Eribulin's exclusive binding to microtubule plus ends results from discrimination between GTP and GDP forms of β-tubulin.

The clinically approved anticancer agent eribulin (Halaven®) exerts cytotoxic antimitotic effects by binding to high affinity sites on exposed plus ends of growing microtubules (MT). Despite X-ray crystallographic mapping of eribulin's binding pocket within β-tubulin's vinca domain, the biophysical basis for eribulin's MT plus end binding exclusivity remains unknown. We performed surface plasmon resonance (SPR) studies of tubulin binding to biotinylated eribulin probes to ask if eribulin discriminates between GTP and GDP forms of β-tubulin, which characterize growing MT plus ends and MT sides, respectively. Tubulin binding to biotin-eribulin proceeded via a single state binding reaction, while binding to biotin-vinblastine occurred via a two-state reaction incorporating a conformational change. Biochemical approaches confirmed tubulin conformational changes induced by vinblastine but not eribulin. SPR competition studies using free eribulin and vinblastine confirmed tubulin binding specificity to cognate biotinylated probes, showing that eribulin binding within the β-tubulin vinca domain is physically and functionally distinct from vinblastine. SPR studies using tubulin containing only GTP or GDP forms of β-tubulin showed that while biotin-eribulin has only slightly higher overall affinity for GTP-tubulin, dissociation from GTP-tubulin was ∼7-fold slower than from GDP-tubulin, establishing that eribulin discriminates between GTP and GDP forms of β-tubulin. In contrast, vinblastine fails to discriminate between these two tubulin forms, consistent with its known binding to both MT ends and sides. Overall, our results establish, for the first time, the biophysical basis for eribulin's MT plus end binding exclusivity as resulting from the ability to discriminate between GTP and GDP forms of β-tubulin.