Recombinant hirudin: kinetic mechanism for the inhibition of human thrombin

Recombinant hirudin variant-2(Lys⁴⁷ ), was found to be a competitiveinhibitor of human -thrombin with respect to peptidyl p-Miitroanilidesubstrates. These results contrast with those of Degryse andcoworkers that suggest that recombinant hirudin variant-2(Lys⁴⁷)inhibited thrombin by a non-competitive mechanism [Degryse etal. (1989) Protein Engng, 2, 459–465], -Thrombin, whichcan arise from -thrombin by autolysis, was shown to have anaffinity for recombinant hirudin variant-2(Lys⁴⁷) that was fourorders of magnitude lower than that of -thrombin. It was demonstratedthat the apparent noncompetitive mechanism observed previouslywas probably caused by a contamination of the thrombin preparationby -thrombin. Comparison of the inhibition of -thrombin by recombinanthirudins variant-2(Lys⁴⁷) and variant-1, which differ from oneanother in eight out of 65 amino acids, indicated that the twovariants have essentially the same kinetic parameters.     

Construction and structure-activity relationships of chimeric prourokinase derivatives with intrinsic thrombin-inhibitory potential

The blood clotting enzyme thrombin plays a central role in theaetiology of occlusive disorders such as stroke and acute myocardialinfarction. During fibrinolytic therapy with plasminogen activators,thrombin is neutralized by anticoagulative drugs. In order tocombine plasminogen-activating and thrombin-inhibitory activitieswe constructed chimeric derivatives of recombinant single-chain,urokinase-type plasminogen activator (rscu-PA) which comprisethe kringle and protease domain of rscu-PA fused via a linkersequence to a thrombin-inhibitory domain. The inhibitory domaincontains a sequence element directed to the active site of thrombinand a sequence taken from either hirudin or the human thrombinreceptor both binding to the fibrinogen recognition site ofthrombin. Analysing different sets of point mutants showed thatthe linker between the protease domain and the active sitedirectedsequence is contributing significantly to the thrombin-inhibitorypotential. Kinetic analysis of thrombin inhibition revealedthat most of the chimeras tested competitively inhibit the thrombin-mediatedcleavage of a peptide substrate in a concentration-dependentmanner; however, in two examples the insertion of one glycineresidue into the active site directed-sequence abolished theblockade of the active site. This supports the conclusion thatthe chimeras with high thrombin-inhibitory potential interactwith the active site and the fibrinogen recognition site ofthrombin.     

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.     

Design, synthesis and analysis of novel bicyclic and bifunctional protease inhibitors

Two novel synthetic inhibitors were designed to combine the advantageous properties of Bowman Birk inhibitor (BBI) and sunflower trypsin inhibitor-1 (SFTI-1). As is the case for BBI, the novel inhibitors have two active sites that give dual independent protease inhibition. However, they also possess a small bicyclic structure, reminiscent of the single-site SFTI-1. It is found that the synthetic inhibitors retain the potent inhibitory properties of the parent structures; they are also found to be relatively resistant to proteolysis. Their inhibition properties and a comparison of their stability to proteolysis relative to SFTI-1 are described. It is found that the new inhibitors do indeed allow bifunctional inhibition, although, unlike BBI, the small size of the inhibitor prevents the simultaneous inhibition of two proteases at the same time.     

Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin

We have previously shown that replacing the P1-site residue(Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lysresults in the acquisition of inhibitory activity toward chymotrypsinor trypsin, respectively. However, the inhibitory activitiesthus induced are not strong. In the present study, we introducedadditional amino acid replacements around the reactive siteto try to make the P1-site mutants more effective inhibitorsof chymotrypsin or trypsin. The amino acid replacement AspTyrat the P2' site of OMCHI3(P1Met) resulted in conversion to a35000-fold more effective inhibitor of chymotrypsin with aninhibitor constant (K_i) of 1.17x10^–11 M. The K_i valueof OMCHI3(P1Met, P2'Ala) indicated that the effect on the interactionwith chymotrypsin of removing a negative charge from the P2'site was greater than that of introducing an aromatic ring.Similarly, enhanced inhibition of trypsin was observed whenthe AspTyr replacement was introduced into the P2' site of OMCHI3(P1Lys).Two additional replacements, AspAla at the P4 site and ArgAlaat the P3' site, made the mutant a more effective inhibitorof trypsin with a K_i value of 1.44x10^–9 M. By contrast,ArgAla replacement at the P3' site of OMCHI3(P1Met, P2'Tyr)resulted in a greatly reduced inhibition of chymotrypsin, andAspAla replacement at the P4 site produced only a small changewhen compared with a natural variant of OMCHI3. These resultsclearly indicate that not only the P1-site residue but alsothe characteristics, particularly the electrostatic properties,of the amino acid residues around the reactive site of the proteaseinhibitor determine the strength of its interactions with proteases.Furthermore, amino acids with different characteristics arerequired around the reactive site for strong inhibition of chymotrypsinand trypsin.     

Highly effective protease inhibitors from variants of human pancreatic secretory trypsin inhibitor (hPSTI): an assessment of 3-D structure-based protein design

The results of a protein design project are used to comparedifferent predictive strategies with respect to proteinproteininteractions. We have been able to generate variants of humanpancreatic secretory trypsin inhibitor (hPSTI) optimized withrespect to the affinity and specificity for human leukocyteelastase relative to trypsin and chymotrypsin, and in particularchymotrypsin. The extremely strong and specific human leukocyteelastase inhibitors were thus developed in three rounds of mutagenesisand two rounds of 3-D modelling; only 24 variants in total weresynthesized, although variations at seven different amino acidpositions were involved (i.e. from 20⁷ possible variants). Anexcellent elastase inhibitor could be designed with the minimumof two amino acid exchanges. The value of structural modellingand actual structure determination is discussed in the lightof the experimental results of the designed protein variantsand the results of tertiary structure determinations of thefree variant and the inhibitorprotease complex. Particular referenceis given to the strategy to be followed in protein design projectsin general and to the development of protease inhibitors inparticular.     

Engineered turns of a recombinant antibody improve its in vivo folding

Using recombinant antibodies functionally expressed by secretionto the periplasm in Escherichia coli as a model system, we identifiedmutations located in turns of the protein which reduce the formationof aggregates during in vivo folding or which influence cellstability during expression. Unexpectedly, the two effects arebased on different mutations and could be separated, but bothmutations act synergistically in vivo. Neither mutation increasesthe thermodynamic stability in vitro. However, the in vivo foldingmutation correlates with the yield of oxidative folding in vitro,which is limited by the side reaction of aggregation. The invivo folding data also correlate with the rate and activationentropy of thermally induced aggregation. This analysis showsthat it is possible to engineer improved frameworks for semi-syntheticantibody libraries which may be important in maintaining librarydiversity. Moreover, limitations in recombinant protein expressioncan be overcome by single amino acid substitutions.     

The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli

In a systematic study of the periplasmic folding of antibodyfragments in Escherichia coli, we have analysed the expressionof an aggregation-prone and previously non-functional anti-phosphorylcholineantibody, T15, as a model system and converted it to a functionalmolecule. Introduction of heavy chain framework mutations previouslyfound to improve the folding of a related antibody led to improvedfolding of T15 fragments and improved physiology of the hostE.coli cells. Manipulation of the complementarity determiningregions (CDR) of the framework-mutated forms of T15 furtherimproved folding and bacterial host physiology, but no improvementwas seen in the wild type, suggesting the existence of a hierarchyin sequence positions leading to aggregation. Rational mutagenesisof the T15 light chain led to the production of functional T15fragments for the first time, with increased levels of functionalprotein produced from V_H manipulated constructs. We proposethat a hierarchical analysis of the primary amino acid sequence,as we have described, provides guidelines on how correctly folding,functional antibodies might be achieved and will allow furtherdelineation of the decisive structural factors and pathwaysfavouring protein aggregation.     

Effects of deletion in the flexible loop of the protease inhibitor SSI (Streptomyces subtilisin inhibitor) on interactions with proteases

The Streptomyces subtilisin inhibitor (SSI) is a proteinaceousprotease inhibitor which inhibits serine proteases by forminga stable Michaelis complex. The flexible loop region (Thr64–Val69)is a very flexible region in an SSI molecule and its importancein interactions with proteases has been suggested, since conformationalchange of this loop was found to occur for the smooth bindingof SSI with various proteases. In this study, mutated SSIs lackingone or two residues in this region were generated and the effectsof deletions on the interaction with proteases were investigated.Deletion was introduced into mutated SSI(Lys73) and SSI(Gly70Lys73)both known to be trypsin inhibitors, to examine the effectsof deletion on interactions with subtilisin BPN' or trypsin.The deletion of one residue (Gly66) caused increased inhibitoryactivity toward trypsin, indicating the protruding flexibleloop hinders binding with trypsin. Reduction of such hindranceby one-residue shortening in this loop is shown to be effectivefor the interaction of SSI(Lys73) with trypsin. In contrast,one-residue shortening had virtually no effect on inhibitiontoward subtilisin BPN'. Differences in the subsite structuresof these proteases may have been the reason for this contrast.The deletion of two residues (Thr64 and Gly66) in this regionconverted SSI into a temporary inhibitor. Structural analysisof the degradation intermediate showed that the peptide bondat the reactive site of doubly deleted SSI was cleaved by subtilisinBPN' after its binding with protease. Thus, the irreversibilityof the cleaved peptide bond at the reactive site of mutatedSSI in the complex with protease may possibly be the cause forits temporary inhibition. Irregular conformation around thereactive site caused by the deletion of two residues in theflexible loop would convert SSI into a temporary inhibitor.Thus, moderate flexibility in the flexible loop region may possiblybe a structural requirement for SSI to function.     

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.     

Effects of hydrophobic amino acid substitution in Pleurotus ostreatus proteinase A inhibitor 1 on its structure and functions as protease inhibitor and intramolecular chaperone

We previously demonstrated that Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) could function as an intramolecular chaperone of subtilisin BPN', as in the case of the propeptide of subtilisin BPN', and that its Phe44 --> Ala mutant, which lost its tertiary structure, could not assist the refolding of subtilisin BPN'. In this study, we examined the effects of hydrophobic amino acid substitutions at other sites and substitutions of Phe44 with other hydrophobic residues on the structure and functions of POIA1. These mutations were introduced into POIA1cm that had been obtained by the substitution of the C-terminal six residues of POIA1 with those of the propeptide of subtilisin BPN'. When Ile32 or Ile64 was substituted with Ala, the tertiary structure of the resultant mutant was markedly destroyed, and the activities as a protease inhibitor and an intramolecular chaperone were significantly lowered. Among the position 44 mutants, the Phe44 --> Val mutant was a much less effective intramolecular chaperone with conversion to a digestible inhibitor, possibly owing to destruction of the tertiary structure. On the other hand, the Phe44 --> Leu or Ile mutant maintained its tertiary structure, and hence could function as a more effective intramolecular chaperone than the Phe44 --> Val mutant. Furthermore, since the Phe44 --> Leu mutant was a more susceptible inhibitor than POIA1cm, the halo formed around a colony of Bacillus cells transformed with a plasmid encoding this mutant was larger than others. These results clearly show the close relationship between the tertiary structure and functions of POIA1 as a protease inhibitor and an intramolecular chaperone, and that a combination of such inhibitory properties and intramolecular chaperone activity of POIA1 might affect the diameter of the halo formed around Bacillus colonies in vivo.     

Comparison of the solution and X-ray structures of barley serine proteinase inhibitor 2

A comparison of the solution n.m.r. structures of barley serineprotease inhibitor 2 (BSPI-2) with the X-ray structures of bothsubtilisin complexed and native BSPI-2 is presented. It is shownthat the n.m.r. and X-ray structures are very similar in termsof overall shape, size, polypeptide fold and secondary structure.The average atomic rms difference between the 11 restraineddynamics structures on the one hand and the two X-ray structureson the other is 1.9±0.2 Å for the backbone atomsand 3.0±0.3 Å for all atoms. The cor respondingvalues for the restrained energy minimized mean dynamics structureare 1.5 and 2.4 Å, respectively.     

Folding and stability of the active N-terminal domain of tissue inhibitor of metalloproteinases-1 and -2

The truncated forms of tissue inhibitor of metalloproteinase-1and -2 (TIMP-1 and -2), comprising the N-terminal active domain,are ideal molecules for structural analysis by intrinsic fluorescenceas each contains a single conserved tryptophan residue. In thispaper we describe studies on their conformational stability,unfolding/refolding kinetics and the environment of the uniquetryptophan as judged by its fluorescence properties in the nativestate and exposure to an external quencher, acrylamide. Twoforms of TIMP-2 were studied: TIMP-2 T21 derived from the full-lengthcDNA clone isolated from a mixed-tumour library, and TIMP-2A21 containing the highly conserved V18IRAK22 sequence. In allthree TIMP proteins the tryptophan environments in the nativestate appeared to be similar, but substantial differences wereseen in their conformational stabilities and refolding kinetics.TIMP-1 was approximately twice as stable as TIMP-2 T21 and 1.4-foldmore stable than TIMP-2 A21. This stability difference betweenTIMP-1 and TIMP-2 was shown to be independent of N-linked glycosylation.TTMP-1 and TIMP-2 A21 both showed simple two-state refoldingkinetics, whereas TIMP-2 T21 refolding was more complex andbiphasic in character. These differences between TIMP-2 T21and A21 suggest that residue 21 is a structurally importantsite in the TIMP protein.All three truncated molecules can beconsidered as stable independent folding domains ideally suitedfor further structural analysis     

Three-dimensional structure of protein C inhibitor predicted from structure of {alpha}1-antitrypsin with computer graphics

The three-dimensional structure of a proteolytically modifiedprotein C inhibitor, a member of the serine protease inhibitorsuperfamily, was constructed with computer graphics based onits amino acid sequence homology with that of the modified 1-antitrypsinwhose structure had been elucidated by X-ray crystallography.The intact form of protein C inhibitor was predicted with an-carbon model based on its hydrophilicity and hydrogen bondpattern. Furthermore, a model of its interaction with activatedprotein C was constructed based on the structure of the complexbetween trypsin and its inhibitor, which had been determinedby X-ray crystallography.     

Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties

A monomeric variant of triosephosphate isomerase (TIM) witha new engineered binding groove has been characterized further.In this variant (ml8bTIM), the phosphate binding loop had beenshortened, causing the binding site to be much more extended.Here, it is reported that in the V233A variant of ml8bTIM (A-TIM),three important properties of the wild-type TIM active sitehave been restored: (i) the structural properties of loop-7,(ii) the binding site of a conserved water molecule betweenloop-7 and loop-8 and (iii) the binding site of the phosphatemoiety. It is shown that the active site of A-TIM can bind TIMtransition state analogs and suicide inhibitors competently.It is found that the active site geometry of the A-TIM complexesis less compact and more solvent exposed, as in wild-type TIM.This correlates with the observation that the catalytic efficiencyof A-TIM for interconverting the TIM substrates is too low tobe detected. It is also shown that the A-TIM active site canbind compounds which do not bind to wild-type TIM and whichare completely different from the normal TIM substrate, likea citrate molecule. The binding of this citrate molecule isstabilized by hydrogen bonding interactions with the new bindinggroove.     

The determination of the three-dimensional structure of barley serine proteinase inhibitor 2 by nuclear magnetic resonance, distance geometry and restrained molecular dynamics

The solution structure of the 64 residue structured domain (residues20–83) of barley serine proteinase inhibitor 2 (BSPI-2)is determined on the basis of 403 interproton distance, 34 øbackbone torsion angle and 26 hydrogen bonding restraints derivedfrom n.m.r. measurements. A total of 11 converged structureswere computed using a metric matrix distance geometry algorithmand refined by restrained molecular dynamics. The average rmsdifference between the final 11 structures and the mean structureobtained by averaging their coordinates is 1.4±0.2 Åfor the backbone atoms and 2.1±0.1 A for all atoms. Theoverall structure, which is almost identical to that found byX-ray crystallography, is disc shaped and consists of a centralfour component mixed parallel and antiparallel ß-sheetflanked by a 13 residue helix on one side and the reactivesite loop on the other.     

Complex additivity of the effects of suppressor mutations in differing protein environments

In the -complementation of -galactosidase, a defective ß-galactosidaseprotein interacts with an autologous peptide fragment (-peptide)to restore enzymatic activity. Within a specific site of a defective-peptide we have previously isolated a large number of mutations,many of which suppress the functional defect. The -peptide wasoriginally defective due to both insertional and substitutionalsequence alteration near its N-terminus, which provided an increasein the sensitivity of detection of (suppressor) secondary mutationswhich conferred improved function. We have now studied the effectsof the suppressor mutations when the primary deleterious mutationsare sequentially reversed. This was done in intact ß-galactosidase,as we have shown that mutations in the -peptide have relatedfunctional effects in the whole protein. Evidence was obtainedshowing that the effects of at least some suppressor mutationswere not simply additive when the mutations are placed intothe original wild-type protein environment. One suppressor appearedto function less effectively in the normal environment, whileanother when tested in the same manner functioned at a relativelyincreased level. This failure to show simple additivity maybe attributable to the physical proximity of the original defectivemutations and the introduced suppressors. Nevertheless, evenin such cases it may be feasible to use a defective proteinas a sensitive starting point for the identification of mutationswhich improve the wild-type protein.     

Involvement of the NH2-terminal region of oryzacystatin-I in cysteine proteinase inhibition

Cystatins are small protein inhibitors of cysteine protein-ases.The relative importance of the N-terminal region of cystatins,and of a conserved glycine within this region, remains uncleardespite several studies. It was found that deletion of the N-terminal21 amino acids abolishes the inhibitory capacity of oryzacystatin-I.The importance of a conserved glycine residue (GlylO) was alsoexamined by replacing it with 11 other amino acids. Three furtherglycine residues (Gly5, -6 and -11) in this N-terminal regionof oryzacystatin-I were similarly mutated. Only those variantsin which GlylO was substituted show any significant change ininhibitory capacity compared with wild-type oryzacystatin-I.The inhibitory characteristics of hybrid cystatin moleculescomprising regions of chicken egg white cystatin and oryzacystatinwere also examined. It is suggested that in common with animalcystatins, the N-terminal region of the plant cystatin, oryzacystatin-I,and in particular the highly conserved GlylO residue are importantfor effective inhibition of papain     

Secretion and in vivo folding of the Fab fragment of the antibody McPC603 in Escherichia coli: influence of disulphides and cis-prolines

Using the well-characterized antibody McPC603 as a model, wehad found that the F_v fragment can be isolated from Escherichiacoli as a functional protein in good yields, whereas the amountof the correctly folded F_ab fragment of the same antibody producedunder identical conditions is significantly lower. In this paper,we analyse the reasons for this difference. We found that avariety of signal sequences function in the secretion of theisolated chains of the F_ab fragment or in the co-secretion ofboth chains in E.coli. The low yield of functional F_ab fragmentis not caused by inefficient expression or secretion in E.coli,but by inefficient folding and/or assembly in the periplasm.We compared the folding yields for the F_v and the F_ab fragmentin the periplasm under various conditions. Several diagnosticframework variants were constructed and their folding yieldsmeasured. The results show that substitutions affecting cis-prolineresidues and those affecting various disulphide bonds in theprotein are by themselves insufficient to dramatically changethe partitioning of the folding pathway to the native structure,and the cause must lie in a facile aggregation of folding intermediatescommon to all structural variants. However, all structural variantscould be obtained in native form, demonstrating the generalutility of the secretory expression strategy.     

Enhancement of protein stability by the combination of point mutations in T4 lysozyme is additive

A number of mutations have been shown previously to stabilizeT4 lysozyme. By combining up to seven such mutations in thesame protein, the melting temperature was incrementally increasedby up to 83°C at pH 5.4 (G = 3.6 kcal/mol). This shows thatit is possible to engineer a protein of enhanced thermostabilityby combining a series of rationally designed point mutations.It is also shown that this stabilization is achieved with onlyminor, localized changes in the structure of the protein. Thisis consistent with the observation that the change in stabilityof each of the multiple mutants is, in each case, additive,i.e. equal to the sum of the stability changes associated withthe constituent single mutants. One of the seven substitutions,Asn116 Asp, changes a residue that participates in substratebinding; not surprisingly, it causes a significant loss in activity.Ignoring this mutation, there is a gradual reduction in activityas successively more mutations are combined.