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Drosophila Ncd reveals an evolutionarily conserved powerstroke mechanism for homodimeric and heterodimeric kinesin-14s.

ABSTRACT: Drosophila melanogaster kinesin-14 Ncd cross-links parallel microtubules at the spindle poles and antiparallel microtubules within the spindle midzone to play roles in bipolar spindle assembly and proper chromosome distribution. As observed for Saccharomyces cerevisiae kinesin-14 Kar3Vik1 and Kar3Cik1, Ncd binds adjacent microtubule protofilaments in a novel microtubule binding configuration and uses an ATP-promoted powerstroke mechanism. The hypothesis tested here is that Kar3Vik1 and Kar3Cik1, as well as Ncd, use a common ATPase mechanism for force generation even though the microtubule interactions for both Ncd heads are modulated by nucleotide state. The presteady-state kinetics and computational modeling establish an ATPase mechanism for a powerstroke model of Ncd that is very similar to those determined for Kar3Vik1 and Kar3Cik1, although these heterodimers have one Kar3 catalytic motor domain and a Vik1/Cik1 partner motor homology domain whose interactions with microtubules are not modulated by nucleotide state but by strain. The results indicate that both Ncd motor heads bind the microtubule lattice; two ATP binding and hydrolysis events are required for each powerstroke; and a slow step occurs after microtubule collision and before the ATP-promoted powerstroke. Note that unlike conventional myosin-II or other processive molecular motors, Ncd requires two ATP turnovers rather than one for a single powerstroke-driven displacement or step. These results are significant because all metazoan kinesin-14s are homodimers, and the results presented show that despite their structural and functional differences, the heterodimeric and homodimeric kinesin-14s share a common evolutionary structural and mechanochemical mechanism for force generation.

SUBMITTER: Zhang P

PROVIDER: S-EPMC4443328 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Drosophila Ncd reveals an evolutionarily conserved powerstroke mechanism for homodimeric and heterodimeric kinesin-14s.

Zhang Pengwei P Dai Wei W Hahn Juergen J Gilbert Susan P SP

Proceedings of the National Academy of Sciences of the United States of America 20150504 20

Drosophila melanogaster kinesin-14 Ncd cross-links parallel microtubules at the spindle poles and antiparallel microtubules within the spindle midzone to play roles in bipolar spindle assembly and proper chromosome distribution. As observed for Saccharomyces cerevisiae kinesin-14 Kar3Vik1 and Kar3Cik1, Ncd binds adjacent microtubule protofilaments in a novel microtubule binding configuration and uses an ATP-promoted powerstroke mechanism. The hypothesis tested here is that Kar3Vik1 and Kar3Cik1, ...[more]

PMID: 25941402

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Project description:Polymyxins, a family of cationic antimicrobial cyclic peptides, act as a last line of defense against severe infections by Gram-negative pathogens with carbapenem resistance. In addition to the intrinsic resistance to polymyxin E (colistin) conferred by Neisseria eptA, the plasmid-borne mobilized colistin resistance gene mcr-1 has been disseminated globally since the first discovery in Southern China, in late 2015. However, the molecular mechanisms for both intrinsic and transferable resistance to colistin remain largely unknown. Here, we aim to address this gap in the knowledge of these proteins. Structural and functional analyses of EptA and MCR-1 and -2 have defined a conserved 12-residue cavity that is required for the entry of the lipid substrate, phosphatidylethanolamine (PE). The in vitro and in vivo data together have allowed us to visualize the similarities in catalytic activity shared by EptA and MCR-1 and -2. The expression of either EptA or MCR-1 or -2 is shown to remodel the surface of enteric bacteria (e.g., Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, etc.), rendering them resistant to colistin. The parallels in the PE substrate-binding cavities among EptA, MCR-1, and MCR-2 provide a comprehensive understanding of both intrinsic and transferable colistin resistance. Domain swapping between EptA and MCR-1 and -2 reveals that the two domains (transmembrane [TM] region and phosphoethanolamine [PEA] transferase) are not functionally exchangeable. Taken together, the results represent a common mechanism for intrinsic and transferable PEA resistance to polymyxin, a last-resort antibiotic against multidrug-resistant pathogens.IMPORTANCE EptA and MCR-1 and -2 remodel the outer membrane, rendering bacteria resistant to colistin, a final resort against carbapenem-resistant pathogens. Structural and functional analyses of EptA and MCR-1 and -2 reveal parallel PE lipid substrate-recognizing cavities, which explains intrinsic and transferable colistin resistance in gut bacteria. A similar mechanism is proposed for the catalytic activities of EptA and MCR-1 and -2. Together, they constitute a common mechanism for intrinsic and transferable polymyxin resistance.

Dataset Information

Drosophila Ncd reveals an evolutionarily conserved powerstroke mechanism for homodimeric and heterodimeric kinesin-14s.

Publications

Drosophila Ncd reveals an evolutionarily conserved powerstroke mechanism for homodimeric and heterodimeric kinesin-14s.

Similar Datasets

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