Structural determinants of microtubule minus end preference in CAMSAP CKK domains

Abstract

CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which has lost minus-end specificity. Near-atomic cryo-electron microscopy structures of HsCKK-and NgCKK-microtubule complexes show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit subtly different footprints on microtubules. Whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. NMR experiments show that HsCKK is remarkably rigid, supporting this remodelling ability. Thus, in contrast to many MAPs, CKK domains can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition.

Nucleotide Binding, Evolutionary Insights, and Interaction Partners of the Pseudokinase Unc-51-like Kinase 4

“…Data were analyzed using the SAINT (statistical analysis of interactomes [Choi et al, 2011;Teo et al, 2014]) algorithm to identify high-confidence proximity interactors (displaying a Bayesian false discovery rate %0.01 Figure 5; Table S1). The proximity interactor detected with the highest number of peptides was calmodulin-regulated spectrin-associated protein 1 (CAMSAP1), a large cytoplasmic scaffolding polypeptide that binds microtubules via its CKK domain, and controls noncentrosomal MT minus-end dynamics (Atherton et al, 2019;Baines et al, 2009). CAMSAP family members are involved in the regulation of a number of key cellular functions, including cell polarity, regulation of neuronal differentiation, and axonal regeneration (Akhmanova and Hoogenraad, 2015;Marcette et al, 2014;Pongrakhananon et al, 2018).…”

The mechanism of kinesin inhibition by kinesin-binding protein

“…For KBP-alone and KBP-KIF15_MD6S data, de novo initial 3D models were created in cryoSPARC2. For KBP-KIF15_MD6S data, a single round of The KIF15_MD6S-MT dataset was processed using our MT RELION-based pipeline (MiRP) as described previously, using low-pass filtered KIF5B_MDdecorated MTs as references 55,56 . KIF15_MD6S 13-protofilament-MTs were the most common MT architecture and were selected after supervised 3D classification in MiRP for analysis.…”

The Centrosome and the Primary Cilium: The Yin and Yang of a Hybrid Organelle

“…These proteins specifically bind and stabilize uncapped MT minus ends and support MT minus-end growth independently of γ-TuRC [268,269,270,271]. Such unique properties of the CAMSAP proteins are mediated by a conserved, family-defining CKK (CAMSAP1, KIAA1078 and KIAA1543) domain, which recognizes subtly specific tubulin conformations at the MT minus end [272,273,274]. Notably, the CAMSAP proteins are found only in animals with differentiated tissues, but not in any other organisms, such as those of non-animal lineages or sponges, which lack tissues [272].…”