Detection of secondary structure elements in proteins by hydrophobic cluster analysis

Hydrophobic cluster analysis (HCA) is a protein sequence comparisonmethod based on -helical representations of the sequences wherethe size, shape and orientation of the clusters of hydrophobicresidues are primarily compared. The effectiveness of HCA hasbeen suggested to originate from its potential ability to focuson the residues forming the hydrophobic core of globular proteins.We have addressed the robustness of the bidimensional representationused for HCA in its ability to detect the regular secondarystructure elements of proteins. Various parameters have beenstudied such as those governing cluster size and limits, thehydrophobic residues constituting the clusters as well as thepotential shift of the cluster positions with respect to theposition of the regular secondary structure elements. The followingresults have been found to support the -helical bidimensionalrepresentation used in HCA: (i) there is a positive correlation(clearly above background noise) between the hydrophobic clustersand the regular secondary structure elements in proteins; (ii)the hydrophobic clusters are centred on the regular secondarystructure elements; (iii) the pitch of the helical representationwhich gives the best correspondence is that of an -helix. Thecorrespondence between hydrophobic clusters and regular secondarystructure elements suggests a way to implement variable gappenalties during the automatic alignment of protein sequences.     

Structure-function relationships in the catalytic and starch binding domains of glucoamylase

Sixteen primary sequences from five sub-families of fungal,yeast and bacterial glucoamylases were related to structuralinformation from the model of the catalytic domain of Aspergillusawamori var. X100 glucoamylase obtained by protein crystallography.This domain is composed of thirteen -belices, with five conservedregions defining the active site. Interactions between methyl-maltoside and active site residues were modelled, and the importanceof these residues on the catalytic action of different glucoamylaseswas shown by their presence in each primary sequence. The overallstructure of the starch binding domain of some fungal glucoamylaseswas determined based on homology to the Cterminal domains ofBacillus cyclodextrin glucosyltransferases. Crystallographyindicated that this domain contains 6–8 ß-strandsand homology allowed the attribution of a disulfide bridge inthe glucoamylase starch binding domain. Glucoamylase residuesThr525, Asn530 and Trp560, homologous to Bacillus stearothermophiluscyclodextrin glucosyltransferase residues binding to maltosein the Cterminal domain, could be involved in raw-starch binding.The structure and length of the linker region between the catalyticand starch binding domains in fungal glucoamylases can varysubstantially, a further indication of the functional independenceof the two domains.     

Reading-frame shift in Saccharomyces glucoamylases restores catalytic base, extends sequence and improves alignment with other glucoamylases

A recent article [Coutinho and Reilly (1994) Protein Engng,7, 749–760] presented the alignment of 14 glucoamylasesby hydrophobic cluster analysis. The catalytic bases of twoof these glucoamylases, from Saccharomyces cerevisiae and Saccharomycesdiastaticus, were not conserved, opening the possibility ofa reading-frame shift error in a segment coding for amino acidsnear the apparent C-termini of the mature proteins. Indeed,an addition of one nucleotide restores the catalytic base, extendsthe sequence by 39 residues and greatly improves the amino acidalignment in this region.     

Demonstration by burst-phase analysis of a robust folding intermediate in the FF domain

The role of intermediates in the folding reaction of single-domainproteins is a controversial issue. It was previously shown bydifferent methods that an on-pathway intermediate is populatedin the presence of sodium sulphate during the folding of theFF domain from HYPA/FBP11. Here we demonstrate using analysisof the amplitudes of kinetic traces that this burst-phase foldingintermediate is present at different salt concentration andat various pH, and is also found in roughly 30 site-directedmutants. The intermediate appears robust to changing conditionsand thus fulfils an important criterion for a productive molecularspecies on the folding reaction pathway.     

Protein engineering of the hydrophobic domain of human factor IX

Vitamin K-dependent plasma proteins contain a highly conservedhydrophobic domain located between the -carboxyglutamic acid(Gla) domain and the first epidermal growth factor (EGF)-likedomain. Here we have used protein engineering of the hydrophobicdomain in human factor IX to investigate its function in intactfactor IX. Mutant proteins were generated by site-directed mutagenesisand in vitro expression in Madin-Darby canine kidney (MDCK)cells. All of our mutants, including one with a deletion ofthe entire hydrophobic domain, were activated by factor XIa,showing that this domain is not required for factor IX activation.The results with the mutant Phe41 Val suggest that the hydrophobicdomain interacts with the adjacent EGF-like domain. Our datafor the Phe41 Asp mutant is consistent with, but cannot prove,a role for this residue in the maintenance of a phospholipid-bindingstructure required for factor IX function.     

Deletion analysis of the starch-binding domain of Aspergillus glucoamylase

The large form of glucoamylase (GAI) from Aspergillus awamori(EC 3.2.1.3 [EC] ) binds strongly to native granular starch, whereasa truncated form (GAII) which lacks 103 C-terminal residues,does not. This C-terminal region, conserved among fungal glucoamylasesand other starch-degrading enzymes, is part of an independentstarch-binding domain (SBD). To investigate the SBD boundariesand the function of conserved residues in two putative substrate-bindingsites, five gluco-amylase mutants were constructed with extensivedeletions in this region for expression in Saccharomyces cerevisiae.Progressive loss of both starch-binding and starch-hydrolyticactivity occurred upon removal of eight and 25 C-terminal aminoacid residues, or 21 and 52 residues close to the N-terminus,confirming the requirement for the entire region in formationof a functional SBD. C-terminal deletions strongly impairedSBD function, suggesting a more important role for one of theputative binding sites. A GAII phenocopy showed a nearly completeloss of starch-binding and starch-hydrolytic activity. The deletionsdid not affect enzyme activity on soluble starch or thermo-stabilityof the enzyme, confirming the independence of the catalyticdomain from the SBD.     

Protein engineering of the relative specificity of glucoamylase from Aspergillus awamori based on sequence similarities between starch-degrading enzymes

Aspergillus glucoamylase catalyzes hydrolysis of D-glucose fromnon-reducing ends of starch with an {small tilde}300-fold {k^JKm) preference for the a-1,4- over the a-l,6-glucosidic linkagedetermined using the substrates maltose and iso-maltose. Itis postulated that as most amylolytic enzymes act on eitherthe a-1,4- or a-l,6-linkages, sequence comparison between active-siteregions should enable the correlation of the substrate bondspecificity with particular residues at key positions. Therefore,the already high bond-type selectivity in Aspergillus glucoamylasecould theoretically be augmented further by three single mutations,Serll9 Tyr, Glyl83 Lys and Serl84 His, in two separate active-siteregions. These mutants all had slight increases in activityas compared with the wild-type enzyme towards the a-l,4-linkedmaltose; this was due to lower Km values as well as small decreasesin activity towards isomaltose. This latter decrease in activitywas a result of higher Km values and a decrease in fc^, forthe Serl84 His mutant As a consequence, the selectivity of thethree glucoamylase mutants for a-1,4- over a-l,6-linked disaccharidesis enhanced 2.3- to 3.5-fold. In addition, the introductionof a cationic side chain in Glyl83 Lys and Serl84 His glucoamylase,broadens the optimal pH range for activity towards acidic aswell as alkaline conditions.     

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.     

Structural characterization by computer experiments of the lipid-free LDL-receptor-binding domain of apolipoprotein E

The structure and dynamics of the lipid-free LDL-receptor-bindingdomain of apolipoprotein E (apoE-RBD) has been investigatedby Molecular Dynamics Simulations. ApoE-RBD in its monomericlipid-free form is a singular four-helix bundle made up of fourelongated amphipathic helices. Analysis of one 1.5 ns moleculardynamics trajectory of apoE-RBD performed in water indicatesthat the lipid-free domain adopts a structure that exhibitscharacteristics found in native proteins: it has very stablehelices and presents a compact structure. Yet its interior exhibitsa larger number of transient atomic-size cavities relative tothat found in other proteins of similar size and its apolarside chains are more mobile. The latter features distinguishthe elongated four-helix bundle as a slightly disordered structure,which shows a structural likeness with some de novo designedfour-helix bundle proteins and shares with the latter a leucine-richresidue composition. We anticipate that these unique propertiescompared with other native helix bundles may be related to thepostulated ability of apoE-RBD to undergo an opening of itsbundle upon interaction with phospholipids. The distributionof empty cavities computed along the trajectory in the interfaceregions between the different pairs of helices reveals thatthe tertiary contacts in one of the interfaces are weaker suggestingthat this particular interface could be more easily rupturedupon lipid association.     

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Fungal glucoamylases contain four conserved regions. One regionfrom the Aspergillus niger enzyme contains three key carboxylicacid residues, the general acid catalytic group, Glu179, alongwith Asp176 and Glu180. Three site-directed mutations, Leu177– His, Trp178 – Arg and Asn182 – Ala, wereconstructed near these acidic groups to reveal the functionof other conserved residues in this region. Leu177 and Trp178are strictly conserved among fungal glucoamylases, while anamide, predominantly Asn, always occurs at position 182. Substitutionsof Leu177 or Trp178 cause significant decreases in k_cat withthe substrates tested. Similar increases in activation energiesobtained with Leu177 – His with both -(1,4)- and -(1,6)-linkedsubstrates indicate Leu177 is located in subsite 1. K_M valuesobtained with the Trp178 – Arg mutation increase for an-(1,6)-linked substrate, but not for -(1,4)-linked substrates.Calculated differences in activation energy between substratesindicate Trp178 interacts specifically with subsite 2. The Asn182 Ala mutation did not change k_cat or K_M values, indicating thatAsn182 is not crucial for activity. These results support amechanism for glucoamylase catalytic activity consisting ofa fast substrate binding step followed by a conformational changeat subsite 1 to stabilize the transition state complex.     

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.     

Identification and elimination by site-directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamori glucoamylase

Both Dative Aspergillus niger glucoamylase and wild-type Aspergillusawamori glucoamylase expressed in Saccharo-myces cerevisiae,which have identical primary structures, undergo hydrolysisat aspartyl bonds at low pH values and elevated temperatures.In native A.niger enzyme the Aspl26–Glyl27 bond was preferentiallycleaved at pH 3.5,while at pH 4.5 cleavage of the Asp257–Pro258and Asp293–Gly294 bonds was dominant. In wild-type A.awamoriglucoamylase, cleavage of the latter was dominant at both pH3.5 and 4.5. Site-directed mutations Aspl26Glu and Glyl27Alain wild-type enzyme decreased specific activities by 60 and30%, respectively, and increased irreversible thermoinactivationrates 3- to 4-fold at pH 4.5. Replacement of Asp257 with Gluand Asp293 with Glu or Gin decreased specific activities by20%, but greatly reduced cleavage of the Asp257–Pro258and Asp293–Gly294 bonds. The Asp257Glu mutant was producedvery slowly and was more thermostable than wild–type glucoamylaseat pH 4.5up to 70°C. Replacement of Asp293 with either Gluor Gln significantly raised protein production and slightlyincreased thermostability at pH 3.5 and 4.5, but not at pH5.     

Synthesis of a squash-type protease inhibitor by gene engineering and effects of replacements of conserved hydrophobic amino acid residues on its inhibitory activity

Cucurbita maxima trypsin inhibitor I (CMTI-I), a member of thesquash-type protease inhibitor family, is composed of 29 aminoacids and shows strong inhibition of trypsin by its compactstructure. To study the structure–function relationshipof this inhibitor using protein engineering methods, we constructedan expression system for CMTI-I as a fused protein with porcineadenylate kinase (ADK). A Met residue was introduced into thejunction of ADK and CMTI-I to cleave the fusion protein withCNBr, whereas a Met at position 8 of authentic CMTI-I was replacedby Leu. Escherichia coli JM109 transformed with the constructedplasmid expressed the fused protein as an inclusion body. Aftercleavage of the expressed protein with CNBr, fully reduced speciesof CMTI-I were purified by reversed-phase HPLC and then oxidizedwith air by shaking. For efficient refolding of CMTI-I, we used50 mM NH₄HCO₃ (pH 7.8) containing 0.1% PEG 6000 at higher proteinconcentration. Strong inhibitory activity toward trypsin wasdetected only in the first of three HPLC peaks. The inhibitorconstant of CMTI-I thus obtained, in which Met8 was replacedby Leu, was 1.4x10-¹⁰ M. The effect of replacement of Met withLeu at position 8 was shown to be small by comparison of theinhibitor constant of authentic CMTI-III bearing Lys at position9 (8.9x10-¹¹ M) with that of its mutant bearing Leu at position8 and Lys at position 9 (1.8x10-¹⁰ M). To investigate the roleof the well conserved hydrophobic residues of CMTI-I in itsinteraction with trypsin, CMTI-I mutants in which one or allof the four hydrophobic residues were replaced by Ala were prepared.The inhibitor constants of these mutants indicated that thosewith single replacements were 5–40 times less effectiveas trypsin inhibitors and that the quadruple mutant was –450times less effective, suggesting that the hydrophobic residuesin CMTI-I contribute to its tight binding with trypsin. However,each mutant was not converted to a temporary inhibitor.     

A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm

We present a novel method that predicts transmembrane domainsin proteins using solely information contained in the sequenceitself. The PRED-TMR algorithm described, refines a standardhydrophobicity analysis with a detection of potential termini(`edges', starts and ends) of transmembrane regions. This allowsone both to discard highly hydrophobic regions not delimitedby clear start and end configurations and to confirm putativetransmembrane segments not distinguishable by their hydrophobiccomposition. The accuracy obtained on a test set of 101 non-homologoustransmembrane proteins with reliable topologies compares wellwith that of other popular existing methods. Only a slight decreasein prediction accuracy was observed when the algorithm was appliedto all transmembrane proteins of the SwissProt database (release35). A WWW server running the PRED-TMR algorithm is availableat http://o2.db.uoa.gr/PRED-TMR/     

The functional importance of Leu15 of human epidermal growth factor in receptor binding and activation

The biological importance of Leu15 of epidermal growth factor(EGF) is suggested by its conservation through evolution, itscritical location in the domain–domain interface of EGFand its close proximity to Arg41, a residue that is crucialfor receptor binding and activation. Mutagenesis of Leu15 ofhuman EGF (hEGF) was employed to examine the role of this residuein the ligand-receptor interaction. The relative receptor affinitiesof the hEGF variants, as determined by radioreceptor competitionassays, varied depending on the amino acid substitution. TheL15F, L15W and L15V hEGF analogues had receptor affinities 45,26 and 18% respectively of wild type hEGF. The L15A and L15Ranalogues displayed receptor affinities of only 2.4 and 1.6%relative to wild type hEGF. No binding of the L15E analoguewas detected. The relative agonist activities, as measured byreceptor tyrosine kinase stimulation assays, generally followeda similar trend. The L15F, L15W and L15V analogues stimulatedthe receptor kinase to a level (V_max) similar to that for wildtype hEGF. A striking difference was observed between the L15Aand L15R variants; although having similar binding affinities,the L15A mutant activated the receptor to only {small tilde}5%of the wild type V_max in contrast to 53% for the L15R mutant¹H-NMR analysis of the L15R and L15A mutants showed only minorstructural alterations that were not sufficient to account forthe dramatic losses in binding and agonist activities. The resultsindicate that both the size and hydrophobicity of the -branchedaliphatic side chain of Leu15 of hEGF are important in the formationof a catalytically active ligand–receptor complex.     

Site-directed and deletion mutational analysis of the receptor binding domain of the interleukin-6 receptor targeted fusion toxin DAB389-IL-6

We have used site-directed and in-frame deletion mutationalanalysis in order to explore the structural features of theIL–6 portion of the diphtheria toxin-related interleukin–6(IL–6) fusion toxin DAB₃₈₉-IL–6 that are essentialfor receptorbinding and subsequent inhibition of protein synthesisin target cells. Deletion of the first 14 amino acids of theIL–6 component of the fusion toxin did not alter eitherreceptor binding affinity or cytotoxk potency. In contrast,both receptor binding and cytotoxic activity were abolishedwhen the C–terminal 30 amino acids of the fusion toxinwere deleted. In addition, we explored the relative role ofthe disulfide bridges within the IL–6 portion of DAB₃₈₉-IL–6in the stabilization of structure required for receptor-binding.The analysis of mutants in which the substitution of eitherCys⁴⁴⁰, Cys⁴⁴⁶, Cys⁴⁶⁹ or Cys⁴⁷⁹ to Ser respectively, demonstratesthat only the disulfide bridge between Cys⁴⁶⁹ and Cys⁴⁷⁹ isrequired to maintain a functional receptor binding domain. Inaddition, the internal in-frame deletion of residues 435–451,which includes Cys⁴⁴⁰ and Cys⁴⁴⁶, was found to reduce, but notabolish receptor binding affinity. These results further demonstratethat the disulfide bridge between Cys⁴⁴⁰ and Cys⁴⁴⁶ is not essentialfor receptor-binding. However, the reduced cytotoxic potencyof DAB₃₈₉-IL6(435–451) suggests that the conformationand/or receptor binding sites associated with this region ofthe fusion toxin is/are important for maintaining the wild typereceptor binding affinity and cytotoxic potency.     

Generation of a Ni(II) binding site by introduction of a histidine cluster in the structure of human glutathione transferase Al-1

Two mutant forms of human glutathione transferase (GST) Al–1with affinity for metal ions were constructed by introductionof His residues by site–directed mutagenesis. A mutant,2–His, contained the mutations Lys84Gln, Asp85His andGlu88His, and another, 5–His, contained the mutationsTyr79His, Asn80His, Lys84His, Asp85His and Glu88HLs. The mutantproteins were obtained in good yields (40–150 mg per 3I culture) by heterologous expression in Escherichia coli. Themutant enzymes possessed novel binding affinities for Ni(II)and Zn(II) ions, as demonstrated by immobilized metal ion affinitychromatography. The mutant with two novel His residues (2–Hismutant) did not bind as tightly to immobilized Nifll) as didthe mutant with five novel His residues (5–His mutant).When tested for affinity to immobilized Zn(II), only the 5–Hismutant remained bound to the column. The affinity of the 5–Hismutant for Ni(II) ions in solution was determined by bindingexperiments in an aqueous polymeric two–phase system.Analysis of the binding curve showed two binding sites per enzymesubunit and a dissociation constant of 6.7 1 . 6 M. The kineticconstants k_cat, K_m and k_cat/k_m for the reaction with glutathioneand l–chloro–2,4–dinitrobenzene were determinedby steady–state kinetic analysis and the parameter valuesfor the mutant forms were found to be indistinguishable fromthose obtained for the wild–type GST Al–1. The differencesin surface charge in the mutant proteins as compared with thewild–type enzyme did not alter the pH dependence of k_cat.The results provide an alternative method for purification offully active recombinant GST Al–1 by the introductionof novel metal binding sites. The data also showed that twoHis residues are sufficient for Ni(II) binding.     

A study of structural determinants in the interleukin-1 fold

The structures of interleukin-1ß, basic fibroblastgrowth factor and Erythrina trypsin inhibitor have been analysedin order to determine whether the hydrophobic core remains conserved,even when the structures have extremely low sequence similarities.We find that there are significant differences in the way eachprotein achieves a satisfactory arrangement of core residuesand that positions which contribute to the core of one structureare not guaranteed to contribute to the integrity of another.Furthermore, the side-chain packing arrangements of these coreresidues vary significantly between the three structures. Duringthis analysis the side-chain rotamers for three independentlydetermined interleukin-1ß structures were also compared.It was found that although buried residues are generally inagreement the remaining residues frequently occupy differentrotamers in the three structures. This suggests that althoughmeaningful studies are possible for buried side-chains the resultsobtained from equivalent analyses of accessible residues shouldbe treated with caution. These results are discussed with specificreference to the optimization of side-chain packing in proteinsof known structure.     

Bipartite organization of the Bacillus subtilis endo-{beta}-1,4-glucanase revealed by C-terminal mutations

The C-terminal boundary of primary sequence of the Bacillussubtilis PAP115 endo-ß-1,4-glucanase (EG) requiredfor stable catalytic activity has been mapped by site-directedmutagenesis using Escherichia coli as host. The 52 kDa cel geneproduct, EG₄₇₀ and a 33 kDa mutant (EG₃₀₀), lacking 170 residuesthrough a nonsense mutation at the leucine-330 codon of thegene, exhibited similar patterns of enzymatic activity and pHoptima using cellooligopentaose as substrate.CD spectra indicatedthat the bulk of the -helical secondary structure in EG₄₇₀ wascontained within EG₃₀₀. However, relative to EG₄₇₀, the specificactivity of EG₃₀₀ was 3- to 4-fold lower with amorphous celluloseas substrate and {small tilde}4-to5-fold higher with carboxymethylcellulose(soluble cellulose).These results along with data which showthat EG₄₇₀ binding capacity to mirocrystalline cellulose is{small tilde} 11 times more than that of EG₃₀₀, demonstratethe importance of residues 330–499 for non-catalytic bindingof cellulose. A construct of the cel gene carrying a deletionof codons 330–499 and an insertion of a nonsense codonat leucine-330, was further used to make mutants EG₂₉₆ and EG₂₉₁with nonsense codon substitutions at arginine and serine-321,respectively.Western analysis using EG-specific antiserum revealedthat relative losses in enzymatic activity of EG₂₉₆ (50%) andEG₂₉₁ (95%) could be accounted for by the extent of their proteolysis,signifying a marked destabilization of these enzymes by removalof only a few amino acids.