Epitope mapping for the monoclonal antibody that inhibits intramolecular electron transfer in flavocytochrome b2.
Flavocytochrome b(2) (yeast L-lactate dehydrogenase) carries one FMN and one protohaem IX on each of its four subunits. The prosthetic groups are bound to separate domains, the haem domain (residues 1-99) and the flavin domain (residues 100-485), which interact for electron transfer between lactate-reduced FMN and haem b(2); in vivo, the latter reduces cytochrome c. In the crystal structure, one haem domain out of two is mobile. Previously we have described a monoclonal antibody, raised against the tetramer, that only recognizes the native haem domain and prevents electron transfer between flavin and haem, while having no effect on flavin reduction by the substrate [Miles, Lederer and Lê (1998) Biochemistry 37, 3440-3448]. In order to understand the structural basis of the uncoupling between the domains, we proceeded to site-directed mutagenesis, so as to map the epitope on the surface of the haem domain. We analysed the effects of 14 mutations at 12 different positions, located mostly in the domain interface or at its edge; we also analysed the effect of replacing protohaem IX with its dimethyl ester. We used as criteria the antibody-mediated inhibition of cytochrome c reduction by flavocytochrome b(2), competitive ELISA tests and surface plasmon resonance. We have thus defined a minimal epitope surface on the haem domain; it encompasses positions 63, 64, 65, 67, 69 and 70 and one or both haem propionates. When the haem and flavin domains are docked for electron transfer, the 65, 67 and 70 side chains, as well as the haem propionates, are excluded from solvent. The present results thus indicate that, when bound, the antibody acts as a wedge between the domains and constitutes a physical barrier to electron transfer.
Lê
K H Diêp
KH
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS UPR 9063, 91198 Gif-sur-Yvette Cedex, France.
Mayer
Martine
M
Lederer
Florence
F
D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001483:Base Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004579:Electron Transport / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis / Q000737:chemistry; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D000939:Epitopes; D021122:Protein Subunits; D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)