Introduction of internal cysteines as conformational probes in yeast phosphoglycerate kinase

Several mutants of yeast phosphoglycerate kinase, each containingonly one internal cysteine residue, were constructed from asingle mutant devoid of cysteine. These cysteines were introducedas local conformational probes in selected buried positions.The enzyme activity, conformational characteristics and stabilityindicated that the mutations introduced only small perturbationsin the molecule. The folding–unfolding process mediatedby guanidine hydrochloride under equilibrium conditions wasstudied by following the variations in ellipticity and the reactivityof the cysteine residue towards 5,5'-dithiobis(nitrobenzoate).The process was found to be reversible except for mutant C97A,V49C,suggesting that this region located in helix I might be crucialin determining an intermediate on the folding pathway. The transitionsobtained by the two signals did not coincide, indicating thatthe local structures, in several parts inside the molecule,are more sensitive to the denaturant than the overall conformation.     

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.     

Structural and functional properties of mutant Arg203Pro from yeast phosphoglycerate kinase, as a model of phosphoglycerate kinase-Uppsala

A pathological variant of human phosphoglycerate kinase, phosphoglyceratekinase-Uppsala, associated with chronic nonspherocytic hemolyticanemia has been found to differ from the normal enzyme by substitutionof an arginine at position 206 (corresponding to position 203in yeast) by a proline. In order to understand the structuraland functional consequences of this mutation, the correspondingmutant in yeast phosphoglycerate kinase was constructed. Thethree-dimensional structure of this mutant was resolved at 2.9Å. Although the overall structure is not modified, smalllocal changes were observed. The kinetic parameters of the mutantwere not found to be greatly affected, the catalytic constantbeing lowered by only 10–20%. The most significant differencewhen compared with the wild-type enzyme is a decrease in stabilityby about 3 kcal/mol. The physiological implications of thisinstability are discussed.     

Site-directed mutagenesis of aspartic acid 372 at the ATP binding site of yeast phosphoglycerate kinase: over-expression and characterization of the mutant enzyme

A new phosphoglycerate kinase over-expression vector, pYE-PGK,has been constructed which greatly facilitates the insertionand removal of mutant enzyme genes by cleavage at newly introducedBamtHI sites. This vector has been used to prepare mutant proteinin appreciable (100 mg) quantities for use in kinetic, crystaUographicand NMR experiments. Aspartate 372 is an invariant amino acidresidue in genes known to code for a functionally active PGK.The function of this acidic residue appears to be to help desolvatethe magnesium ion compfexed with either ADP or ATP when thissubstrate binds to the enzyme. Both crystallographk and nuclearmagnetic resonance experiments show that the replacement ofthe residue with asparagine has only minimal effects on theoverall structure. The substitution of the charged carboxylgroup with that of the neutral amide affects the binding ofthe nucleotide substrate as predicted but not, as might havebeen expected, the binding of 3-phospho-glycerate. The overallvelocity of the enzymic reaction (V_max) is reduced 10-fold bythe substitution of aspartic acid 372 by an asparagine residue(D372N). This reduction in V_max is considerably less than onewould expect from its known position within the structure ofthe enzyme. This result therefore poses questions about ourunderstanding of charged groups at the active centres of enzymesand of the reason for their apparent conservation.     

Site-directed mutations of arginine 65 at the periphery of the active site cleft of yeast 3-phosphoglycerate kinase enhance the catalytic activity and eliminate anion-dependent activation

The function of arginine 65, a conserved residue located atthe periphery of the active site cleft in yeast 3-phospho-glyceratekinase (PGK), has been investigated by site-directed mutagenesis.Mutant enzymes with glutamine, serine and alanine at position65 all have very similar kinetic properties. The maximum velocities,determined in the absence of sulfate anion, are - 100% higherthan the V_max of wild-type PGK. The K_m values are increased2- to 3-fold for ATP and 5- to 6-fold for 3-phosphoglycerate(3PG). These results demonstrate that arginine 65 is not essentialfor catalysis. In contrast to wild-type enzyme, the mutantsare not activated by sulfate ions. In addition, steady-statekinetic experiments indicate that the mutants are no longeractivated by high concentrations of either 3PG or ATP. The dissociationconstants for anions were determined by spectral titrationsof the R65Q mutant labeled with a chromophoric probe. The K_dfor 3PG is increased 6-fold, as compared to wild-type PGK, whereasthe K_d for ATP is essentially unchanged. The KA for sulfateis decreased < 2-fold. The suppression of substrate- andsulfate-dependent activation suggests that arginine 65 participatesin the regulatory mechanism responsible for activation of theenzyme.     

Characterization of engineered anti-p185HER-2 (scFv-CH3)2 antibody fragments (minibodies) for tumor targeting

An engineered antibody fragment (minibody; scFv-C(H)3gamma(1) dimer, M(r) 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti- p185(HER-2) 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (V(L)-linker-V(H) and V(H)-linker-V(L)) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 C(H)3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20-60 mg/l, demonstrated binding to the human p185(HER-2) overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (K(D) approximately 2-4 nM) were equivalent to that for the parental 10H8 mAb (K(D) approximately 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (+/-1.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (beta-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen-antibody system.     

Ligand-induced conformational changes in wild-type and mutant yeast pyruvate kinase

A mutant form of pyruvate kinase in which serine 384 has beenmutated to proline has been engineered in the yeast Saccharomycescerevisiae. Residue 384 is located in a helix in a subunit interfaceof the tetrameric enzyme, and the mutation was anticipated toalter the conformation of the helix and hence destabilize theinterface. Previous results indicate that the mutant favoursthe T quarternary conformation over the R conformation, andthis is confirmed by the results presented here. Addition ofphosphoenol-pyruvate (PEP), ADP and fructose-1,6-bisphosphate(Fru 1,6-P₂) singly to the wild-type and mutant enzymes resultsin a significant quenching of tryptophan fluorescence (12–44%),and for Fru-1,6-P₂, a red shift of 15 nm in the emission maximum.Fluorescence titration experiments showed that PEP, ADP andFru-1,6-P₂ induce conformations which have similar ligand-bindingproperties in the wild-type and mutant enzymes. However, theFru-1,6-P₂ induced conformation is demonstrably different fromthose induced by either ADP or PEP. The enzymes differ in theirsusceptibility to trypsin digestion and N-ethylmaleimide inhibition.The thermal stability of the enzyme is unaltered by the mutantion.Far-UV CD spectra show that both enzymes adopt a similar overallsencondary structure in solution. Taken together, the resultssuggest that the Ser384-Pro mutaion causes the enzyme to adopta diffenrent tertiary and/or quaternary structure from the wild-typeenzyme and affects the type and extent of the conformationalchanges induced in the enzyme upon ligand binding. A simplifiedminimal reaction mechanism is proposed in which the R and Tstates differ in both affinity and K_cat. Thus, in terms of themodels of cooperativity and allsoteric interaction, pyruvatekinase is both a K and a V system.     

Association of complementary fragments and the elucidation of protein folding pathways

Natural or engineered sites for chemical cleavage can be usedto generate complementary fragments of well characterized proteins.The peptide fragments represent a unique tool for studying earlyevents in protein folding, since these are usually the mostinaccessible to the experimentalist. Analysis of the isolatedfragments in nondenaturing conditions together with the determinationof the structure of the folded non-covalent complexes and, mostimportantly, the kinetic analysis of the resulting second-orderassociation/folding reaction can give a more complete pictureof a folding pathway. The contribution of fragments to the understandingof some well characterized protein folding pathways is discussed.     

Engineering yeast alcohol dehydrogenase. Replacing Trp54 by Leu broadens substrate specificity

Analysis of a crystal structure of alcohol dehydrogenase (Adh)from horse liver suggests that Trp54 in the homologous yeastalcohol dehydrogenase prevents the yeast enzyme from efficientlycatalysing the oxidation of long-chain primary alcohols withbranching at the 4 position (e.g. 4-methyl-1-pentanol, cinnamylalcohol). This residue has been altered to Leu by site-directedmutagenesis. The alteration yields an enzyme that serves asan effective catalyst for both longer straight-chain primaryalcohols and branched chain alcohols.     

The folding pathway of an engineered circularly permuted PDZ domain

To understand the role of sequence connectivity in the folding pathway of a multi-state protein, we have analysed the folding kinetics of an engineered circularly permuted PDZ domain. This variant has been designed with the specific aim of posing two of the strands participating in the stabilisation of an early folding nucleus as contiguous elements in the primary structure. Folding of the circularly permuted PDZ2 has been explored by a variety of different experimental approaches including stopped-flow and continuous-flow kinetics, as well as ligand-induced folding experiments. Data reveal that although circular permutation introduces a significant destabilisation of the native state, a folding intermediate is stabilised and accumulated prior folding. Furthermore, quantitative analysis of the observed kinetics indicates an acceleration of the early folding events by more than two orders of magnitude. The results support the importance of sequence connectivity both in the mechanism and the speed of protein folding.     

Thermostability promotes the cooperative function of split adenylate kinases

Proteins can often be cleaved to create inactive polypeptides that associate into functional complexes through non-covalent interactions, but little is known about what influences the cooperative function of the ensuing protein fragments. Here, we examine whether protein thermostability affects protein fragment complementation by characterizing the function of split adenylate kinases from the mesophile Bacillus subtilis (AKBs) and the hyperthermophile Thermotoga neapolitana (AKTn). Complementation studies revealed that the split AKTn supported the growth of Escherichia coli with a temperature-sensitive AK, but not the fragmented AKBs. However, weak complementation occurred when the AKBs fragments were fused to polypeptides that strongly associate, and this was enhanced by a Q16L mutation that thermostabilizes the full-length protein. To examine how the split AK homologs differ in structure and function, their catalytic activity, zinc content, and circular dichroism spectra were characterized. The reconstituted AKTn had higher levels of zinc, greater secondary structure, and >10(3)-fold more activity than the AKBs pair, albeit 17-fold less active than full-length AKTn. These findings provide evidence that the design of protein fragments that cooperatively function can be improved by choosing proteins with the greatest thermostability for bisection, and they suggest that this arises because hyperthermophilic protein fragments exhibit greater residual structure compared to their mesophilic counterparts.     

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A₂ His48 isat hydrogen-bonding distance to Asp99. This Asp-His couple isassumed to act together with a water molecule as a catalytictriad. Asp99 is also linked via an extended hydrogen bondingsystem to the side chains of Tyr52 and Tyr73. To probe the functionof the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipaseA₂, the Asp99 residue was replaced by Asn, and each of the twotyrosines was separately replaced by either a Phe or a Gln.The catalytic and binding properties of the Phe52 and Phe73mutants did not change significantly relative to the wild-typeenzyme. This rules out the possibility that either one of thetwo Tyr residues in the wild-type enzyme can function as anacyl acceptor or proton donor in catalysis. The Gln73 mutantcould not be obtained in any significant amounts probably dueto incorrect folding. The Gln52 mutant was isolated in low yield.This mutant showed a large decrease in catalytic activity whileits substrate binding was nearly unchanged. The results suggesta structural role rather than a catalytic function of Tyr52and Tyr73. Substitution of asparagine for aspartate hardly affectsthe binding constants for both monomeric and micellar substrateanalogues. Kinetic characterization revealed that the Asn99mutant has retained no less than 65% of its enzymatic activityon the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine,probably due to the fact that during hydrolysis of monomericsubstrate by phospholipase A₂ proton transfer is not the rate-limitingstep. The Asp to Asn substitution decreases the catalytic rateon micellar 1,2-dioctanoyl-sn-glycero-3-phosphocholine 25-fold.To explain this remaining activity we suggest that in the mutantthe Asn99 orients His48 in the same way as Asp99 orients His48in native phospholipase A₂ and that the lowered activity iscaused by a reduced stabilization of the transition state.     

Site-directed mutagenesis of the putative active site residues of 3C proteinase of coxsackievirus B3: evidence of a functional relationship with trypsin-like serine proteinases

Picornavirus 3C proteinases (3C^pro) are cysteine proteinasesbut recent sequence analyses have shown that they are relatedto trypsin-like serine proteinases. Two models of 3C^pro structurehave been presented. Both models indicate that residues His40and Cysl47 are members of the catalytic triad but the modelsdiffer in the designation of the third member of the catalytictriad, which is assigned as either Glu71 or Asp85. To test theimportance of these four residues in the catalytic activityof 3C^pro of coxsackievirus B3, a member of the enterovirus subgroupof the picornavirus family, single amino acid substitutionswere introduced at each of the four sites. All of these mutationsresulted in the reduction or inactivation of autocatalytic cleavageof the 3C precursor protein expressed in Escherichia coli, suggestingthat all of these residues are essential for the proteolyticreaction. The substitution of Cysl47 with Ala abolished 3C^proactivity while the mutant in which Cysl47 was replaced withSer retained reduced proteolytic activity both in cis and intrans. Our results strongly support the proposal that Cysl47of 3C^pro functions as a nucleophile analogous to Serl95 of trypsin-likeserine proteinases.     

Protein modification from mutational analysis of an autologous peptide fragment

In the -complementation of ß-galactosidase an N-terminalpeptide fragment (-peptide) of the wild-type enzyme interactswith a defective ß-galactosidase enzyme to restorecapacity for subunit assembly and activity. We have used previouslya random mutagenesis and screening approach to identify a pentapeptideresidue tract in the -peptide that was highly tolerant of residuesubstitution, with some mutations conferring improved function.This tract is of clear importance for -peptide function butis apparently dispensible in the intact parental enzyme. Toinvestigate this further, we selected tract mutations and placedthem into intact ß-galactosidase, at the correspondingN-terminal position as in the -peptide. We then tested whethersuch specific tract sequences conferred properties to the wholeenzyme which could be predicted from the behaviour of the defectiveenzyme complemented with the corresponding mutant -peptide.This was shown for mutations which positively or negativelyaffected enzyme stability. Additionally, a subset of mutationswhich affected complementation efficiency in vivo were predictedto affect the formation of higher-order structures in the intactprotein, and this was observed experimentally. Mutations whichdecreased peptide complementation dramatically decreased thelevel of formation of multimers in the intact protein and amutation which increased peptide complementation produced markedenhancement of multimer formation in a protein with a preexistingimpairment in higher-order structure formation. Such subtleeffects are difficult to detect directly in the whole proteinby randomization/selection approaches, but in the complementingpeptide the role of the residues within the pentapeptide tractis effectively amplified. Identification of residue tracts exhibitingfunctional tolerance to amino acid substitution in an activepeptide fragment can thus be combined with transferral of potentiallyuseful mutant peptide sequences back into the intact protein.Manipulation of a complementation system in this manner affordsa sensitive approach towards targeted improvement of proteins.     

Essentiality of Lys-329 of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum as demonstrated by site-directed mutagenesis

The unusual chemical properties of active-site Lys-329 of ribulosebisphosphate carboxylase/oxygenase from Rhodo-spirillum rubrumhave suggested that this residue is required for catalysis.To test this postulate Lys-329 was replaced with glycine, serine,alanine, cysteine, arginine, glutamic acid or glutamine by site-directedmutagenesis. These single amino acid substitutions do not appearto induce major conformational changes because (i) intersubunitinteractions are unperturbed in that the purified mutant proteinsare stable dimers like the wild-type enzyme and (ii) intrasubunitfolding is normal in that the mutant proteins bind the competitiveinhibitor 6-phosphogluconate with an affinity similar to thatof wild-type enzyme. In contrast, all of the mutant proteinsare severely deficient in carboxylase activity (< 0.01% ofwild-type) and are unable to form the exchange-inert complex,characteristic of the wild-type enzyme, with the transitionstateanalogue carboxyarabinitol bisphosphate. These results underscorethe stringency of the requirement for a lysyl side-chain atposition 329 and imply that Lys-329 is involved in catalysis,perhaps stabilizing a transition state in the overall reactionpathway.     

Mutation of a highly conserved aspartate residue in subdomain IX abolishes Fer protein-tyrosine kinase activity

Before the structure of cAMP-dependent protein kinase had beensolved, sequence alignments had already suggested that severalhighly conserved peptide motifs described as kinase subdomainsI through XI might play some functional role in catalysis. Crystalstructures of several members of the protein kinase superfamilyhave suggested that the nearly invariant aspartate residue withinsubdomain IX contributes to the conformational stability ofthe catalytic loop by forming hydrogen bonds with backbone amideswithin subdomain VI. However, substitution of this aspartatewith alanine or threonine in some protein kinases have indicatedthat these interactions are not essential for activity. In contrast,we show here that conversion of this aspartate to arginine abolishedthe catalytic activity of the Fer protein-tyrosine kinase whenexpressed either in mammalian cells or in bacteria. Structuralmodeling predicted that the catalytic loop of the FerD743R mutantwas disrupted by van der Waal's repulsion between the side chainsof the substituted arginine residue in subdomain IX and histidine-683in subdomain VI. The FerD743R mutant model predicted a shiftin the peptide backbone of the catalytic loop, and an outwardrotation of histidine-683 and arginine-684 side chains. However,the position and orientation of the presumptive catalytic base,aspartate-685, was not substantially changed. The proposed modelexplains how substitutions of some, but not all residues couldbe tolerated at this nearly invariant aspartate in kinase subdomainIX.     

Bacterial expression, purification and preliminary kinetic description of the kinase domain of v-fps

The gene coding for the tyrosine protein kinase domain of v-fpswas subcloned into a plasmid vector expressing glutathione-S-transferase(GST). This new vector expresses a fusion protein in Escherichiacoli composed of the kinase domain linked with GST at the N-terminus(GST-kin). A portion of the total expressed protein was solubleupon cell lysis and was purified by affinity chromatographyusing glutathione cross-linked agarose. GST-kin (M_r 57 000)is a phosphoprotein as judged by ³²P autoradiography, consistentwith the known autophosphorylation site within the kinase core[Weinmaster et aL (1984) Cell, 37, 559–568]. Cleavageof the fusion protein with thrombin and purification on phosphocelluloseresin yielded the pure kinase domain (M_r 33 000). The activityof the kinase domain is indistinguishable from that of GST-kinusing the peptide substrate EEEIYEEIE, indicating that Nterminalfusion has no effect on the kinase domain. GSTkin phosphorylatesa second peptide, EAEIYEAIE, with improved catalytic efficiency.Initial velocity data are consistent with a random bireactantmechanism with no substrate synergism observed in the ternarycomplex. Steady-state kinetic analyses reveal that this peptideis phosphorylated, with a k_cat of 3.6 s^–1, a K_peptideof 500 µM and a K_ATP of 250 µM. The expression,purification and preliminary kinetic analysis of the kinasedomain of v-fps provide the first step in the application ofstructurefunction studies for this oncoprotein     

Comparison of conservation within and between the Ser/Thr and Tyr protein kinase family: proposed model for the catalytic domain of the epidermal growth factor receptor

The protein kinase family can be subdivided into two main groupsbased on their ability to phosphorylate Ser/Thr or Tyr substrates.In order to understand the basis of this functional difference,we have carried out a comparative analysis of sequence conservationwithin and between the Ser/Thr and Tyr protein kinases. A multiplesequence alignment of 86 protein kinase sequences was generated.For each position in the alignment we have computed the conservationof residue type in the Ser/Thr, in the Tyr and in both of thekinase subfamilies. To understand the structural and/or functionalbasis for the conservation, we have mapped these conservationproperties onto the backbone of the recently determined structureof the cAMP–dependent Ser/Thr kinase. The results showthat the kinase structure can be roughly segregated, based uponconservation, into three zones. The inner zone contains residueshighly conserved in all the kinase family and describes thehydrophobic core of the enzyme together with residues essentialfor substrate and ATP binding and catalysis. The outer zonecontains residues highly variable in all kinases and representsthe solvent–exposed surface of the protein. The thirdzone is comprised of residues conserved in either the Ser/Thror Tyr kinases or in both, but which are not conserved betweenthem. These are sandwiched between the hydrophobic core andthe solvent-exposed surface. In addition to analyzing overallconservation hi the kinase family, we have also looked at conservationof its substrate and ATP binding sites. The ATP site is highlyconserved throughout the kinases, whereas the substrate bindingsite is more variable. The active site contains several positionswhich differ between the Ser/Thr and Tyr kinases and may beresponsible for discriminating between hydroxyl bearing sidechains. Using this information we propose a model for Tyr substratebinding to the catalytic domain of the epidermal growth factorreceptor (EGFR).