Structural Basis for Microtubule Binding by the Anti-Parallel Microtubule Crosslinker PRC1 (Protein Regulator of Cytokinesis-1)

Abstract

Successful cytokinesis is essential for maintaining genome stability. The central spindle, an array of antiparallel microtubules formed prior to cytokinesis, is required for complete furrow ingression to form two daughter cells. Members of the conserved MAP65 family (Ase1, AtMAP-65, PRC1) play a key role in organizing microtubules in the central spindle. Invitro studies indicate that members of this protein family can crosslink microtubules with an anti-parallel orientation bias. We have combined structural methods and single molecule fluorescence to understand the structural basis for biased crosslinking by the human MAP65 protein PRC1.We describe the crystal structure of the conserved domain of PRC1. This domain is capable of binding microtubules (Kd ∼ 6 uM), but shows no sequence similarity to other known microtubule binding proteins. This domain has a spectrin fold, a motif ubiquitous in proteins interacting with the actin cytoskeleton, but not seen previously in microtubule binding proteins. Cryo-EM and helical image analysis yielded a 3D map of PRC1 bound to microtubules. Docking the crystal structure into the EM map gives us a structural view of PRC1-microtubule interactions.PRC1 also has a proteolytically sensitive Lys/Arg rich domain that is thought to regulate microtubule binding. Single-molecule assays reveal that the presence of this domain allows the protein to undergo long diffusional sliding on microtubules. Constructs in which this domain is truncated show reduced interaction-lifetime and crosslinking efficiency.These results suggests that the two domains act together to achieve biased anti-parallel crosslinking. We propose a model where oriented binding and polarity bias is achieved by the spectrin domain and the positively charged flexible domain enhances the probability of biased crosslinking by increasing the dwell time of PRC1 on microtubules via a diffusive binding mechanism.