X-ray structures of the antigen-binding domains from three variants of humanized anti-p185HER2 antibody 4D5 and comparison with molecular modeling.
The X-ray structures of 1 Fv and 2 Fab humanized anti-p185HER2 antibody fragments (IgG1-kappa) have been determined at a resolution between 2.7 A and 2.2 A. The antibodies are three different versions of a human antibody framework onto which the antigen recognition loops from a murine antibody (4D5) have been grafted. The sequences of the three versions differ in the framework region at positions L55, H78 and H102. The version 8 Fv fragment crystallizes in space group P2(1) with cell parameters a = 37.6 A, b = 63.4 A, c = 90.2 A, beta = 98.2 degrees, with two molecules per asymmetric unit, and has been refined against data 10.0 A-2.2 A to an R-factor of 18.3%. Versions 4 and 7 Fabs crystallize in space group P1 with cell parameters a = 39.2 A, b = 80.2 A, c = 86.1 A, alpha = 113.1 degrees, beta = 92.7 A, gamma = 102.6 A and two molecules per asymmetric unit. Version 4 has been refined against data 10.0 A-2.5 A resolution to an R-factor of 17.9%. Version 7 has been refined against data 10 A-2.7 A to an R-factor of 17.1%. The X-ray structures have been used to assess the accuracy of structural predictions made via molecular modeling, and they confirm the structural role of certain framework residues and the conformations of five of six complementarity determining regions (CDRS). The average deviation of the model from the X-ray structures is within the range observed among the X-ray structures for 81% of the C alpha atoms. Of the hydrogen bonds common to the X-ray structures, 94% of the main-chain-main-chain and 79% of the main-chain-side-chain ones were predicted by the model. The side-chain conformation was predicted correctly for 79% of the buried residues. The third CDR in the heavy chain is variable, differing by up to 8 A between molecules within an asymmetric unit. The structural relationship between variable domains of light and heavy chains is not significantly altered by the absence of constant domains in the Fv molecule. The antigen-binding potential of an unusual light chain sequence has been confirmed. The arginine at position 66 interacts with the first light chain CDR, but in a fashion somewhat different than predicted. A substitution of a leucine for an alanine side-chain directed between the beta-sheets has only relatively small and local effects.(ABSTRACT TRUNCATED AT 400 WORDS)
Eigenbrot
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Department of Protein Engineering, Genentech, Inc., South San Francisco.
Randal
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Presta
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Carter
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Kossiakoff
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D001666:Binding Sites, Antibody; D003198:Computer Simulation; D006801:Humans; D006860:Hydrogen Bonding; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000276:immunology*; D011487:Protein Conformation; D018719:Erb-b2 Receptor Tyrosine Kinases; D017386:Sequence Homology, Amino Acid; D014961:X-Ray Diffraction
D000906:Antibodies; D007135:Immunoglobulin Variable Region; D009856:Oncogene Proteins, Viral; D018719:Erb-b2 Receptor Tyrosine Kinases