Improvement of Fc-erythropoietin structure and pharmacokinetics by modification at a disulfide bond

Erythropoietin (Epo) is a cytokine that controls the production of red blood cells (RBCs). Epo acts continuously on RBC precursors to prevent apoptosis, so it is important to maintain high levels of Epo activity when treating anemic patients. We describe here modified human Epo [Epo(NDS)] with mutations His32Gly, Cys33Pro, Trp88Cys and Pro90Ala that result in the rearrangement of the disulfide bonding pattern from Cys29-Cys33 to Cys29-Cys88 and that, in the context of an Fc-Epo(NDS) fusion protein, lead to significantly improved properties. Fc-Epo was secreted from NS/0 myeloma cells as about 35% high molecular weight aggregates, was unstable upon removal of N-linked oligosaccharides and showed poor pharmacokinetics and little efficacy in mice. In contrast, a corresponding Fc-Epo(NDS) was secreted almost exclusively as a unit dimer, was relatively stable to removal of N-linked oligosaccharides, had much improved pharmacokinetic properties and had a significantly improved effect on RBC production. These results indicate that rearrangement of the disulfide bonding pattern in a therapeutic protein can have a significant effect on pharmacokinetics and, potentially, the dosing schedule of a protein drug.     

Expression and characterization of Lactobacillus 30a histidine decarboxylase in Escherichia coli

The genes coding for histidine decarboxylase from a wild-typestrain and an autoactivation mutant strain of Lactobacillus30a have been cloned and expressed in Escherichia coli. Themutant protein, G58D, has a single Asp for Gly substitutionat position 58. The cloned genes were placed under control ofthe ß-galactosidase promoter and the products arenatural length, not fusion proteins. The enzyme kinetics ofthe proteins isolated from E. coli are comparable to those isolatedfrom Lactobacillus 30a. At pH 4.8 the K_m of wild-type enzymeis 0.4 mM and the k_cat = 2800 min^–1; the correspondingvalues for G58D are 0.5 mM and 2750 min^–1. The wild-typeand G58D have autoactivation half-times of 21 and 9 h respectivelyunder pseudophysiological conditions of 150 mM K⁺ and pH 7.0.At pH 7.6 and 0.8 M K⁺ the half times are 4.9 and 2.9 h. Therelatively slow rate of autoactivation for purified proteinand the differences in cellular and non-cellular activationrates, coupled with the fact that wild-type protein is readilyactivated in wild-type Lactobacillus 30a but poorly activatedin E. coli, suggest that wild-type Lactobacillus 30a containsa factor, possibly an enzyme, that enhances the activation rate.     

Site-directed alteration of Glu197 and Glu66 in a pyruvoyl-dependent histidine decarboxylase

Site-directed mutagenesis has been used to explore the roleof two carboxylates in the active site of histidine decarboxylasefrom Lactobacillus 30a. The most striking observation is thatconversion of Glu197 to either Gln or Asp causes a major decreasein catalytic rate while enhancing substrate binding. This isconsistent with models based on X-ray diffraction results whichsuggest that the acid may protonate a reaction intermediateduring catalysis. The Asp197 protein undergoes a suicide reactionwith substrate, apparently triggered by inappropriate protonationof the intermediate. This leads to decarboxylation-dependenttransamination which converts the pyruvoyl cofactor to an alanine,inactivating the enzyme. Conversion of Glu66 to Gln affectsparameters of kinetic cooperativity. The mutation fixes theHill number at – 1.5, midway between the pH-dependentvalues of the wild-type enzyme.     

Breakdown in the relationship between thermal and thermodynamic stability in an interleukin-1{beta} point mutant modified in a surface loop

Sequence variants of the ß-barrel protein interleukin-1ßhave been analyzed for their stabilities toward irreversiblethermal inactivation by monitoring the generation of light scatteringaggregates on heating. The derived temperatures for the onsetof aggregation (T_agg values) correlate well with the free energiesof unfolding of these proteins with the exception of one variant,Lys97—Val (K97V), which undergoes aggregation at a temperature7°C lower than expected based on its thermodynamic stability.This lower than expected thermal stability may be due to generationof an aggregation-prone unfolding intermediate at a temperaturelower than the T_m of the global transition. This hypothesisis supported by the location of residue 97 in the long 86–99loop which has structural features suggesting it may comprisea small, independent folding unit or microdomain. The excellentcorrelation of thermal and thermodynamic stabilities of sevenof the eight variants tested is consistent with accepted modelsfor thermal inactivation of proteins. At the same time the poorfit of the K97V variant underscores the risk in using thermalstability data in quantitative analysis of mutational studiesof the folding stability of proteins.     

The application of hydrogen bonding analysis in X-ray crystallography to help orientate asparagine, glutamine and histidine side chains

Protein X-ray crystallography produces an electron density mapthat rarely detects individual hydrogen atoms or distinguishesbetween carbon, nitrogen and oxygen atoms in the electron density.This makes it difficult to orientate the side chains of Asn,Gln and His, which appear symmetrical in the electron density;their orientation is usually judged on the basis of hydrogenbonding. Based on the observation that almost all buried donorsand acceptors are satisfied, we have developed a simple algorithmto compare the alternative conformations of these residues and,where possible, identify the most favourable. In a cross-sectionof protein structures we found a few side chains (15.0% of Asnand Gln and 9.9% of His) which would be more favourable in thealternative orientation. We have also found that this proportionrises slightly with worsening resolution.     

Rapid crystallization of T4 lysozyme by intermolecular disulfide cross-linking

In an attempt to facilitate crystallization, engineered cysteineswere used to promote formation of a ‘back–to–back’dimer that occurs in different crystal forms of wild–typeand mutant T4 lysozymes. The designed double mutant, N68C/A93C,in which the surface residues Asn68 and Ala93 were replacedby cysteines, formed dimers in solution and crystallized isomorphouslyto wild–type, but at a much faster rate. Overall, themutant structure remained very similar to wildtype despite theformation of two intermolecular disulfide bridges. The crystalsof cross–linked dimers had thermal factors somewhat lowerthan wild–type, indicating reduced mobility, but did notdiffract to noticeably higher resolution. Introduction of thesame cross-links was also used to obtain crystals in a differentspace group of a T4 lysozyme mutant that could not be crystallizedpreviously. The results suggest that the formation of the lysozymedimer is a critical intermediate in the formation of more thanone crystal form and that covalent cross–Unking of theintermediate accelerates nucleation and facilitates crystalgrowth. The disulfide crosslinks are located on the ‘back’of the molecule and formation of the cross–linked dimerappears to leave the active sites completely unobstructed. Nevertheless,the cross–linked dimer is completely inactive. One explanationfor this behavior is that the disulfide bridges prevent hinge-bendingmotion that may be required for catalysis. Another possibilityis that the formation of the dimer increases the overall bulkof the enzyme and prevents its access to the susceptible glycosidkbonds within the cell wall substrate     

Structure activity relationships of monocyte chemoattractant proteins in complex with a blocking antibody

Monocyte chemoattractant proteins (MCPs) are cytokines that direct immune cells bearing appropriate receptors to sites of inflammation or injury and are therefore attractive therapeutic targets for inhibitory molecules. 11K2 is a blocking mouse monoclonal antibody active against several human and murine MCPs. A 2.5 A structure of the Fab fragment of this antibody in complex with human MCP-1 has been solved. The Fab blocks CCR2 receptor binding to MCP-1 through an adjacent but distinct binding site. The orientation of the Fab indicates that a single MCP-1 dimer will bind two 11K2 antibodies. Several key residues on the antibody and on human MCPs were predicted to be involved in antibody selectivity. Mutational analysis of these residues confirms their involvement in the antibody-chemokine interaction. In addition to mutations that decreased or disrupted binding, one antibody mutation resulted in a 70-fold increase in affinity for human MCP-2. A key residue missing in human MCP-3, a chemokine not recognized by the antibody, was identified and engineering the preferred residue into the chemokine conferred binding to the antibody.     

Identification of the reactive cysteine in clostridial glutamate dehydrogenase by site-directed mutagenesis and proof that this residue is not strictly essential

Cys320 of clostridial glutamate dehydrogenase, a residue closeto the coenzyme binding sitehas been replaced by serine.Themutant enzyme was successfully overproduced and purified byusing the normal protocol for the wild-type enzyme and alsobehaved indistinguishably from wild-type enzyme on native andSDS-PAGE. The specific activity was significantly enhanced inassays at both pH 7 (+90%) and pH 8 (+38%). Detailed initial-ratekinetics revealed that at pH 7 this increase was mainly attributableto a higher maximum rate, since the K_m values for both substrateswere marginally increased. In the mutant enzyme the inactivatingreaction with DTNB that characterizes the wild-type enzyme iscompletely eliminated. This proves that inactivation of thewild-type enzyme is due to modification of Cys320, that neverthelessCys320 is not strictly essential for catalytic activity andthat the remaining cysteine residue at position 144 is inaccessibleto DTNB. Provision of an engineered subunit with a correct nativestructure but with its DTNB titre decreased from 1 to 0 mol/molnow offers a valuable tool for counting subunits in hybrid oligomers     

Contribution of a salt bridge to binding affinity and dUMP orientation to catalytic rate: mutation of a substrate-binding arginine in thymidylate synthase

Invariant arginine 179, one of four arginines that are conservedin all thymidylate synthases (TS) and that bind the phosphatemoiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP),can be altered even to a negatively charged glutainic acid withlittle effect on k_cat. In the mutant structures, ordered wateror the other phosphate binding arginines compensate for thehydrogen bonds made by Arg179 in the wild-type enzyme and thereis almost no change in the conformation or binding site of dUMP.Correlation of dUMP K_ds for TS R179A and TS R179K with the structuresof their binary complexes shows that the positive charge onArg179 contributes significantly to dUMP binding affinity. k_cat/Kmfor dUMP measures the rate of dUMP binding to TS during theordered bi-substrate reaction, and in the ternary complex dUMPprovides a binding surface for the cofactor. k_cat/Km reflectsthe ability of the enzyme to accept a properly oriented dUMPfor catalysis and is less sensitive than is K_d to the changesin electrostatics at the phosphate binding site.     

Structure and humanization of a rat monoclonal Fab to human interleukin-5

The X-ray crystal structure of a rat monoclonal Fab JES1-39D10,raised against recombinant human interleukin-5, has been determinedwith the use of molecular replacement techniques and refinedat 2.7 Å resolution by simulated annealing. The overallstructure is similar to a murine Fab HyHEL-10 that is specificfor hen egg white lysozyme. An interesting feature of the structureis the presence of leucine residues to support the H1 complementarity-determiningregion (CDR) loop. To our knowledge this is the first Fab crystalstructure containing this unusual HI loop support pattern. Theactivity of three humanized versions of 39D10 is explained byanalysis of Fv interface residues and H1 support patterns of39D10 and the human template HLL.     

The creation of a novel fluorescent protein by guided consensus engineering

Consensus engineering has been used to increase the stability of a number of different proteins, either by creating consensus proteins from scratch or by modifying existing proteins so that their sequences more closely match a consensus sequence. In this paper we describe the first application of consensus engineering to the ab initio creation of a novel fluorescent protein. This was based on the alignment of 31 fluorescent proteins with >62% homology to monomeric Azami green (mAG) protein, and used the sequence of mAG to guide amino acid selection at positions of ambiguity. This consensus green protein is extremely well expressed, monomeric and fluorescent with red shifted absorption and emission characteristics compared to mAG. Although slightly less stable than mAG, it is better expressed and brighter under the excitation conditions typically used in single molecule fluorescence spectroscopy or confocal microscopy. This study illustrates the power of consensus engineering to create stable proteins using the subtle information embedded in the alignment of similar proteins and shows that the benefits of this approach may extend beyond stability.     

Two-dimensional surface display of functional groups on a beta-helical antifreeze protein scaffold

We tested a disulfide-rich antifreeze protein as a potential scaffold for design or selection of proteins with the capability of binding periodically organized surfaces. The natural antifreeze protein is a beta-helix with a strikingly regular two-dimensional grid of threonine side chains on its ice-binding face. Amino acid substitutions were made on this face to replace blocks of native threonines with other amino acids spanning the range of beta-sheet propensities. The variants, displaying arrays of distinct functional groups, were studied by mass spectrometry, reversed-phase high performance liquid chromatography, thiol reactivity and circular dichroism and NMR spectroscopies to assess their structures and stabilities relative to wild type. The mutants are well expressed in bacteria, despite the potential for mis-folding inherent in these 84-residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded beta-helical scaffold, thermally stable and remarkably tolerant of amino acid substitutions, is therefore useful for design and engineering of macromolecules with the potential to bind various targeted ordered material surfaces.     

Apomyoglobin as a molecular recognition surface: expression, reconstitution and crystallization of recombinant porcine myoglobin in Escherichia coli

Recombinant porcine myoglobin has been produced in Escherichiacoli using the cII fusion expression system of Nagai and Th?gersen[Nature, 309, 810–812 (1984)]. After processing and reconstitutionwith haem, the protein is gel-electrophoretically and spectrophotometricallyindistinguishable from native pig myoglobin. Large crystalsof both native and recombinant porcine myoglobin were grownfrom 50 mM sodium phosphate, pH 7.1, 80% ammonium sulphate.The crystals belong to space group C2 (a = 156.9 ?, b = 42.0?, c = 92.2 ?, ß = 127.9?) and diffract to a nominal2.5 ? resolution. We plan to explore apomyoglobin as a bindingsurface in studies combining site-directed mutagenesis and X-rayanalysis. These experiments will be extended by studying thebinding of haem analogues to the mutant apoproteins.     

Display of {beta}-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-{beta}-lactamase fusions

Bacterial cell-surface exposure of foreign peptides and solubleproteins has been achieved recently by employing a fusion proteinmethodology. An Lpp'–OmpA(46–159)–Bla fusionprotein has been shown previously to display the normally periplasmicenzyme ß-lactamase (Bla) on the cell surface of theGram-negative bacterium Escherichia coli. Here, we have investigatedthe role of the OmpA domain of the tripartite fusion proteinin the surface display of the passenger domain (Bla) and havecharacterized the effects of the fusion proteins on the integrityand permeability of the outer membrane. We show that in additionto OmpA(46–159), a second OmpA segment, consisting ofamino acids 46–66, can also mediate the display of Blaon the cell surface. Other OmpA domains of various lengths (aminoacids 46–84, 46–109, 46–128, 46–141and 46–145) either anchored the Bla domain on the periplasmicface of the outer membrane or caused a major disruption of theouter membrane, allowing the penetration of antibodies intothe cell. Detergent and antibiotic sensitivity and periplasmicleakage assays showed that changes in the permeability of theouter membrane are an unavoidable consequence of displayinga large periplasmic protein on the surface of E.coli. This isthe first systematic report on the effects that cell surfaceengineering may have on the integrity and permeability propertiesof bacterial outer membranes.     

Comparison of backbone structures of glucose isomerase from Streptomyces and Arthrobacter

The C backbones of the glucose isomerase molecules of Streptomycesrubiginosus and Arthrobacter have been determined by X-ray crystallographyand compared. Each molecule is a tetramer of eight-stranded/ß barrels, and the mode of association of the tetramersis identical in each case. The Arthrobacter electron densityshows four additional amino acids at the carboxyl terminus.There is also an insertion of six amino acids at position 277,and two individual insertions at about positions 348 and 357(numbering according to the Streptomyces structure). There isa close structural homology throughout the whole molecule, whichis most accurate up to position 325. The r.m.s. displacementfor 315 homologous C positions up to this position is 0.92 Å.     

The S variant of human {alpha}1-antitrypsin, structure and implications for function and metabolism

The S variant of the human ₁-antitrysin with E-264 – V,is responsible for a mild ₁-antitrypsin deficiency quite commonin the European population. S protein specifically cleaved atthe susceptible peptide bond was crystallized and its crystalstructure determined and refined to 3.1 Å resolution.The S variant crystallizes isomorphous to the normal M variant.The difference Fourier electron density map shows the E –V change as outstanding residual density. In addition, smallstructural changes of the main polypeptide chain radiate fromthe site of mutation and affect parts far removed from it. Bythe mutation, internal hydrogen bonds and salt linkages of E-264to Y-38 and K-487, respectively, are lost. They cause the far-reachingslight distortions and are probobly related to the reduced thermalstability of the S mutant. They may also be responsible forslower folding of the polypeptide chain and the clinical symptomsof ₁-antitrypsin deficiency. In a theoretical study by moleculardynamics methods simulations of the M and S proteins were madeand the results analysed with respect to structrual and dynamicproperties and compared with the experimental results. Thereis a significant correlation between experimental and theoreticalresults in some respects.     

Enzyme IIIlac of the staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: site-specific mutagenesis of histidine residues, biochemical characterization and 1H-NMR studies

The lactose-specific pbosphocarrier protein enzyme III of thebacterial phosphoenol-pyruvate-dependent phosphotransferasesystem of Staphylococcus aureus was modified by sitespecificmutagenesis on the corresponding lacF gene in order to replacethe histidine residues 78 and 82 of the amino acid sequencewith a serine residue. Wild-type and both mutant genes wereoverexpressed in Escherichia coli and the gene products werepurified to homogeneity. The conformation of wild-type and mutantproteins were monitored by ¹H-NMR spectroscopy. In vitro phosphorylationstudies on mutant lactose-specific enzyme III, as well as evidencefrom NMR spectroscopy, lead to the conclusion that His78 isthe activesite for phosphorylation of lactose-specific enzymeIII by phospho-HPr (histidine-containing protein). The roleof His82 probably is the enhancement of velocity and efficiencyof the phosphotransfer from lactose-specific enzyme in to lactosespecifkenzyme II. This result refutes the conclusion of former workbased on data by protelytk cleavage and sequencing of the ³²P-labeledpeptide of lactose-specific enzyme DTI that His82 is the active-sitefor phosphorylation.     

Structure-function analysis of tissue-type plasminogen activator by linker-insertion, point and deletion mutagenesis

We undertook a structure–function analysis of human tissueplasminogen activator (tPA) using linker-scanning and deletionmutagenesis. Synthetic oligonucleotide linkers were introducedinto the tPA cDNA at pre-existing restriction enzyme sites.This generated a series of tPA variants which contained smallprimary sequence alterations consisting of point mutations,deletions or insertions. The majority of the linker-insertionvariants demonstrate a significant reduction in amidolytic andfibrinolytic activity in comparison to wild-type tPA. The exceptionsare the variants with linker-inserts placed at the BglII(115)and StyI(277) sites of the tPA cDNA (4SLEG5 and 57LEA58 respectively),which encode insertions at the boundaries of the finger domain.The variants with linker-inserts in the light chain (proteasedomain) of tPA are the lowest in enzymatic activity. Particularlysensitive to mutation are highly conserved amino acids. Heavychain deletion variants were constructed from point mutantsat the domain boundaries of tPA. Deletion of the kringle domainslowers the fibrinolytic activity to a greater extent than deletionof the finger or growth factor domains. We conclude that alterationsin any domain of the tPA molecule, and particularly in the highlyconserved residues within these domains, can affect fibrinolyticactivity.     

Structural effects induced by removal of a disulfide-bridge: the X-ray structure of the C30A/C51A mutant of basic pancreatic trypsin inhibitor at 1.6 A

The X-ray structure of a variant of basic pancreatic trypsininhibitor (BPTI) has been analyzed to determine the structuralaccommodation resulting from removal of a disulfide crosslinkin a protein. The disulfide removed, Cys30–Cys51, hasbeen implicated in both the folding pathway of the protein andits overall thermal stability. In the variant studied, C30A/C51A,the disulfide cysteines were replaced by less bulky alanines.The atomic displacements observed for C30A/C51A indicate a setof concerted shifts of two segments of chain, which togethersignificantly diminish a packing defect at the site of the removedcysteine sulfur atoms. The observed structural changes are distributedasymmetrically around the sites of mutation, indicating thatthe adjacent ß-sheet is more resistant to the perturbationthan the -helix on the opposite side of the disulfide bond.The thermal parameters of groups involved in the structuralaccommodation are not significantly altered. A comparison ofthe X-ray structures reported for native BPTI determined inthree different crystal forms indicates that the magnitude ofits conformational variability exceeds that of the structuralchanges caused by the disulfide removal. This emphasizes thenecessity of using isomorphous crystal systems to determinethe relatively small effects due to mutation.     

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.