Crystal structure of the disulfide-stabilized Fv fragment of anticancer antibody B1: Conformational influence of an engineered disulfide bond

Orna Almog, Itai Benhar, George Vasmatzis, Maria Tordova, Byungkook Lee, Ira Pastan, Gary L. Gilliland

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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 (BldsFv) crystallizes in space group P6122 with the unit cell parameters a = b = 80.1 Å, and c = 138.1 Å. The crystal structure of the BldsFv 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 and in bond angle of 2.74°. Comparisons of the BldsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of BldsFv 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.

Original languageEnglish (US)
Pages (from-to)128-138
Number of pages11
JournalProteins: Structure, Function and Genetics
Volume31
Issue number2
DOIs
StatePublished - May 1 1998
Externally publishedYes

Fingerprint

Immunoglobulin Variable Region
Disulfides
Crystal structure
Antibodies
Inclusion Bodies
Epitopes
Binding Sites
Complementarity Determining Regions
Antigens
Immunoglobulin Fragments
Guanidine
Tyrosine
Chlorides
Neoplasms
Monoclonal Antibodies
Cells
Carbohydrates
Carcinoma
Peptides

Keywords

  • Antibody
  • Antitumor
  • Protein stability
  • Protein structure
  • Single chain Fv
  • Variable domains
  • X-ray crystallography

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Structural Biology

Cite this

Crystal structure of the disulfide-stabilized Fv fragment of anticancer antibody B1 : Conformational influence of an engineered disulfide bond. / Almog, Orna; Benhar, Itai; Vasmatzis, George; Tordova, Maria; Lee, Byungkook; Pastan, Ira; Gilliland, Gary L.

In: Proteins: Structure, Function and Genetics, Vol. 31, No. 2, 01.05.1998, p. 128-138.

Research output: Contribution to journalArticle

Almog, Orna ; Benhar, Itai ; Vasmatzis, George ; Tordova, Maria ; Lee, Byungkook ; Pastan, Ira ; Gilliland, Gary L. / Crystal structure of the disulfide-stabilized Fv fragment of anticancer antibody B1 : Conformational influence of an engineered disulfide bond. In: Proteins: Structure, Function and Genetics. 1998 ; Vol. 31, No. 2. pp. 128-138.
@article{732f44bdef1a48e8a6ee0d50782bd1ee,
title = "Crystal structure of the disulfide-stabilized Fv fragment of anticancer antibody B1: Conformational influence of an engineered disulfide bond",
abstract = "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 (BldsFv) crystallizes in space group P6122 with the unit cell parameters a = b = 80.1 {\AA}, and c = 138.1 {\AA}. The crystal structure of the BldsFv has been determined at 2.1-{\AA} 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 and in bond angle of 2.74°. Comparisons of the BldsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of BldsFv is a deep depression 10-{\AA} wide and 17-{\AA} 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.",
keywords = "Antibody, Antitumor, Protein stability, Protein structure, Single chain Fv, Variable domains, X-ray crystallography",
author = "Orna Almog and Itai Benhar and George Vasmatzis and Maria Tordova and Byungkook Lee and Ira Pastan and Gilliland, {Gary L.}",
year = "1998",
month = "5",
day = "1",
doi = "10.1002/(SICI)1097-0134(19980501)31:2<128::AID-PROT3>3.0.CO;2-I",
language = "English (US)",
volume = "31",
pages = "128--138",
journal = "Proteins: Structure, Function and Bioinformatics",
issn = "0887-3585",
publisher = "Wiley-Liss Inc.",
number = "2",

}

TY - JOUR

T1 - Crystal structure of the disulfide-stabilized Fv fragment of anticancer antibody B1

T2 - Conformational influence of an engineered disulfide bond

AU - Almog, Orna

AU - Benhar, Itai

AU - Vasmatzis, George

AU - Tordova, Maria

AU - Lee, Byungkook

AU - Pastan, Ira

AU - Gilliland, Gary L.

PY - 1998/5/1

Y1 - 1998/5/1

N2 - 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 (BldsFv) crystallizes in space group P6122 with the unit cell parameters a = b = 80.1 Å, and c = 138.1 Å. The crystal structure of the BldsFv 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 and in bond angle of 2.74°. Comparisons of the BldsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of BldsFv 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.

AB - 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 (BldsFv) crystallizes in space group P6122 with the unit cell parameters a = b = 80.1 Å, and c = 138.1 Å. The crystal structure of the BldsFv 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 and in bond angle of 2.74°. Comparisons of the BldsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of BldsFv 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.

KW - Antibody

KW - Antitumor

KW - Protein stability

KW - Protein structure

KW - Single chain Fv

KW - Variable domains

KW - X-ray crystallography

UR - http://www.scopus.com/inward/record.url?scp=0032080531&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032080531&partnerID=8YFLogxK

U2 - 10.1002/(SICI)1097-0134(19980501)31:2<128::AID-PROT3>3.0.CO;2-I

DO - 10.1002/(SICI)1097-0134(19980501)31:2<128::AID-PROT3>3.0.CO;2-I

M3 - Article

C2 - 9593187

AN - SCOPUS:0032080531

VL - 31

SP - 128

EP - 138

JO - Proteins: Structure, Function and Bioinformatics

JF - Proteins: Structure, Function and Bioinformatics

SN - 0887-3585

IS - 2

ER -