Efficient expression and characterization of isolated structural domains of yeast phosphoglycerate kinase generated by site-directed mutagenesis

The two domains of yeast phosphoglycerate kinase were producedby recombinant techniques. The N-domain was obtained by theintroduction of a termination codon at the position coding forPhe185, and the C-domain by a deletion in the gene of the codingsequence between Serl and Leu186. Both domains were efficientlyexpressed in yeast, the level for the C-domain being greaterthan that for the N-domain. Both domains were found to havea quasi-native structure; the C-domain retained its abilityto bind nucleotides. Small local differences were detected indomain structure compared to that in the whole enzyme, probablydue to the lack of interdomain stabilizing interactions. Nevertheless,such an approach provides direct evidence for independent foldingof domains in a two-domain protein.     

Nuclear magnetic resonance studies of isolated structural domains of yeast phosphoglycerate kinase

The structural integrity and substrate binding properties ofthe two genetically engineered domains of yeast phosphoglyceratekinase were investigated using one- and two-dimensional nuclearmagnetic resonance techniques. Both domains were found to foldwith regions of native-like structure, with the N-domain showinggreater conformational flexibility than the C-domain. The ‘basicpatch’ region of the N-domain is, however, clearly perturbedby removal of the C-domain. This is most likely due to the absenceof stabilizing interactions between the C-terminal peptide (including-helices XIII and XIV) and the N-domain. The C-domain is ableto bind nucleotide with an affinity only three times less thanthat of the native protein.     

Expression of active domains of a human folate-dependent trifunctional enzyme in Escherichia coli

The cDNA encoding the human trifunctional enzyme methylenetetrahydrofolatedehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolatesynthetase was engineered to contain a prokaryotic ribosomebinding site and was expressed under the bacteriophage T7 RNApolymerase promoter in Escherichia coli. Site-directed mutagenesiswas used to prepare constructs that encode separately the dehydrogenase/cyclohydrolase(D/C) domain as amino acid residues 1–301, and the synthetase(Syn) domain as residues 304–935. Both domains formedactive enzymes thereby demonstrating their ability to fold independently.The full-length enzyme, D/C and Syn domains were expressed atlevels 4-, 55- and 3-fold higher than the specific activitiesfound in liver. Additional mutagenesis and independent expressionof domains further defined the interdomain region to includeamino acids 292–310. The D/C domain was purified to homogeneityby a single affinity chromatographic step, and the full-lengthprotein in a twostep procedure. The kinetic properties of theD/C domain appear unaltered from those of the trifunctionalenzyme.     

The SBASE domain library: a collection of annotated protein segments

SBASE is a database of annotated protein domain sequences representingvarious structural, functional, ligand binding and topogenicsegments of proteins. The current release of SBASE contains27 211 entries which are provided with standardized names inorder to facilitate retrieval. SBASE is cross-referenced tothe major protein and nucleic acid databanks as well as to thePROSITE catalog of protein sequence patterns [Bairoch, A. (1992)Nucleic Acids Res., 20, Suppl., 2013–2118]. SBASE canbe used to establish domain homologies through database searchusing programs such as FASTA [Lipman and Pearson (1985) Science,227, 1436–1441], FASTDB [Brutlag et al. (1990) Comp. Appl.Biosci., 6, 237–245] or BLAST3 [Altschul and Lipman (1990)Proc. Natl. Acad. Sci. USA, 87, 5509–5513], which is especiallyuseful in the case of loosely defined domain types for whichefficient consensus patterns cannot be established. The useof SBASE is illustrated on the DNA binding protein Brain-4.The database and a set of search and retrieval tools are freelyavailable on request to the authors or by anonymous ‘ftp’file transfer from <ftp.icgeb.trieste.it>.     

An investigation of protein subunit and domain interfaces

Protein structures were collected from the Brookhaven Databaseof tertiary architectures that displayed oligomeric association(24 molecules) or whose polypeptide folding revealed domains(34 proteins). The subunit and domain interfaces for these proteinswere respectively examined from the following aspects: percentagewater-accessible surface area buried by the respective associations,surface compositions and physical characteristics of the residuesinvolved in the subunit and domain contacts, secondary structuralstate of the interface amino acids, preferred polar and non-polarinteractions, spatial distribution of polar and non-polar residueson the interface surface, same residue interactions in the oligomeric:contacts, and overall cross-section and shape of the contactsurfaces. A general, consistent picture emerged for both thedomain and subunit interfaces.     

Ligand requirements for Ca2+ binding to EGF-like domains

Site-specific mutagenesis studies of the first epidermal growthfactor-like (EGF-like) domain of human clotting factor IX suggestthat the calcium-binding site present in this domain (dissociationconstant K_d=1.8 mM at pH 7.5 and ionic strength I=0.15) involvedthe carboxylate residues Asp47, Asp49 and Asp64. To furthercharacterize the ligands required for calcium binding to EGF-likedomains, two new mutations, Asp47 - Asn and Asp49 - Asn, wereintroduced into the domain by peptide synthesis. ¹H-NMR spectroscopywas used to obtain the dissociation constants for calcium bindingto these mutations. Calcium binding to the Asp49- Asn modifieddomain is only mildly affected (K_d=6 mM, I=0.15), whereas bindingto the Asp47- Asn modified domain is severely reduced (K_d=42mM, I=0.15). From these data, it is proposed that the anionicoxygen atoms of the side chains of residues 47 and 64 are essentialfor calcium binding, whereas the side chain ligand for calciumat residue 49 can be a carboxyamide oxygen. As a control, theintroduction of the modification Glu78- Asp in a region of thedomain not believed to be involved in calcium binding had verylittle effect on the K_d for calcium (K_d=2.6 mM, I=0.15). Finally,the effect of an Asp47- Gly substitution found in the naturalhaemophilia B mutant, factor IX_Alabama, was investigated. Thispeptide has a markedly reduced affinity for calcium (K_d=37 mM,I=0.15), suggesting that the defect in factor IX_Alabama is dueto impaired calcium binding to its first EGF-like domain.     

Grafting of discontinuous sites: a protein modeling strategy

A strategy for modeling continuous as well as discontinuoussites in protein structures has been developed. Central to thismodeling strategy is the search algorithm of FITSITE, a programto search a given target structure for suitable combinationsof backbone positions mirroring as closely as possible the geometricrelationships of a source structural motif of interest. Alltarget sites detected by FITSITE are further refined to mimicthe source geometry. The side-chain rotamer library conceptfails to precisely describe side chains involved in coordinativebonding (e.g. metal binding sites). Therefore an algorithm usingdetailed database bonding parameter information was appliedfor the side-chain construction. The FITSITE program and thesubsequent processing of the program output are presented ina test case. The Rop protein, a four-helix bundle structure,served as the target protein. It was searched for candidatesites to model a variety of metal binding sites, with structuresextracted from Brookhaven Protein Database entries. The preliminaryprotein models were investigated for structural overlaps withneighboring residues by interactive computer graphics; if required,additional changes were performed. A set of parameters for energyminimization with AMBER (including metal ions) was developed,and the completed Rop variants were energy minimized. Finally,12 potentially metal binding Rop variants were selected forproduction via genetic engineering.     

Internalization and translocation of a new chimeric protein composed of Pseudomonas aeruginosa exotoxin A and mouse dihydrofolate reductase as a model system

In an attempt to introduce a large peptide that is not normallytranslocated across membranes into the cytosol of eukaryoticcells, we created a new chimeric protein termed CEDH betweenPseudomonas aeruginosa exotoxin A (ETA) and a variant enzymeof Mus musculus dihydrofolate reductase (DHFR) with reducedaffinity for antifolates, ETA^1–413.DHFR^1–187.ETA^609–613.We have defined, genetically constructed and expressed the chimericprotein in Escherichia coli. We showed that the CEDH chimericprotein, purified to homogeneity on an immunoaffinity resin,confers a methotrexate-resistant phenotype to Chinese hamsterovary cells. Furthermore, the chimeric protein allowed the growthof dihydrofolate reductase-deficient Chinese hamster ovary cellsin the absence of hypoxanthine and thymidine. These resultsdemonstrated that the chimeric protein exhibited enzyme activityand possessed the tightly folded native structure, and thatthe DHFR protein can be selectively internalized and translocatedvia domains of exotoxin A. These data show that the ETA systemis an efficient system for the delivery of a variety of largepolypeptides into the cytosol without stress to the target cells,and extends the use of this delivery system to proteins thatare not normally translocated across membranes.     

Folding and conformational studies on SCR1-3 domains of human complement receptor 1

Short consensus repeats SCR3 and SCR1-3 are soluble recombinantproteins, consisting of the third and first three N-terminaldomains of complement receptor 1, respectively, which retainsome anti-complement activity. The conformational stabilitiesand folding/unfolding of SCR3 and SCR1-3 have been studied usingcircular dichroism and equilibrium and pre-equilibrium fluorescencespectroscopy. Denaturation by guanidinium hydrochloride (GdnHCl)is rapid and completely reversible. Reduction of disulphidebridges in the folded proteins by ß-mercaptoethanolleads to an increase in fluorescence intensity. The fluorescenceintensity of the folded proteins is {small tilde}7.5% of thatof the respective unfolded proteins. The data can be approximatedto a two-state transition between native and denatured formsof the proteins. SCR3 has a conformational stability in waterof 12–13 kJ/mol whereas that of SCR1-3 is 19.5–19.9kJ/mol depending upon the technique utilized. The heat capacitychange associated with the unfolding of SCR1-3 was obtainedby a series of GdnHCl unfolding experiments over a range oftemperatures and was found to be 6.6 kJ/K.mol or 33.8 J/K.mol_residue.The refolding process of SCR3 was found to be simple, describedby a single exponential equation, whereas that of SCR1-3 wasfound to be complex and could be fitted to a double exponentialequation indicating the presence of folding intermediates.     

Uncoupling of catalysis and colipase binding in pancreatic lipase by limited proteolysis

In the intestine, the hydrolysis of triglycerides by pancreaticlipase is performed only in the presence of colipase, whosefunction is to anchor lipase to the bile-salt-coated lipid interface.Biochemical and crystallographk data on porcine and human Upaseshave shown that the molecule is made of two well-delimited domains.In order to get more information on the role of the domainsin catalysis and colipase binding, we performed limited proteolysison lipase from various species and obtained different patternsof cleavage. In the case of porcine and human Upases, only theC-terminal domain (12 kDa) could be obtained after chymotrypticattack, whereas in the horse enzyme the cleavage of the Leu410-Thr411bond gave rise to a large N-terminal (45 kDa) and a small C-terminal(4 kDa) fragment. The isolated porcine and human C-terminaldomains were completely inactive towards emulsified tributyrin,though were able to bind colipase. Conversely, the horse 45kDa fragment retained the lipase activity but failed to correctlybind colipase. This work definitely proves that catalysis andcoUpase binding are separate events involving topographicallydistinct regions of the molecule and focuses attention on therole of the C-terminal domain in colipase binding     

Structural domains of P450-containing monooxygenase systems

All known P450-containing monooxygenase systems share commonstructural and functional domain architecture. Apart from P450itself, these systems can comprise several fundamentally differentprotein components or domains, all of which are shared by othermulticomponent/multidomain enzyme systems with various functions:FAD flavoprotein or domain, FMN domain, Fe₂S₂ ferredoxin, Fe₃S₄ferredoxin, and cytochrome b₅. Either FMN domain, ferredoxinsor cytochrome bs serve as the electron transport intermediatebetween the FAD domain and P450. The molecular evolution ofboth P450-containing systems and of each particular componentdoes not follow phylogeny in general. Gene fusion and horizontalgene transfer events can lead to the appearance of novel redoxchains in the same manner that artificial chimeric proteinscan be constructed by humans. Recent studies using genetic andprotein engineering techniques to investigate the separate domainsand their interaction are described.     

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.     

pH-sensitive interactions between IgG and a mutated IgG-binding protein based upon two B domains of Protein A from Staphylococcus aureus

A fusion protein, consisting of the N-terminal 81 amino acidsfrom an inactive bovine DNase I (Q38,E39–E38,Q39) andtwo sequential synthetic IgG-binding domains based upon domainB of Protein A from Staphylococcus aureus has been shown tobind to porcine IgG with a similar affinity and pH profile toProtein A. The same residue in each B domain (Tyr111 and Tyr169)has been mutated by cassette mutagenesis to Ser, Glu, His, Lysor Arg and the effect of the mutation on binding interactionswith porcine IgG investigated. The evidence presented suggeststhat the interactions at the B domain are highly sensitive tothe presence of a charged residue.     

Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability

Folding stabilities of camelized human antibody VH domains werestudied through the determination of their melting points inthermodenaturation experiments. The melting point of a VH domainoriginating from a synthetic library of human VHs, which hadbeen optimized for the use as small recognition units throughthe mimicking of camelid antibody heavy chains occurring naturallywithout light chain, was 56.6C compared with 71.2C of theoriginal human VH. Its stability was improved (melting point61.6C) through three mutations to mimic camelid VHs even further:Va137 was replaced by phenylalanine and two cysteines were introducedat positions 33 and 100b. The resulting VH folded properly andformed a second intradomain disulphide between the extra cysteines.The new mutations were then built constitutively into a phage-displayVH library, from which antigen-specific VHs were selected. Twowere analysed for stability with melting points of 72.6 and75.3C. Thus secondary camelization enabled the isolation ofVHs with improved folding stabilities exceeding even that ofthe original human VH. This indicates an effect on folding stabilityfor some mutations specific in the light chain lacking camelidheavy chains.     

Large-scale expression, purification and characterization of small fragments of thrombomodulin: the roles of the sixth domain and of methionine 388

Fragments of human thrombomodulin (TM) have been expressed inlarge quantities in the Pichia pastoris yeast expression systemand purified to homogeneity. Fermentation of P.pastoris resultedin yields of 170 mg/1 TM. Purification to homogeneity resultedin an overall 10% yield, so that quantities of –20 mgpurified fragments can be readily obtained. Smaller fragmentsof TM, such as the individual fourth or fifth domains, werenot active, nor were equimolar mixtures of the two domains.These results demonstrate that the fourth and fifth epidermalgrowth factor (EGF)–like domains together comprise thesmallest active fragment of TM. The fragment containing thefourth and fifth EGFlike domains (TMEGF(4–5)] had 10%the specific activity of rabbit TM. Comparison of the M388Lmutant TMEGF(4–5) fragment with the same mutant TMEGF(4–5–6)fragment showed that the fragment with the sixth domain hada 10–fold better Km value for thrombin than the fragmentthat did not contain the sixth domain; this factor completelyaccounts for the higher specific activity of the fragments containingthe sixth domain. Comparison of the wild–type and M388Lmutants showed that the M388L mutation resulted in a 2–foldincrease in k_cat for the activation of protein C by the thrombin–TMfragment complex, completely accounting for the 2–foldincrease in specific activity of these mutant fragments.     

Regulation of protein tyrosine phosphatase 1C: opposing effects of the two src homology 2 domains

The regulatory roles of the two src homology 2 (SH2) domainsof protein tyrosine phosphatase 1C were investigated by comparingrecombinant full-length PTP1C with mutants in which either theN-terminal SH2 (N-SH2) domain (PTP1CANSH2), the C-terminal SH2(C-SH2) domain (PTP1CACSH2) or both SH2 domains were deleted(PTP1CANSH2ACSH2). This revealed that the SH2 domains have opposingand independent effects on activity: strong inhibition by N-SH2(42-fold) and weak activation by C-SH2 (2.1-fold). C-SH2 causedactivation across a wide pH range while N-SH2 inhibited mostat neutral and high pH through a shift of the basic limb ofthe pH profile of kmt/Km, apparently via perturbation of anactive-site pKa value. A phosphotyrosyl peptide derived fromthe erythro-poietin receptor caused an {small tilde}30-foldactivation of PTP1C and PTP1CACSH2 but had no effect on PTP1CANSH2or PTP1CANSH2ACSH2, indicating that binding of this peptideto N-SH2 abolished its inhibition. Since C-SH2 separates N-SH2from the catalytic domain in full-length PTP1C and activationis observed for PTP1CACSH2, it appears that the inhibitory effectof N-SH2 is independent of the position in the sequence andthat intermolecular interactions may also be possible     

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     

Ribosome display of mammalian receptor domains

Many mammalian receptor domains, among them a large number of potential therapeutic target proteins, are highly aggregation-prone upon heterologous expression in bacteria. This severely limits functional studies of such receptor domains and also their engineering towards improved properties. One of these proteins is the Nogoreceptor, which plays a central role in mediating the inhibition of axon growth and functional recovery after injury of the adult mammalian central nervous system. We show here that the ligand binding domain of the Nogoreceptor folds to an active conformation in ternary ribosomal complexes, as formed in ribosome display. In these complexes the receptor is still connected, via a C-terminal tether, to the peptidyl tRNA in the ribosome and the mRNA also stays connected. The ribosome prevents aggregation of the protein, which aggregates as soon as the release from the ribosome is triggered. In contrast, no active receptor was observed in phage display, where aggregation appears to prevent incorporation of the protein into the phage coat. This strategy sets the stage for rapidly studying defined mutations of such aggregation-prone receptors in vitro and to improve their properties by in vitro evolution using the ribosome display technology.     

Repertoires of aggregation-resistant human antibody domains

We recently described a method for the generation of a large human domain antibody repertoire involving combinatorial assembly of CDR building blocks from a smaller repertoire comprising a high frequency of aggregation-resistant antibody domains. Here we show that the frequency of aggregation-resistant domains in the combinatorial repertoire remained high. Furthermore, one of the antigen-binding domains selected from the combinatorial repertoire retained its binding properties through 25 cycles of thermal denaturation, suggesting that antibody domains can be created that rival the heat-resistance of thermophilic proteins such as Taq polymerase.     

Cellulose-binding domains: potential for purification of complex proteins

The endoglucanase CenA and the exoglucanase Cex from Cellulomonasfimi each contain a discrete cellulose-binding domain (CBD),at the amino-terminus or carboxyl-terminus respectively. Thegene fragment encoding the CBD can be fused to the gene of aprotein of interest. Using this approach hybrid proteins canbe engineered which bind reversibly to cellulose and exhibitthe biological activity of the protein partner. Alkaline phosphatase(PhoA) from Escherichia coli, and a ß-glucosidase(Abg) from an Agrobacterium sp. are dimeric proteins. The fusionpolypeptides CenA-PhoA and Abg-CBC_cex are sensitive to proteolysisat the junctions between the fusion partners. Proteolysis resultsin a mixture of homo- and heterodimers; these bind to celluloseif one or both of the monomers carry a CBD, e.g. CenA-PhoA/CenA-PhoAand CenA-PhoA/PhoA. CBD fusion polypeptides could be used inthis way to purify polypeptides which associate with the fusionpartner.