Kinesin-binding protein remodels the kinesin motor to prevent microtubule binding.

Kinesins are regulated in space and time to ensure activation only in the presence of cargo. Kinesin-binding protein (KIFBP), which is mutated in Goldberg-Shprintzen syndrome, binds to and inhibits the catalytic motor heads of 8 of 45 kinesin superfamily members, but the mechanism remains poorly defined. Here, we used cryo­electron microscopy and cross-linking mass spectrometry to determine high-resolution structures of KIFBP alone and in complex with two mitotic kinesins, revealing structural remodeling of kinesin by KIFBP. We find that KIFBP remodels kinesin motors and blocks microtubule binding (i) via allosteric changes to kinesin and (ii) by sterically blocking access to the microtubule. We identified two regions of KIFBP necessary for kinesin binding and cellular regulation during mitosis. Together, this work further elucidates the molecular mechanism of KIFBP-mediated kinesin inhibition and supports a model in which structural rearrangement of kinesin motor domains by KIFBP abrogates motor protein activity.

Effects of Nucleotide and End-Dependent Actin Conformations on Polymerization.

The regulation of actin is key for controlled cellular function. Filaments are regulated by actin-binding proteins, but the nucleotide state of actin is also an important factor. From extended molecular dynamics simulations, we find that both nucleotide states of the actin monomer have significantly less twist than their crystal structures and that the ATP monomer is flatter than the ADP form. We also find that the filament's pointed end is flatter than the remainder of the filament and has a conformation distinct from G-actin, meaning that incoming monomers would need to undergo isomerization that would weaken the affinity and slow polymerization. Conversely, the barbed end of the filament takes on a conformation nearly identical to the ATP monomer, enhancing ATP G-actin's ability to polymerize as compared with ADP G-actin. The thermodynamic penalty imposed by differences in isomerization for the ATP and ADP growth at the barbed end exactly matches experimental results.

Two Deafness-Causing Actin Mutations (DFNA20/26) Have Allosteric Effects on the Actin Structure.

Point mutations in γ-cytoplasmic actin have been shown to result in autosomal-dominant, nonsyndromic, early-onset deafness. Two mutations at the same site, K118M and K118N, provide a unique opportunity to compare the effects of two dissimilar amino acid substitutions that produce a similar phenotype in humans. K118 resides in a helix that runs from K113 to T126, and mutations that alter the position, dynamics, and/or biochemistry of this helix can result in a wide range of pathologies. Using a combination of computational and experimental studies, both employing yeast actin, we find that these mutations at K118 result in changes in the structure and dynamics of the DNase-I loop, alterations in the structure of the H73 loop as well as the side-chain orientations of W79 and W86, changes in nucleotide exchange rates, and significant shifts in the twist of the actin monomer. Interestingly, in the case of K118N, the twist of the monomer is nearly identical to that of the F-actin protomer, and in vitro polymerization assays show that this mutation results in faster polymerization. Taken together, these results indicate that mutations at this site give rise to a series of small changes that can be tolerated in vivo but result in misregulation of actin assembly and dynamics.