Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1, and MAP7D3 act redundantly to enable kinesin-1–dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubule-binding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared with MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be cotransported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules.

Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7/ensconsin family proteins. We found that all four mammalian MAP7 family members bound to kinesin-1, and MAP7, MAP7D1 and MAP7D3 acted redundantly to enable kinesin-1-dependent transport in HeLa cells. Microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions, was inhibited in cells co-depleted of these three MAP7 proteins. In vitro, purified MAP7 and MAP7D3 increased microtubule landing rate and processivity of KIF5B-560.The same was true for MAP7D3 C-terminus, which weakly bound to microtubules and exchanged rapidly on motile KIF5B-560 motors. A C-terminal MAP7 fragment lacking microtubule affinity increased KIF5B-560 recruitment to microtubules in vitro and in cells, and partially rescued kinesin-1-dependent transport in the absence of full-length MAP7 proteins. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules. SummaryA combination of experiments in cells and in vitro reconstitution assays demonstrated that mammalian MAP7 family proteins act redundantly to activate kinesin-1 and promote its microtubule binding and processivity by transiently associating with the stalk region of the motor.All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Target-directed dynamic combinatorial chemistry (DCC) is an emerging technique for the efficient identification of inhibitors of pharmacologically relevant targets. In this contribution, we present an application for a bacterial target, the lectin FimH, a crucial virulence factor of uropathogenic E. coli being the main cause of urinary tract infections. A small dynamic library of acylhydrazones was formed from aldehydes and hydrazides and equilibrated at neutral pH in presence of aniline as nucleophilic catalyst. The major success factors turned out to be an accordingly adjusted ratio of scaffolds and fragments, an adequate sample preparation prior to HPLC analysis, and the data processing. Only then did the ranking of the dynamic library constituents correlate well with affinity data. Furthermore, as a support of DCC applications especially to larger libraries, a new protocol for improved hit identification was established.

Highlights d The MTH1 inhibitor TH588 synergizes with Plk1 inhibition to drive cancer cell death d VISAGE implicates the mitotic spindle, not MTH1, as the target of drug synergy d TH588 binds the colchicine binding site of b-tubulin blocking microtubule assembly d The cancer cell spindle is particularly vulnerable to Plk1 + microtubule inhibitors

Tubulin plays essential roles in vital cellular activities and is the target of awide range of proteins and ligands.Here, using ac ombined computational and crystallographic fragment screening approach,w ea ddressed the question of how many binding sites exist in tubulin. We identified 27 distinct sites,o fw hich 11 have not been described previously,a nd analyzed their relationship to knownt ubulin-protein and tubulin-ligand interactions.W ef urther observed an intricate pocket communication network and identified 56 chemically diverse fragments that bound to 10 distinct tubulin sites.O ur results offer au nique structural basis for the development of novel small molecules for use as tubulin modulators in basic researcha pplications or as drugs.F urthermore,o ur method lays down aframework that may help to discover new pockets in other pharmaceutically important targets and characterize them in terms of chemical tractability and allosteric modulation.