Semi-empirical simulation of Zn/Cd binding site preference in the metal binding domains of mammalian metallothionein

Metallothionein, a two-domain protein, naturally binds sevengram atoms of divalent ions such as Zn and Cd. Four of the metals(Ml, M5, M6 and M7) are found in the -domain and three (M2,M3 and M4) in the ß-domain. Previous studies haveshown that metals in the -domain are more readily exchangeable,and the level of avidity is site specific. By semi-empiricalMNDO modified neglect of diatomic overlap calculations, we foundthe tendency of binding energy for Cd to be M3 > M2 >M4 in the ß-cluster and M5 > M7 > Ml, M6 inthe -cluster. Thus, the replacement of Zn by Cd can be expectedto follow the order M4 M2 M3 in the ß-domain andMS M7 M1 or M6 in the -domain. This is reflected by energydifferences computed with a series of simulated structures derivedfrom either X-ray crystallography or NMR coordinates.     

Recombinant C-terminal domain of pancreatic lipase retains full ability to bind colipase

The organization of the pancreatic lipase in two well defineddomains has been correlated to a specific function for eachdomain, catalytic activity for the N-terminal domain and colipasebinding for the C-terminal domain. In order to see if such anorganization implies that the two domains can behave as separateentities, we expressed the N- and C-terminal domains in insectcells. The recombinant proteins secreted in the cell supernatantspresent the expected molecular properties. However, whereasthe C-terminal domain retains its function of colipase binding,the N-terminal domain appears to be unable to ensure catalysis.The lack of activity of the recombinant N-terminal domain couldresult either from a (partially) incorrect folding or from anincapacity to function by itself. These results suggest that,although both are structurally well defined, the two domainsof the pancreatic lipase behave differently when they are expressedas separate entities.     

Expression and characterization of apolipoprotein(a) kringle IV types 1, 2 and 10 in mammalian cells

We have designed expression constructs containing sequencescorresponding to apolipoprotein(a) kringle FV types 1, 2 and10 and used these constructs to transfect human embryonic kidneycells. We have also expressed a mutant form of kringle FV type2 in which the N-linked glycosylation site has been removedby replacement of an asparagine residue with an alanine. Immunoprecipitationanalysis of [³⁵S]Cys-labeled transfected cell culture supernatantsresulted in the observation of two bands for kringle IV type1 (M_r 30 000 and 26 000), two bands for kringle IV type 2 (M_r25 000 and 22 000), two bands for kringle IV type 10 (M_r 27000 and 23 000) and one band for the glycosylation mutant (M_r22 000). In all cases, observed molecular weights greatly exceededthose predicted from amino acid sequence, suggesting the presenceof both N- and O-linked glycans. None of the recombinant singlekringles were observed to bind to fibrinogen as determined byELISA or by co-immunoprecipitation in the case of kringle IVtype 10 and only kringle IV type 10 was able to bind to lysine-Sepharose.These data suggest that apo(a) binding to fibrinogen/fibrinmay require motif(s) in addition to apo(a) kringle IV type 10.     

Independent self-assembly of cadmium-binding {alpha}-fragment of metallothionein in Escherichia coli without participation of {beta}-fragment

We examined the independent self-assembly of the - and ß-fragmentsof human metallothionein (MT) into cadmiumbinding conformationin an Escherichia coli expression system, in addition to wild-typeMT expression. The expressed -fragment formed independentlythe structure of a metal-binding cluster without the aid ofthe ß-fragment. The -fragment and wild-type MT expressedin E.coli were purified and analyzed for their biochemical andspectroscopic properties. The apparent cadmium binding of the-fragment was approximately 12-fold greater than that for thewild-type MT, whereas in other respects the studied biochemicalproperties were similar. In contrast, we were unable to obtainany independently expressed ß-fragment as the cadmium-bindingform in this study. Possible explanations for this phenomenonare discussed.     

Structural models of the evolutionarily conservative central domain of silk-moth chorion proteins

Silk-moth chorion proteins belong to a small number of families:A, B, C, Hc-A and Hc-B. The central domain is an evolutionarilyconservative region in each family, of variable length and compositionbetween families. This domain shows dear 6-fold periodicitiesfor various amino acid residues, e.g. glycine. The periodicities,together with the well-documented prevalence of ß-sheetand ß-turn secondary structure of chorion proteins,strongly support a structural model in which four-residue ß-strandsalternate with ß-turns, forming a compact antiparallel,probably twisted ß-sheet. Conformational analysis,aided by interactive graphics refinement and recent experimentalfindings, further suggest that this structure consists of ß-strands,alternating with I' and II' ß-turns, and apparentlyforms the basis for the molecular and supramolecular assemblyof chorion.     

Alcohol-induced changes of {beta}-lactoglobulin - retinol-binding stoichiometry

It has been demonstrated using CD that ethanol induces importantsecondary structure changes of ß-lactoglobulin. CDspectra indicate that ß-lactoglobulin secondary structure,which is mainly composed of ß-strands, becomes mostly-helical under the influence of the solvent polarity changes.The midpoint of ß-strand/-helix transition in ß-lactoglobulinis observed at dielectric constant {small tilde}60 (35% ethanol;v/v). According to CD measurements, the ethanol-dependent secondarystructure changes are reversible. The alkylation of lysines-NH₂ in ß-lactoglobulin weakens the central ß-barrelstructure, since the ß-strand/-helix transition midpointof alkylated ß-lactoglobulin is shifted to lower ethanolconcentration (25% ethanol; v/v). ß-Lactoglobulinstructural changes are triggering the dissociation of the ß-lactoglobulin- retinol complex as judged from complete quenching of its fluorescencein ethanol concentration >30% (v/v). However, in 20% ethanol(v/v), ß-lactoglobulin still retains most of its nativesecondary structure as shown by CD and, in this condition, oneß-lactoglobulin molecule binds an additional secondretinol molecule. This suggests that the highly populated speciesobserved around 20% ethanol (v/v) might represent an intermediatestate able to bind two molecules of retinol.     

A mutational analysis of receptor binding sites of interleukin-1{beta}:differences in binding of human interleukin-1{beta} muteins to human and mouse receptors

The 3-D crystal structure of interleukin-1ß(IL-1ß)has been used to define its receptor binding surface by mutationalanalysis. The surface of IL-1ß was probed by site-directedmutagenesis. A total of 27 different IL-1ß muteinswere constructed, purified and analyzed. Receptor binding measurementson mouse and human cell lines were performed to identify receptoraffinities. IL-1ß muteins with modified receptor affinitywere evaluated for structural integrity by CD spectroscopy orX-ray crystallography. Changes in six surface loops, as wellas in the C- and N-termini, yielded muteins with lower bindingaffinities. Two muteins with intact binding affinities showed10- to 100-fold reduced biological activity. The surface regioninvolved in receptor binding constitutes a discontinuous areaof 1000 Å2 formed by discontinuous polypeptide chain stretches.Based on these results, a subdivision into two distinct localareas is proposed. Differences in receptor binding affinitiesfor human and mouse receptors have been observed for some muteins,but not for wild-type IL-1ß. This is the first timea difference in binding affinity of IL-1ß muteinsto human and mouse receptors has been demonstrated     

Strong inhibition of fibrinogen binding to platelet receptor {alpha}IIb{beta}3 by RGD sequences installed into a presentation scaffold

In order to probe the structural constraints on binding of RGDsequences to the platelet receptor _IIbß₃ we have usedrecombinant DNA techniques to install the RGD sequence into‘presentation scaffolds’, small proteins of known3-D structure chosen to present guest sequences in constrainedorientations. Using Escherichia coli expression systems we madesequence variants in which loop residues of the immunoglobulinV_L domain REI and of human interleukin-1ß were replaced(without changing polypeptide length) by the RGD sequence atpositions predicted, based on small molecule studies, to orientthe RGD moiety into an active conformation. These variants donot compete for fibrinogen binding to _IIbß₃ up toalmost 1 mM concentration. Unfolded or proteolytically fragmentedforms of these same proteins do compete, however, showing thatthe RGD sequences in the mutants must be prohibited from bindingby constraints imposed by scaffold structure. To suppress theeffects of such structural constraints we constructed two sequencevariants in which RGD-containing sequences 42–57 or 44–55from the snake venom platelet antagonist kistrin were inserted(this increasing the length of the loop) into the third complementaritydetermining loop of REI. Both of these variants compete stronglyfor fibrinogen binding with IC₅₀s in the nM range. These results,plus data on kistrin-related peptides also presented here, suggestthat the molecular scaffold REI is capable of providing to aninstalled sequence a structural context and conformation beneficialto binding. The results also suggest that in order to bind wellto _IIbß₃, RGD sequences in protein ligands must eitherproject significantly from the surface of the scaffold and/orretain a degree of conformational flexibility within the scaffold.Molecular scaffolds like REI should prove useful in the elucidationof structure-function relationships and the discovery of newactive sequences, and may also serve as the basis for noveltherapeutic agents.     

Molecular dynamics simulations of the whey protein {beta}-lactoglobulin

Molecular dynamics simulations have been used to model the motionsand conformational behavior of the whey protein bovine ß-lactoglobulin.Simulations were performed for the protein by itself and complexedto a single retinol ligand located in a putative interior bindingpocket. In the absence of the retinol ligand, the backbone loopsaround the opening of this ulterior pocket shifted inward topartially close off this cavity, similar to the shifts observedin previously reported molecular dynamics simulations of theuncomplexed form of the homologous retinol binding protein.The protein complexed with retinol does not exhibit the sameconformational shifts. Conformational changes of this type couldserve as a recognition signal allowing in vivo discriminationbetween the free and retinol complexed forms of the 3-lactoglobulinmolecule. The unusual bending of the single a-helix observedin the simulations of retinol binding protein were not observedin the present calculations     

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.     

A single Fc binding domain-alkaline phosphatase gene fusion expresses a protein with both IgG binding ability and alkaline phosphatase enzymatic activity

A recombinant gene fusion was created and cloned using a previouslyconstructed gene encoding a monodomain IgG Fc binding proteinand the gene coding for bacterial alkaline phosphatase. Theconstruct was able to express and secrete a fusion protein thatexhibited both IgG binding and alkaline phosphatase enzymaticactivities. Greater than 60% of the protein demonstrating bothbiological activities was detected from periplasmic space preparations.Nanogram concentrations of the Fc binding-alkaline phosphatasefusion protein allowed primary IgG antibody detection withoutthe use of conjugated secondary antibodies. Removal of the domaincoding for alkaline phosphatase resulted in decreased resistanceof the protein to proteolytic degradation and the loss of IgGFc binding ability. Using affinity-purified fusion protein,the specificity of binding to IgG, IgM and IgA was examined;binding was strong to IgG and barely detectable against IgMor IgA. Affinity for binding of the fusion protein to IgG (k_d= 6.7 x10^-8 M) was determined to be equal to or greater thanpreviously reported for protein A.     

Gene synthesis and functional expression of a protein exhibiting monodomain IgG Fc binding

A gene encoding a bacterial IgG Fc binding domain was designedand synthesized. The synthetic DNA fragment was cloned 3' toan inducible trpE promoter such that expression of the genein Escherichia coli produced abundant Fc binding protein fusedto the first seven amino acids of the trpE protein. The recombinantprotein contained a single Fc binding domain and demonstratedefficient binding to'human IgG in Western blot analysis. Thisprotein degraded rapidly following cell lysis in the absenceof protease inhibitors, but could be effectively protected bythe addition of protease inhibitor. After purification of theprotein by IgG affinity chromatography, IgG Fc binding abilitywas retained for at least 24 h at either 23 or 37°C andon heating for 15 min at temperatures up to 65°C. No immunoprecipitationwas observed in interactions between the monodomain Fc bindingprotein and IgG molecules. Unlike staphylococcal protein A,no detectable binding of the monodomain IgG Fc binding proteinwas observed to either IgM or IgA. Truncated proteins, expressedfrom a series of 3' deletions of the synthetic gene, were usedto estimate the minimum portion of a monodomain Fc binding proteinthat retained Fc binding ability.     

Production and properties of a factor X-cellulose-binding domain fusion protein

A fusion protein, FX–CBD_Cex, which comprises factor Xwith a cellulose-binding domain (CBD_Cex) fused to its C-terminus,was produced in BHK cells. It was purified from the culturemedium by affinity chromatography on cellulose. FX–CBD_Cexcould be activated to FXa–CBD_Cex with Russell viper venom.FXa–CBD_Cex was as active as FXa against a chromogenicsubstrate and against proteins containing the Ile–Glu–Gly–Argsequence hydrolysed by FXa. FXa–CBD_Cex retained its activitywhen adsorbed to cellulose.     

In vivo system for the detection of low level activity barnase mutants

In previous studies, the replacement of His35 in the pore-formingprotein -hemolysin (HL) with Leu, lie, Pro, Arg, Ser, Thr orCys yielded inactive polypeptides. Here, we show that modificationof the inactive single-cysteine mutant HL-H35C with iodoacetamide,to form H35CamC, generates significant pore-forming activity.The closely related polypeptides H35N and H35Q have, respectively,essentially no activity and greatly reduced activity. The modifiedresidue in H35CamC, S-carboxamidomethylcysteine, mimicks histidinein volume, polarity and hydrogen bonding potential, but is unableto ionize. Unmodified H35C is defective in the final step ofpore formation: the conversion of an inactive heptameric membrane-boundassembly intermediate to a structure containing open channels.It is this step in assembly, that is ameliorated in H35CamC.     

High-level bacterial expression, purification and characterization of human calreticulin

To investigate its cellular function and role in autoimmunedisease pathogenesis, we have bacterlally expressed human calreticulin,a major calcium-binding protein in the endoplasmic reticulumand a human autoantigen. This is the First report describingthe heterologous expression of calreticulin from any source.The recombinant calreticulin constituted {small tilde}32% ofthe soluble Escherichia coli proteins, and was purified to apparenthomogeneity by ion exchange and hydrophobic liquid chromatography.As does the bona fide protein, the recombinant calreticulinbinds calcium and undergoes changes in its conformation uponZn²⁺ binding. We take this as a strong indication that the foldingof the E.coli-expressed calreticulin is very similar, if notidentical, to that of the authentic protein. Moreover, the bacteriallyexpressed calreticulin readily reacted with anti-human and anti-rabbitantibodies, and the anti-recombinant calreticulin antibodiesimmunoreacted with HeLa calreticulin. The availability of thisexpression system will allow us to carry out site-specific anddeletion mutagenesis analysis in structure-function studiesof calreticulin.     

Domain organization of the extracellular region of CD45

CD45 is a large, heavily glycosylated, transmembrane proteinphosphotyrosine phosphatase found on all nucleated cells ofhaematopoietic origin. In lymphocytes, the cytoplasmic phosphataseis necessary for efficient signalling through the antigen receptorbut in contrast little is known about the interactions of theextracellular region of the molecule. This consists of a mucin-likeregion, a novel cysteine-containing region and a region containingthree putative fibronectin type III domains. To confirm thisorganization and to identify parts potentially important forfunction, we have expressed fragments of the extracellular domainof rat CD45 as recombinant soluble proteins. Proteins correspondingto two, three and four domains of CD45 were expressed in secretedforms. Single domains and constructs for proteins with truncationsof the predicted domains were not expressed. This is consistentwith the proposed structural organization. Determination ofthe positions of the disulphide bonds in the N-terminal cysteine-containingregion and the first fibronectin type III domain identifiednovel disulphide bonds within the fibronectin type III domainand an unusual inter-domain disulphide linkage. Circular dichroismspectroscopy indicated that this region of rat CD45 has mainlyß-strand secondary structure and no -helical content.These studies support the proposed domain organization of CD45.     

Efficient production of recombinant human factor VIII by co-expression of the heavy and light chains

We have developed a high-level expression system for human bloodcoagulation factor VID (FVUI) consisting of a 90 kDa heavy (H-)chainand an 80 kDa light (L-)chain. Two expression plasmids wereprepared, one expressing the H-chain and the other expressingthe L-chain. These recombinant plasmids were designed to produceeach chain linked to short additional ammo acid residues derivedfrom the FVm precursor sequence. Furthermore, Kozak's translationinitiation consensus sequence was introduced into the startcodon for the H-chain. These modifications have dramaticallyincreased the levels of expression of these chains. Chinesehamster ovary (CHO) cells co-transfected with these two recombinantplasmids were subjected to gene amplification and cloning. Thefinal cell line, designated CTC-CF8, secretes 15 IU/day/106cells of active FVm which is indistinguishable from plasma-derivedFVm in its structure and biochemical properties. This systemis suitable for large-scale production of pathogen-free recombinanthuman FVm which can be used for the treatment of haemophiliaA patients.     

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.