NDLI: Binding of an Antibody Mimetic of the Human Low Density Lipoprotein Receptor to Apolipoprotein E Is Governed through Electrostatic Forces STUDIES USING SITE-DIRECTED MUTAGENESIS AND MOLECULAR MODELING

Content Provider	Journal of Biological Chemistry (JBC)
Author	Raffaı̈, Robert Weisgraber, Karl H. MacKenzie, Roger Rupp, Bernhard Rassart, Eric Hirama, Tomoko Innerarity, Thomas L. Milne, Ross
Abstract	Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp52, Glu53, and Asp56 are essential for high-affinity binding. Although Asp31 (CDRH1), Glu58 (CDRH2), and Asp97 (CDRH3) did not appear to be critical, the Asp97 → Ala variant acquired reactivity with apoE2. A Thr57 → Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8·apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.
Related Links	http://www.jbc.org/content/275/10/7109.abstract
Ending Page	7116
Starting Page	7109
Page Count	8
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	10
Volume Number	275
DOI	10.1074/jbc.275.10.7109
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2000-03-10
Access Restriction	Open
Subject Keyword	Low density lipoprotein receptor (LDLR) Monoclonal antibody (mAb) Heavy chain complementarity-determining region (CDRH) Light chain complementarity-determining region (CDRL) Polymerase chain reaction (PCR) Recombinant Fab (rFab) Polyacrylamide gel electrophoresis (PAGE) Enzyme-linked immunosorbent assay (ELISA) Phosphate-buffered saline (PBS) Wild-type (WT) Surface plasmon resonance (SPR) PROTEIN STRUCTURE AND FOLDING
Alternative Title	Binding of an Antibody Mimetic of the Human Low Density Lipoprotein Receptor to Apolipoprotein E Is Governed through Electrostatic Forces
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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Binding of an Antibody Mimetic of the Human Low Density Lipoprotein Receptor to Apolipoprotein E Is Governed through Electrostatic Forces STUDIES USING SITE-DIRECTED MUTAGENESIS AND MOLECULAR MODELING

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Binding of an Antibody Mimetic of the Human Low Density Lipoprotein Receptor to Apolipoprotein E Is Governed through Electrostatic Forces STUDIES USING SITE-DIRECTED MUTAGENESIS AND MOLECULAR MODELING