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Query: UNIPROT:P06889 (Mol)
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Rodent monoclonal antibodies have been "humanized" or "reshaped" for therapy by transplanting the antigen-binding loops from their variable domains onto the beta-sheet framework regions of human antibodies. However, additional substitutions in the human framework regions are sometimes required for high affinity antigen binding. Here we describe antigen binding by a reshaped antibody derived from the mouse anti-lysozyme antibody D1.3, and several variants in which point mutations had been introduced into framework positions to improve its affinity. The affinities were determined from the relaxation kinetics of reactant mixtures using quenching of fluorescence that occurs upon formation of the antibody-antigen complex. The dissociation constant of lysozyme ranged from 3.7 nM (for D1.3) to 260 nM. Measurement of antibody-antigen association kinetics using stopped-flow showed that D1.3 and most of the reshaped antibodies had bimolecular rate constants of 1.4 x 10(6) s-1 M-1, indicating that differences in equilibrium constant were predominantly due to different rates of dissociation of lysozyme from immune complexes. Mutations in a triad of heavy chain residues, 27, 29 and 71, contributed 0.9 kcal/mol in antigen binding free energy, and a Phe to Tyr substitution of light chain residue 71 contributed an additional 0.8 kcal/mol. The combined effect of all these mutations brought the affinity of the reshaped antibody to within a factor of 4 of D1.3. All of these substitutions were in the beta-sheet framework closely underlying the complementarity-determining regions, and do not participate in a direct interaction with antigen. The informed selection of residues in such positions may prove essential for the success of loop transplants in antibodies. Variation of these sites may also have a role in shaping the diversity of structures found in the primary repertoire, and in affinity maturation.
J Mol Biol 1992 Mar 20
PMID:Antibody framework residues affecting the conformation of the hypervariable loops. 156 Apr 63

The structure of the tri-N-acetylchitotriose inhibitor complex of hen egg-white lysozyme has been refined at 1.75 A resolution, using data collected from a complex crystal with ligand bound at less than full occupancy. To determine the exact value of the inhibitor occupancy, a model comprising unliganded and sugar-bound protein molecules was generated and refined against the 1.75 A data, using a modified version of the Hendrickson & Konnert least-squares procedure. The crystallographic R-factor for the model was found to fall to a minimum at 55% bound sugar. Conventional refinement assuming unit occupancy was found to yield incorrect thermal and positional parameters. Application of the same refinement procedures to an earlier 2.0 A data set, collected independently on different complex crystals by Blake et al. gave less consistent results than the 1.75 A refinement. From an analysis of the high resolution structure a detailed picture of the protein-carbohydrate interactions in the non-productive complex has emerged, together with the conformation and mobility changes that accompany ligand binding. The specificity of interaction between the protein and inhibitor, bound in subsites A to C of the active site, is seen to be generated primarily by an extensive network of hydrogen bonds, both to the protein itself and to bound solvent molecules. The latter also play an important role in maintaining the structural integrity of the active site cleft in the apo-protein.
J Mol Biol 1992 Apr 05
PMID:Refinement of an enzyme complex with inhibitor bound at partial occupancy. Hen egg-white lysozyme and tri-N-acetylchitotriose at 1.75 A resolution. 156 48

An attempt has been made to design modified core-packing arrangements in bacteriophage T4 lysozyme. Alternative replacements of the buried residues Leu99, Met102, Val111 and Phe153 were selected using packing calculations and energy minimization. To test the design procedure, a series of multiple mutants was constructed culminating in the replacement L99F/M102L/V111I/F153L. These variants decrease the stability of T4 lysozyme by approximately 0 to 2 kcal/mol. The crystal structures of a number of the variants were determined. In the variant in which Val111 was replaced by Ile, alpha-helix 107-114 moved by approximately 1.5 A, breaking the hydrogen bond between the backbone carbonyl group of Thr109 and the backbone amide group of Gly113. This conformational change was not anticipated by the design procedure. Compensating interactions of magnitude up to 1.1 kcal/mol occur for some sets of mutations, while other sets display nearly additive stability changes. Within experimental error, the stability of the double mutant V111F/F153L is additive, with delta delta G different by only 0.1 kcal/mol from the sum of the two single mutants. The quadruple mutant L99F/M102L/V111I/F153L is destabilized by 0.5 kcal/mol, compared to delta delta G = -1.6 kcal/mol for the sum of the four single mutants. Multiple mutants show smaller overall structural changes from wild-type than M102L or V111I alone. Co-operative changes in structure and stability can be rationalized in terms of specific structural differences between single and multiple mutants. Genuine repacking of the hydrophobic core of T4 lysozyme with minimal effects on structure, stability and activity thus appears to have been achieved.
J Mol Biol 1992 Apr 20
PMID:Design and structural analysis of alternative hydrophobic core packing arrangements in bacteriophage T4 lysozyme. 156 71

In the chromatin domain of the chicken lysozyme gene of myeloid and oviduct cells, which both have the potential to activate the gene, a developmentally stable DNase I-hypersensitive site is formed around 6.1 kb upstream of the gene. This implies that this DNA region, which has previously been demonstrated to function as a transcriptional enhancer element in myeloid cells, is intimately involved in the cell-type-specific activation of the lysozyme gene locus. Deletion analysis identifies a 157-bp minimal fragment that confers the same promacrophage-specific enhancer activity as the originally described 562-bp -6.1-kb enhancer fragment. By introducing specific point mutations, we demonstrate in transient gene transfer experiments that the minimal fragment consists of at least six adjacent elements, each substantially contributing to enhancer function. The compact multifactorial enhancer complex includes a nuclear factor I (NF-I)/TGGCA binding site, homologies to AP1, and octanucleotide or enhancer core consensus motifs. Point mutation of the NF-I binding site results in the loss of NF-I binding in vitro and enhancer activity in vivo after gene transfer. Surprisingly, four overlapping oligonucleotides, each consisting of at least two elements of the -6.1-kb enhancer, confer myeloid-cell-specific enhancer activity. We found several myeloid-cell-specific DNA-binding proteins interacting with the -6.1-kb enhancer, a result consistent with that described above. Therefore, we suggest that more than a single trans-acting factor mediates the cell type specificity of the -6.1-kb enhancer.
Mol Cell Biol 1992 May
PMID:The -6.1-kilobase chicken lysozyme enhancer is a multifactorial complex containing several cell-type-specific elements. 156 54

As part of a study of the genes involved in antibacterial defense in Drosophila melanogaster, we have isolated genomic clones harboring a family of chicken-type lysozyme genes, using a lepidopteran lysozyme cDNA as probe. The locus was mapped to the cytological location 61F1-4 on the third chromosome and two of the genes at this locus, LysD and LysP, were analyzed in detail. In contrast to the bacteria-induced lysozymes in the hemolymph of many insects, the transcription levels of both Drosophila genes decrease after bacterial injections into the hemocoel. Apparently, these gene products, like the specifically adapted lysozymes in mammalian foregut fermenters, have been recruited for the digestion of bacteria present in fermenting food. The LysD gene is expressed in an anterior section of the midgut during all feeding stages of development in both larvae and adults. The LysP gene is only active in the adult where it is expressed in the salivary glands. The transcription units for both genes are very compact and they lack introns. Lysozyme D is unusual in that it is predicted to have an acidic isoelectric point whereas lysozyme P appears to be a typical basic lysozyme.
Mol Gen Genet 1992 Apr
PMID:The lysozyme locus in Drosophila melanogaster: different genes are expressed in midgut and salivary glands. 158 5

A dynamic structure refinement method for X-ray crystallography, referred to as the normal mode refinement, is proposed. The Debye-Waller factor is expanded in terms of the low-frequency normal modes whose amplitudes and eigenvectors are experimentally optimized in the process of the crystallographic refinement. In this model, the atomic fluctuations are treated as anisotropic and concerted. The normal modes of the external motion (TLS model) are also introduced to cover the factors other than the internal fluctuations, such as the lattice disorder and diffusion. A program for the normal mode refinement (NM-REF) has been developed. The method has first been tested against simulated diffraction data for human lysozyme calculated by a Monte Carlo simulation. Applications of the method have demonstrated that the normal mode refinement has: (1) improved the fitting to the diffraction data, even with fewer adjustable parameters; (2) distinguished internal fluctuations from external ones; (3) determined anisotropic thermal factors; and (4) identified concerted fluctuations in the protein molecule.
J Mol Biol 1992 May 20
PMID:Normal mode refinement: crystallographic refinement of protein dynamic structure. I. Theory and test by simulated diffraction data. 159 30

The dynamic structure of a protein, human lysozyme, is determined by the normal mode refinement of X-ray crystal structure. This method uses the normal modes of both internal and external motions to distinguish the real internal dynamics from the external terms such as lattice disorder, and gives an anisotropic and concerted picture of atomic fluctuations. The refinement is carried out with diffraction data of 5.0 to 1.8 A resolution, which are collected on an imaging plate. The results of the refinement show: (1) Debye-Waller factor consists of two parts, highly anisotropic internal fluctuations and almost isotropic external terms. The former is smaller than the latter by a factor of 0.72 in the scale of B-factor. Therefore, the internal dynamics cannot be recognized directly from the apparent electron density distribution. (2) The internal fluctuations show basically similar features as those predicted by the normal mode analysis, with almost the same amplitude and a similar level of anisotropy. (3) Correlations of fluctuations are detected between two lobes forming the active site cleft, which move simultaneously in opposite directions. This corresponds to the hinge-bending motion of lysozyme.
J Mol Biol 1992 May 20
PMID:Normal mode refinement: crystallographic refinement of protein dynamic structure. II. Application to human lysozyme. 159 31

Differential scanning calorimetry experiments as a function of pH have been carried out for native hen egg white lysozyme and a three-disulphide derivative (CM6,127-lysozyme). The results indicate that the enthalpy (delta H298) and heat capacity changes (delta Cp) for unfolding are closely similar for the two proteins. This shows that the substantial reduction (25 degrees C at pH 3.8) in Tm resulting from removal of the 6-127 disulphide bond can, to a good approximation, be attributed totally to an increase in the entropy difference between the native and denatured states. The significance of this result for understanding the factors influencing the stability of folded proteins is discussed.
J Mol Biol 1992 Jun 20
PMID:Thermodynamic consequences of the removal of a disulphide bridge from hen lysozyme. 161 99

The transcription and translation signals of the S-layer gene (slpA) from Thermus thermophilus HB8 have been used to express a thermostable kanamycin adenyl transferase gene in this organism. The chimaeric resistance gene was inserted in vitro into slpA to produce different inactive forms of the gene, which were used to transform T. thermophilus HB8. After 48 hours of incubation at 70 degrees C, only two constructions that contained the kat gene flanked by Thermus sequences from both sides of slpA were able to produce protein layer (P100)-defective mutants. The mutants obtained with both constructions showed identical protein patterns, in which a major 50 kDa protein and two other minor proteins were tentatively identified as P100 fragments, expressed from the extreme 5' end of slpA. They also exhibited important phenotypic defects, such as slow growth in liquid broth, a tendency to aggregate as 'rotund bodies', a twisted filamentous shape, and an extreme sensitivity to lysozyme, suggesting protective and shaping roles for the S-layer in T. thermophilus HB8. These results also demonstrate for the first time the feasibility of using selective antibiotic-resistance markers in extreme thermophiles.
Mol Microbiol 1992 Jun
PMID:Insertional mutagenesis in the extreme thermophilic eubacteria Thermus thermophilus HB8. 162 84

To study the nature of antibody-antigen interactions, we have determined the variable gene sequences of the anti-cytochrome c immunoglobulin G1 (IgG1) monoclonal antibody E8, and obtained diffraction-quality crystals of the E8 antigen-binding fragment (Fab), both free and bound to its antigen, horse cytochrome c. The FabE8 crystals belong to space group P21 with unit cell dimensions of a = 45.0 A, b = 85.1 A, c = 63.3 A and beta = 105.5 degrees, have one FabE8 molecule per asymmetric unit and diffract to at least 2.1 A resolution. Crystals of the FabE8-cytochrome c complex belong to space group P212121 with unit cell dimensions of a = 84.3 A, b = 73.3 A and c = 94.9 A, accommodate one complex per asymmetric unit and diffract to 2.4 A resolution. In the nucleotide-derived amino acid sequences, the light-chain variable domain (VL) but not the heavy-chain variable domain (VH) of E8 is nearly identical to that of the anti-lysozyme antibody D1.3, differing by only five amino acid residues. Only one of these interacts with lysozyme in the D1.3-lysozyme crystal structure. Six negative and four positive charges in the VH complementarity determining regions of E8 complement four positive and three negative charges in the E8 epitope on cytochrome c. These data suggest that only a subset of the residues in an antibody-protein interface may be critical for binding and that the VH may play a dominant role in antigenic recognition.
J Mol Biol 1991 Sep 20
PMID:Biochemical implications from the variable gene sequences of an anti-cytochrome c antibody and crystallographic characterization of its antigen-binding fragment in free and antigen-complexed forms. 165 53


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