Special Randall seminar: A structural model for microtubule minus-end recognition and protection by CAMSAP proteins

A structural model for microtubule minus-end recognition and protection by CAMSAP proteins

Speaker:  Dr Joe Atherton,  The Institute of Structural and Molecular Biology, Birkbeck University of London

Host: Roberto Steiner

Abstract: Microtubules are polar polymers, with minus and plus ends exhibiting differential dynamics and regulated by different cofactors. The evolutionarily conserved calmodulin-regulated spectrin-associated protein (CAMSAP) family are minus-end binding proteins that modulate minus-end microtubule dynamics. All CAMSAPs share a characteristic conserved CKK microtubule-binding domain, which defines their minus end specificity. However, the mechanism of this specificity is not understood. To shed light on this question, we determined the CKK structure by X-ray crystallography and characterised CAMSAP1 and 3’s CKK binding site on microtubules by cryo-EM. The CAMSAP CKK binds at a previously undescribed binding site at the microtubule intra-dimer inter-protofilament interface. TIRF microscopy was used to confirm the contributions of conserved residues at the CKK-microtubule interface. The nucleotide state of tubulin was also shown not to influence minus-end specificity. Interestingly, CKK binding imposes a right-handed ‘supertwist’, on microtubule protofilaments, even on the taxol-stabilized microtubules used for our reconstructions. Further cryo-EM investigations, including of a CKK mutant, and another CKK with reduced minus-end specificity, supports the possibility that subtle alterations in CKK positioning relative to tubulin polymer confer minus-end specificity. Cryo-electron tomography of minus-ends reveals curved lattice to sheet regions that retain lateral protofilament interactions and present unique minus-end tubulin conformations to which CAMSAPs may preferentially bind.