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Crystal structure of the disulfide‐stabilized Fv fragment of anticancer antibody B1: Conformational influence of an engineered disulfide bond
Author(s) -
Almog Orna,
Benhar Itai,
Vasmatzis George,
Tordova Maria,
Lee Byungkook,
Pastan Ira,
Gilliland Gary L.
Publication year - 1998
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(19980501)31:2<128::aid-prot3>3.0.co;2-i
Subject(s) - chemistry , stereochemistry , immunoglobulin light chain , immunoglobulin fab fragments , crystal structure , crystallography , recombinant dna , antibody , epitope , complementarity determining region , peptide sequence , biochemistry , biology , gene , immunology
A recombinant Fv construct of the B1 monoclonal antibody that recognizes the Lewis Y ‐related carbohydrate epitope on human carcinoma cells has been prepared. The Fv is composed of the polypeptide chains of the V H and V L domains expressed independently and isolated as inclusion bodies. The Fv is prepared by combining and refolding equimolar amounts of guanidine chloride solubilized inclusion bodies. The Fv is stabilized by an engineered interchain disulfide bridge between residues V L 100 and V H 44. This construct has a similar binding affinity as that of the single‐chain construct (Benhar and Pastan, Clin. Cancer Res. 1:1023–1029, 1995). The B1 disulfide‐stabilized Fv (B1dsFv) crystallizes in space group P6 1 22 with the unit cell parameters a = b = 80.1 Å, and c = 138.1 Å. The crystal structure of the B1dsFv has been determined at 2.1‐Å resolution using the molecular replacement technique. The final structure has a crystallographic R‐value of 0.187 with a root mean square deviation in bond distance of 0.014 Å and in bond angle of 2.74°. Comparisons of the B1dsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of B1dsFv is a deep depression 10‐Å wide and 17‐Å long with the walls of the depression composed of residues, many of which are tyrosines, from complementarity determining regions L1, L3, H1, H2, and H3. Model building studies indicate that the Lewis Y tetrasaccharide, Fuc–Gal–Nag–Fuc, can be accommodated in the antigen combining site in a manner consistent with the epitope predicted in earlier biochemical studies (Pastan, Lovelace, Gallo, Rutherford, Magnani, and Willingham, Cancer Res. 51:3781–3787, 1991). Thus, the engineered disulfide bridge appears to cause little, if any, distortion in the Fv structure, making it an effective substitute for the B1 Fab. Proteins 31:128–138, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.