumed to be highlyconcerned with the substrate, -L-glutamyl-L-cysteine (-Glu-Cys),binding by X-ray crystallography and affinity labeling studies.Using site-directed mutagenesis, we investigated functionalroles of those residues for -Glu-Cys binding. The mutant enzymesof Arg86 and Asn283 altered their kinetic parameters, especiallythe Michaelis constants of -Glu-Cys. In the case of Asn283,the residue is not likely to have an essential role in -Glu-Cysbinding but its side chain would extend to make a van der Waalscontact with bound -Glu-Cys. Chemical modification of a cysteineresidue with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) showed Arg86would not only be much responsible for -Glu-Cys binding butwould also have a role in maintaining the structural integrityof the enzyme. The other mutant enzymes showed little defectin their kinetic parameters of -Glu-Cys.     

A Pore-forming protein with a protease-activated trigger

Hemolysin (HL) is a 293 amino acid pore-forming toxin, whichis secreted as a water-soluble monomer by Staphylococcus aureus.By forming a hexameric pore, HL damages the plasma membranesof target cells. Previous studies established that HL proteinswith nicks near the midpoint of a central glycine-rich loopare held together by a domain-domain interaction and are hemolyticallyactive. In contrast, HL proteins comprising two HL truncationmutants that overlap in the central loop have no or greatlyreduced pore-forming activity, even though the two chains againform a tight complex. Based on these findings, overlap mutantshave now been designed that are activated when redundant aminoacids in the loop are removed by proteases. Further, the identityof the activating enzyme can be specified by additional mutagenesisof the protease recognition site in the overlap sequence. Mutantsof aHL that are activated by tumor-associated proteases mightbe useful components of immunotoxins     

The differences in conformation between {alpha}-human atrial natriuretic polypeptide, {alpha}-hANP, and its derivative, Met(O)-{alpha}-hANP, in solution

The differences in conformation between -human atrial natriureticpolypeptide (-hANP) and its inactive analog, Met(O)--hANP, havebeen analyzed by nuclear magnetic resonance spectroscopy. Allproton resonances for both peptides were assigned by means ofthe sequential assignment procedure. The three-dimensional structureof -hANP in solution had previously been determined by distancegeometry calculation using distance constraints derived fromnuclear Overhauser effects (NOEs). Here, the three-dimensionalstructure of Met(O)--hANP was determined. The conformationaldifferences between these two molecules were as follows: threesegments of -hANP, Serl–Cys7, Arg11–Ala17 and Gln18–Tyr28,have some ordered structures. In Met(O)--hANP the Gln18-Tyr28region has a similar conformation, while the remaining two regionsdo not have the ordered structure found in -hANP. It is suggestedthat the conserved conformation of the Gln18–Tyr28 regionis required for binding to the ANP receptor and that the slightbiological activity of Met(O)-a-hANP is due to loss of the orderedstructures evoked in the Serl–Cys7 and Arg11–Ala17regions of -hANP.     

Structure of {alpha}-{alpha}-hairpins with short connections

This paper presents the results of detailed stereochemical analysisof structures and sequences of --hairpins with short connections.It is shown that --hairpins of each given type have very similarpatterns of hydrophobic, hydrophilic and glycine residues intheir amino acid sequences. These results can be used in theprediction of --hairpin conformation as well as in protein designand engineering.     

The thermostability of DNA-binding protein HU from bacilli

The primary and tertiary structures of DNA-binding protein HUfrom Bacillus stearothermophilus are already known. The primarystructure has been previously determined for HU from the closelyrelated B.globigü and the determinations of the sequencesfrom B.caldolyticus and B.subtilis are described here. Thesebacteria have optimum growth temperatures of > 70C (B.caldolyticus),65C (B.stearothermophilus), 37C (B.subtilis) and 30C (B.globigü).in vitro measurements from circular dichroic spectra describedhere give T_m values reflecting these growth temperatures, of68, 64, 43 and 41C respectively. We discuss here the relativethermostability of the four proteins in terms of the amino aciddifferences between the sequences and the three-dimensionalmodel of the B.stearothermophilus HU. The current model forthe interaction of the protein with DNA is only discussed interms of its relevance with regard to thermostability.     

Chemical synthesis of {gamma}-echistatin analogues: elucidation of status of C-terminal (45-49)

Three analogues of -echistatin, des(45–49)--echistatin,des(46–49)-y-echistatin and des(47–49)--echistatin,were synthesized by solid-phase methodology and their biologicalactivities were measured and compared. The results reveal thatwithout the C-terminal (45–49) of -echistatin, the foldingof the protein to the final active structure is not interferedwith and Lys-45 influences the inhibition of platelet aggregation.