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.     

Cloning of rabbit interleukin-1{beta}: differential evolution of IL-1{alpha} and IL-1{beta} proteins

We have cloned the rabbit IL-1ß cDNA, which encodesa 268 amino acid precursor similar in length to other sequencedIL-1 precursors. Comparison of all published IL-1 and IL-1ßsequences respectively indicates that the IL-1 gene family isevolving faster than the IL-1ß family, and that thetwo genes diverged –270 million years ago. Surprisingly,there are differences in the regions preferentially conservedwithin the two families. The IL-1 family is most conserved atthe amino terminus whereas the IL-1ß family is mostconserved in the carboxy-terminal half. This is despite thefact that the carboxy-terminal half encodes the active portionof both molecules and would be expected to adopt a similar ß-sheetstructure in IL-1 as in the published X-ray structure of matureIL-1ß. These findings suggest that differences inthe function and properties of the IL-1 and IL-1ßprecursor molecules may have been conserved. These differencesmay therefore provide an explanation for the existence of twoIL-1 molecules.     

An 8-fold {beta}{alpha} barrel protein with redundant folding possibilities

Protein sequences containing redundant segments of secondarystructure at both termini have the choice a priori of foldinginto several possible circularly permuted variants of the wild-typetertiary structure. To test this hypothesis the gene of phosphoribosylanthranilate isomerase from yeast, which is a single-domain8-fold ß barrel protein, was modified to produce a10-fold ß homologue in Escherichia coli. It containeda duplicate of the two C-terminal ß units of supersecondarystructure fused to its N-terminus. Most of the protein was recoveredfrom the insoluble fraction of disrupted cells by dissolutionin guanidinium chloride solutions and refolding. Pristine proteinwas purified from the soluble fraction. The purified (ß)₁₀proteins were enzymically almost fully active. Absorbance, fluorescenceand circular dichroism spectra as well as the reversible unfoldingbehaviour of both proteins were also very similar to the propertiesof the original (ß)₈ protein. Digestion with endopeptidasesconverted both the pristine and the refolded (ß)₁₀variant to the same large fragment that had the N-terminal sequenceand mol. wt of the wild-type ß)₈ protein. The datasuggest that the folding of the (ß)₁₀ variant is controlledthermodynamically both in vivo and in vitro.     

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)-unit inE.coli triosephosphate isomerase (TIM), a typical (ß)-barrelprotein, assuming that the pseudosymmetrical ß-barrelcan be divided into eight successive loop/ß-strand/loop/-helixmotifs. We replaced the eighth (ß)-unit of E.coliTIM with the corresponding chicken (ß)-unit. The substitution,involving the replacement of 10 of the 23 residues of this (ß)-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.     

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.     

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 (/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 (/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 -amylase numbering), was used asthe sequence-structural template. The isolated sequence stretchesof the 12 (/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(/ß)₈-barrel     

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.     

Recombinant-derived interleukin-1{alpha} stabilized against specific deamidation

Recombinant-derived human interleukln-1 (IL-1), purified fromEscherichia coli, was resolved by isoelectric focusing on polyacrylamidegels into two species of isoelectric points (pI) 5.45 and 5.20,which constituted 75% and 25% of the total IL-1 protein respectively.The pI 5.45 and pI 5.20 species were separated by chromatofocusingand subjected to N-terminal sequence analysis. The pI 5.45 speciescontained the expected Asn residue at position 36 of the matureprotein sequence whereas the pI 5.20 species contained an Aspresidue at the same position. A mutant protein in which Asn-36was substituted for a Ser residue was isolated from E.coli andshown to be homogeneous on isoelectric focusing analysis witha pI = 5.45. ¹H-n.m.r. and circular dichroism analyses of wild-typeand the mutant IL-1 indicated a similar conformation which wasalso indicated by the identical receptor binding affinitiesof IL-1 with Asn, Asp or Ser in position 36. The mutant proteinwas stabilized against specific base catalysed and temperature-induceddeamidation, and may be more suitable than the wild-type positionfor physical and structural studies.     

Independent self-assembly of cadmium-binding {alpha}-fragment of metallothionein in Escherichia coli without participation of {beta}-fragment

We examined the independent self-assembly of the - and ß-fragmentsof human metallothionein (MT) into cadmiumbinding conformationin an Escherichia coli expression system, in addition to wild-typeMT expression. The expressed -fragment formed independentlythe structure of a metal-binding cluster without the aid ofthe ß-fragment. The -fragment and wild-type MT expressedin E.coli were purified and analyzed for their biochemical andspectroscopic properties. The apparent cadmium binding of the-fragment was approximately 12-fold greater than that for thewild-type MT, whereas in other respects the studied biochemicalproperties were similar. In contrast, we were unable to obtainany independently expressed ß-fragment as the cadmium-bindingform in this study. Possible explanations for this phenomenonare discussed.     

Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the {alpha}/{beta}-barrel Proteins

We have attempted to construct an artificial polypeptide thatfolds like the eight-stranded parallel ß-barrel structures.Our approach consists of repeating eight times a unit peptidedesigned to adopt a ‘ß-strand/-helix’pattern. A first ‘test’ sequence for this structuralunit was deduced from a series of parameters defined after ananalysis of three natural /ß-barrel proteins and includingprincipally the lengths of the secondary structure elements,the /ß packing and the fitting on average Garnierprofiles. The gene encoding this structural unit was synthesized,cloned and expressed in Escherichia coli either as a monomeror as direct repeats of 2–12 units. Preliminary structuralcharacterization of the 7-, 8- and 9-fold unit polypeptidesby circular dichroism measurements indicates the presence ofthe predicted amount of -helix in the three proteins. Furtheranalysis by urea-gradient gel electrophoresis demonstrates that,in the conditions tested, only the 8-fold unit polypeptide formsa compact structure through a cooperative and rapid two-statefolding transition involving long-range molecular interactions.     

Altered specificities of genetically engineered {alpha}1 antitrypsin variants

Seven active site variants of human ₁-antitrypsin (₁AT) wereproduced in Escherichia coli following site-specific mutagenesisof the ₁AT complementary DNA. ₁AT (Ala ³⁵⁸), ₁AT (Ile³⁵⁸ and₁AT (Val³⁵⁸), were efficient inhibitors of both neutrophil andpancreatic elastases, but not of cathepsin G. ₁AT (Ala³⁵⁸, Val³⁵⁸)and ₁AT (Phe³⁵⁸ specifically inhibited pancreatic elastase andcathepsin G respectively. The most potent inhibitor of neutrophilelastase was ₁AT (Leu³⁵⁸), which also proved to be effectiveagainst cathepsin G. The ₁AT (Arg³⁵⁸) variant inactivated thrombinwith kinetics similar to antithrombin III in the presence ofheparin. Electrophoretic analysis showed that SDS-stable highmol. wt complexes were formed between the mutant inhibitorsand the cognate proteases in each case. These data indicatethat effective inhibition occurs when the ₁AT P1 residue (position358) corresponds to the primary specificity of the target protease.Moreover, alteration of the P3 residue (position 356) can furthermodify the reactivity of the inhibitor. Two of the variantshave therapeutic potential: ₁AT (Leu³⁵⁸ may be more useful thanplasma ₁AT in the treatment of destructive lung disorders and₁ (Arg³⁵⁸ could be effective in the control of thrombosis.     

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.     

Model building of disulfide bonds in proteins with known three-dimensional structure

As an aid in the selection of sites in a protein where a disulfidebond might be engineered, a computer program has been developed.The algorithm starts with the generation of Cß positionsfrom the N, C and C atom coordinates available from a three-dimensionalmodel. A first set of residue pairs that might form a disulfidebond is selected on the basis of Cß–Cßdistances between residues. Then, for each residue in this set,S positions are generated, which satisfy the requirement that,with ideal values for the C–Cß and Cß–Sbond lengths and for the bond angle at Cß, the distancebetween S of residue 1 and Cß of residue 2 in a pair(determined by the bond angle at S2) is at, or very close toits ideal value. Usually two acceptable S positions are foundfor each half cystine, resulting in up to four different conformationsfor the disulfide bond. Finally, these conformations are subjectedto an energy minimization procedure to remove large deviationsfrom ideal geometry and their final energies are calculated.User input determines which final conformations are energeticallyacceptable. These conformations are written to a file to allowfurther analysis and e.g. inspection on a computer graphicsdevice.     

Study of cysteine residues in the {alpha} subunit of Escherichia coli tryptophan synthase. 2. Role in enzymatic function

To understand the functional roles of Cys residues in the subunitof tryptophan synthase from Escherichia coli, single mutantsof the subunit, in which each of the three Cys residues wassubstituted with Ser, Gly, Ala or Val, were constructed by site-directedmutagenesis. The effects of the substitutions on the functionof tryptophan synthase were investigated by activity measurements,calorimetric measurements of association with the ßsubunit and steadystate kinetic analysis of catalysis. Althoughthe three Cys residues are located away from the apparentlyimportant parts for enzymatic activity, substitutions at position81 by Ser, Ala or Val caused decreases in the intrinsic activityof the subunit. Furthermore, Cys81Ser and Cys81Val reducedstimulation activities in the and ß reactions dueto formation of a complex with the ß subunit. Thelower stimulation activities of the mutant proteins were notcorrelated with their abilities to associate with the ßsubunit but were correlated with decreases in k_cat. The presentresults suggest that position 81 plays an indirectly importantrole in the activity of the subunit itself and the mutual activationmechanism of the complex.     

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     

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.     

Semi-empirical simulation of Zn/Cd binding site preference in the metal binding domains of mammalian metallothionein

Metallothionein, a two-domain protein, naturally binds sevengram atoms of divalent ions such as Zn and Cd. Four of the metals(Ml, M5, M6 and M7) are found in the -domain and three (M2,M3 and M4) in the ß-domain. Previous studies haveshown that metals in the -domain are more readily exchangeable,and the level of avidity is site specific. By semi-empiricalMNDO modified neglect of diatomic overlap calculations, we foundthe tendency of binding energy for Cd to be M3 > M2 >M4 in the ß-cluster and M5 > M7 > Ml, M6 inthe -cluster. Thus, the replacement of Zn by Cd can be expectedto follow the order M4 M2 M3 in the ß-domain andMS M7 M1 or M6 in the -domain. This is reflected by energydifferences computed with a series of simulated structures derivedfrom either X-ray crystallography or NMR coordinates.     

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.     

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.     

Molecular model-building of amylin and {alpha}-calcitonin gene-related polypeptide hormones using a combination of knowledge sources

Amylin is the major component of the amyloid found in the pancreasesof noninsulin-dependent diabetics (type 2 diabetes). It is a37 amino acid polypeptide and has been shown to have 46% sequenceidentity with the neuropeptide -calcitonin gene-related peptide(-CGRP). Both amylin and -CGRP are known to be potent inhibitorsof glycogen synthesis in stripped rat soleus muscle. Secondarystructure prediction and tertiary structure model-building showthe two polypeptides to have an -helix/ß-strand motifsimilar to that observed in the insulin B-chain. The resultshave been supported by CD spectroscopy, although there is nosequence similarity between insulin and amylin/-CGRP. Aggregationstates have been predicted based on the dimeric and hexamericarrangements seen in porcine insulin. Rat and hamster amylinhave a changed sequence motif in the ß-strand whichresults in lack of amyloid formation and type 2 diabetes. This,we propose, is caused by disruption of hydrogen bonding whichprevents the formation of the dimer.