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Proc Amer Assoc Cancer Res, Volume 45, 2004 5441 HTI-286, a synthetic analog of hemiasterlin, is a novel tripeptide that depolymerizes microtubules. The binding-sites on tubulin of antimitotic peptides such as hemiasterlin and dolastatin-10 are not well characterized. To determine the binding site for HTI-286 two radiolabeled benzophenone photoaffinity probes of HTI-286 were utilized. We have already established that photoprobe 1 binds within residues 314-338 of α-tubulin. Here we describe the binding of photoprobe 2 (N,β,β-trimethyl-L-phenylalanyl-4-benzoyl-N-[(1S,2E)-3-carboxy-1-isopropyl-2-butenyl]-N,β,β-trimethyl-L-phenylalaninamide). The affinity of probe 2 for bovine brain tubulin was determined to be 3.6 μM, 6-fold higher than that for HTI-286. HTI-286 and probe 2 inhibited tubulin polymerization in a cell-free system by 88% and 69%, respectively. Probe 2 was 20-fold less potent in its ability to inhibit cell growth (IC50 = 22nM). Radiolabeled probe 2 exclusively labeled the α-subunit in bovine brain and HeLa cell tubulin preparations. Photolabeling of tubulin was inhibited by unlabeled photoaffinity analog, HTI-286, vinblastine, and dolastatin-10. However, some concentrations of paclitaxel and colchicine enhanced binding of probe 2. Digestion of labeled tubulin with trypsin, formic acid, Lys C, and CNBr suggests that probe 2 binds N-terminal to residue 306 (formic acid cleavage site), and is likely to reside within amino acids 204-280 in α-tubulin. These and other data from our laboratory suggest that the HTI-286 binding site maps to α-tubulin at the interdimer region. This region has not been reported as a binding site for any other known tubulin-binding drug. The C-terminal (2-4 kDa) regions of α- and β-tubulin are susceptible to cleavage by subtilisin under non-denaturing conditions. Subtilisin cleaves β-tubulin more rapidly than α-tubulin. Vinblastine and dolastatin-10 binding induces polymerization of tubulin into non-microtubule structures that pellet at 100,000 g . Vinblastine-induced polymerization can block limited proteolysis (at room temperature) with subtilisin of α-tubulin, but not β-tubulin. We observed that HTI-286 does not cause tubulin to polymerize into structures that pellet at 100,000 g , but nevertheless protects the subtilisin cleavage site in α-tubulin. This suggests that the mechanism of protection of α-tubulin by HTI-286 is distinct from that seen by vinblastine and dolastatin-10. We propose that the protection of subtilisin-cleavage is due to a change in the conformation of the C-terminus upon binding of HTI-286 to α-tubulin. Taken together these data strongly suggest that HTI-286 binds to α-tubulin in the interdimer region.