Tubulin-tyrosine ligase has a binding site on beta-tubulin: a two-domain structure of the enzyme.
Tubulin-tyrosine ligase and alpha beta-tubulin form a tight complex which is conveniently monitored by glycerol gradient centrifugation. Using two distinct ligase monoclonal antibodies, several subunit-specific tubulin monoclonal antibodies, and chemical cross-linking, a ligase-binding site was identified on beta-tubulin. This site is retained when the carboxy-terminal domains of both tubulin subunits are removed by subtilisin treatment. The ligase-tubulin complex is also formed when ligase is added to alpha beta-tubulin carrying the monoclonal antibody YL 1/2 which binds only to the carboxyl end of tyrosinated alpha-tubulin. The beta-tubulin-binding site described here explains the extreme substrate specificity of ligase, which does not act on other cellular proteins or carboxy-terminal peptides derived from detyrosinated alpha-tubulin. Differential accessibility of this site in tubulin and in microtubules seems to explain why ligase acts preferentially on unpolymerized tubulin. Ligase exposed to V8-protease is converted to a nicked derivative. This is devoid of enzymatic activity but still forms the complex with tubulin. Gel electrophoresis documents both 30- and a 14-kD domains, each which is immunologically and biochemically distinct and seems to cover the entire molecule. The two domains interact tightly under physiological conditions. The 30-kD domain carries the binding sites for beta-tubulin and ATP. The 14-kD domain can possibly form an additional part of the catalytic site as it harbors the epitope for the monoclonal antibody ID3 which inhibits enzymatic activity but not the formation of the ligase-tubulin complex.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D005260:Female; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D010453:Peptide Synthases / Q000378:metabolism*; D011485:Protein Binding; D013552:Swine; D014404:Tubulin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D046911:Macromolecular Substances; D014404:Tubulin; D010453:Peptide Synthases; C023213:tyrosyltubulin ligase