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Architecture of the Mto1/2 microtubule nucleation complex

Thakur, H. C.; Lynch, E. M.; Borek, W. E.; Bao, X. X.; Ashraf, S.; Zou, J.; Rappsilber, J.; Cook, A. G.; Sawin, K. E.

2019-08-31 cell biology
10.1101/754457 bioRxiv
Show abstract

Proteins that contain a Centrosomin Motif 1 (CM1) domain are key regulators of{gamma} -tubulin complex-dependent microtubule nucleation, but how they are organized in higher-order structures is largely unknown. Mto1[bonsai], a truncated functional version of the Schizosaccharomyces pombe CM1 protein Mto1, interacts with Mto2 to form an Mto1/2[bonsai] complex in vivo. Here we show that recombinant Mto1/2[bonsai] forms higher-order multimers in vitro and that Mto2 alone can also multimerize. We demonstrate that Mto2 multimerization involves two separate homodimerization domains, the near N-terminal domain (NND) and the twin-cysteine domain (TCD). The TCD crystal structure reveals a stable homodimer with a novel dimerization interface. While the NND homodimer is intrinsically less stable, using crosslinking mass spectrometry we show that within Mto1/2[bonsai] complexes, it can be reinforced by additional cooperative interactions involving both Mto2 and Mto1[bonsai]. We propose a model for Mto1/2[bonsai] complex architecture that is supported by functional analysis of mutants in vivo.

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