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.     

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.     

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.     

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.     

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.     

Hyperthermostable mutants of Bacillus licheniformis {alpha}-amylase: multiple amino acid replacements and molecular modelling

We have identified previously two critical positions for thethermostability of the highly thermostable -amylase from Bacilluslicheniformis. We have now introduced all 19 possible aminoacid residues to these two positions, His 133 and Ala209. Themost favourable substitutions were to Ile and Val, respectively,which both increased the half-life of the enzyme at 80°Cby a factor of 3. At both positions a stabilizing effect ofhydrophobic residues was observed, although only in the caseof position 133 could a clear correlation be drawn between thehydrophobicity of the inserted amino acid and the gain in proteinstability. The construction of double mutants showed a cumulativeeffect of the most favourable and/or deleterious substitutions.Computer modelling was used to generate a 3-D structure of thewild-type protein and to model substitutions at position 209,which lies in the conserved (/ß)₈ barrel domain of-amylase; Ala209 would be located at the beginning of the thirdhelix of the barrel, in the bottom of a small cavity facingthe fourth helix. The model suggests that replacement by, forexample, a valine could fill this cavity and therefore increaseintra- and interhelical compactness and hydrophobic interactions.     

The use of alanine scanning mutagenesis to determine the role of the N-terminus of the regulatory chain in the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase

The location of the first seven residues of the regulatory chainof Escherichia coli aspartate transcarbamoylase has been identifiedby X-ray crystallography to be near the binding site of theregulatory nucleotides. In order to determine the function ofthe N-terminus of the regulatory chain of aspartate transcarbamoylasein heterotropic regulation, alanine scanning mutagenesis wasused. Specifically, Thr2r, His3r, Asp4r, Asn5r, Lys6r and Leu7rwere each replaced with alanine. Analyses of these mutant enzymesindicate that none of these substitutions significantly alterthe catalytic properties of the enzyme. However, three of themutant enzymes, Asp4r Ala, Lys6r Ala and Leu7r Ala, exhibitednotable changes in their response to the regulatory nucleotides,while mutations at Thr2r, His3r and Asn5r exhibited only minorchanges in their heterotropic responses. For the Asp4r Alaenzyme, the responses to ATP and CTP were reduced 30 and 40%respectively, compared with the wild-type enzyme. For the Lys6r Ala enzyme, the response to ATP was reduced 70%, while theCTP response was reduced 50%. In the case of the Leu7r Alaenzyme, a 30 and 20% reduction in response to ATP and CTP respectively,was observed. The synergistk inhibition by UTP in the presenceof CTP for the Lys6r Ala enzyme was reduced 40% compared withthat of the wild type enzyme. For the Leu7r Ala enzyme, thesynergistic inhibition was abolished. In addition, UTP decreasedthe CTP binding affinity of the Leu7r Ala enzyme. Analysisof the kinetic data from these mutant enzymes suggests thatresidues Thr2r, His3r and Asn5r have little effect on the heterotropicmechanism, while residues Asp4r, Lys6r and Leu7r play a moresignificant role in the heterotropic response of the enzymetoward the nucleotides. Furthermore, residue Leu7r appears tobe directly involved in the mechanism for synergistic inhibitionof aspartate transcarbamoylase. In this study alanine scanningmutagenesis has provided a rapid method of identifying thoseresidues in the N-terminal region of the regulatory chain ofaspartate transcarbamoylase important for heterotropic regulation.     

Synthesis and characterization of a recombinant fragment of human {alpha}-fetoprotein with antigenic selectivity versus albumin

A DNA sequence coding for human -fetoprotein amino acid sequence38–119 was synthesized and cloned in a bacterial expressionvector. The -fetoprotein sequence was selected as the leasthomologous to albumin, since the two proteins have an overallamino acid identity of %. A chimeric protein was obtained whichwas purified by preparative electrophoresis and characterizedin its primary structure by fast atom bombardment mass spectrometry.About 70% of the -fetoprotein sequence was physically mappedand found to correspond to the amino acids encoded in the syntheticgene. The use of this recombinant protein allowed the selectionof monoclonal antibodies recognizing both the recombinant fragmentand native -fetoprotein. These antibodies should allow the developmentof an immunoassay for -fetoprotein with absolute selectivityversus albumin. This might result in more sensitive clinicaldeterminations, avoiding the possibility of cross-reactions.     

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     

Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor {alpha}

A gene encoding human tumour necrosis factor (TNF-) has beenchemically synthesized, cloned and expressed to yield a biologicallyactive protein in Escherichia coli. The 480-bp gene was assembledby enzymic ligation of 32 oligonucleotides, cloned directlyinto M13mp18 for sequence verification and expressed in thebroad host range high-level expression vector pMMB66EHST. Expressedrecombinant TNF- was shown to have the correct molecular weight,processed N-terminal sequence, antibody cross-reactivity andtumour cell killing activity. The expression product of thesynthetic gene has been purified to homogeneity by a two-stepion-exchange procedure and the purified material shown to beactive.     

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.     

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     

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.     

Stability, activity and flexibility in {alpha}-lactalbumin

-Lactalbumins and the type-c lysozymes are homologues with similarfolds that differ in function and stability. To determine ifthe lower stability of -lactalbumin results from specific substitutionsrequired for its adaptation to a new function, the effects oflysozyme-based and other substitutions on thermal stabilitywere determined. Unblocking the upper cleft in -lactalbuminby replacing Tyr103 with Ala, perturbs stability and structurebut Pro, which also generates an open cleft, is compatible withnormal structure and activity. These effects appear to reflectalternative enthalpic and entropic forms of structural stabilizationby Tyr and Pro. Of 23 mutations, only three, which involve substitutionsfor residues in flexible substructures adjacent to the functionalsite, increase stability. Two are lysozyme-based substitutionsfor Leu110, a component of a region with alternative helix andloop conformations, and one is Asn for Lys114, a residue whosemicroenvironment changes when -lactalbumin interacts with itstarget enzyme. While all substitutions for Leu110 perturb activity,a Lys114 to Asn mutation increases T_m by more than 10°Cand reduces activity, but two other destabilizing substitutionsdo not affect activity. It is proposed that increased stabilityand reduced activity in Lys114Asn result from reduced flexibilityin the functional site of -lactalbumin.     

Increasing the thermostability of a neutral protease by replacing positively charged amino acids in the N-terminal turn of {alpha}-helices

The 247–260 and 289–299 -helices of Bacillus subtilisneutral protease have a lysine in their N-terminal turn. Theselysines were replaced by Ser or Asp in order to improve electrostaticinteractions with the -helix dipole. After replacing Lys bySer at positions 249 or 290, the thermostability of the enzymewas increased by 0.3 and 1.0°C, respectively. The Asp249and Asp290 mutants exhibited a stabilization of 0.6 and 1.2°C,respectively. The results show the feasibility of stabilizingenzymes by introducing favourable residues at the end of -helices.     

Activation of the low oxygen affinity-inducing potential of the Asn108({beta}){->}Lys mutation of Hb-Presbyterian on intramolecular {{alpha}}{{alpha}}-fumaryl cross-bridging

The Asn108ßLys mutation in hemoglobin (HbPresbyterianmutation) endows a low O₂ affinity-inducing propensity to theprotein. Introduction of a fumaryl cross-bridge between itstwo 99 lysine residues also induces a low O₂ affinity into HbA.We have now engineered an -fumaryl cross-bridge into Hb-Presbyterianto determine the synergy or additivity, if any, that can beachieved between these two low O₂ affinity-inducing structuralperturbations. Despite the presence of the additional -aminogroup of Lys108(ß) within the central cavity, the-amino group of Lys99() of deoxy Hb-Presbyterian retained highselectivity for -fumaryl cross-bridging, with an overall efficiencycomparable to that with HbA. The -fumaryl cross-linking of Hb-Presbyterianreduced its O₂ affinity much more significantly than that observedwith HbA, indicating a synergy between the two low O₂ affinity-inducingstructural perturbations. Apparently, the -fumaryl cross-bridgein Hb-Presbyterian activates part of the latent low O₂ affinity-inducingpotential of Lys108(ß) that is generally activatedin the presence of chloride. The synergy between the Asn108(ß)Lysmutation and the -fumaryl cross-bridging was conserved in thepresence of chloride, but not in the presence of DPG. Furthermore,in the presence of chloride and DPG, -fumaryl Hb-Presbyterianaccessed a low O₂ affinity T-state that is accessed by HbA,-HbA and Hb-Presbyterian only in the presence of IHP. Isoelectricfocusing analysis suggested that the -fumaryl cross-linkingof Hb-Presbyterian induces changes in the ionization behaviorof one or more of the functional groups neighboring Lys99()and Lys108(ß) [presumably His103() and/or Glu101(ß)]to compensate for the extra positive charge of Lys108(ß).Molecular modeling studies identified two potential chloridebinding sites per ß dimer within the middle of thecentral cavity of -fumaryl HbA involving residues His103(),Arg104(ß) and Asn108(ß). The affinity ofthese sites is increased in -fumaryl Hb-Presbyterian as a resultof the Asn108(ß)Lys mutation. Thus, the results ofthe present study suggest that the enhanced neutralization ofthe positive charges in the middle of the central cavity ofHb achieved by these two electrostatic modifications, one (the-fumaryl cross-bridge) acting directly and the other (the Presbyterianmutation) acting indirectly through the mediation of chlorideion binding, facilitates the - fumaryl-Hb Presbyterian to accessa low O₂ affinity T-state structure much more readily than eitherHb-Presbyterian or -fumaryl HbA.     

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.     

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.