Biology subjects

Krementsova, E. B.

Publications and source records attributed to Krementsova, E. B..

5 recordsLinked to original sources

bioRxiv2020

Structure of Full Length Plasmodium Myosin A and its light chain PfELC, dual targets against malaria parasite pathogenesis

Parasites from the genus Plasmodium are the causative agents of malaria. The mobility, infectivity and ultimately pathogenesis of this parasite relies on a macromolecular complex, called the glideosome. At the core of the glideosome is an essential and divergent Myosin A motor (PfMyoA), a first order drug target against malaria. Here we present the full-length structure of PfMyoA in two states of its motor cycle. We report novel interactions that are essential for motor priming and the mode of recognition of its two light chains (PfELC and MTIP) by two degenerate IQ motifs. Kinetic and motility assays using PfMyoA variants, along with molecular dynamics, demonstrate how specific priming and atypical sequence adaptations tune the motor’s mechano-chemical properties. Supported by evidence for an essential role of the PfELC in malaria pathogenesis, these structures provide a blueprint for the design of future antimalarials targeting both the glideosome motor and its regulatory elements.HighlightsThe first structures of the full length PfMyoA motor in two states of its motor cycle.A unique priming of the PfMyoA lever arm results from specific lever arm/motor domain interactions, which allows for a larger powerstroke to enhance speed.Sequence adaptations within the motor domain and degenerate IQ motifs in the lever arm dictate PfMyoA motor properties.PfELC is essential for blood cell invasion and is a weak link in the assembly of a fully functional motor, providing a second novel target for antimalarial drug design.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2019

MAP7 recruits kinesin-1 to microtubules to direct organelle transport

Microtubule-associated proteins (MAPs) play well-characterized roles in regulating microtubule polymerization, dynamics, and organization. In addition, MAPs control trans-port along microtubules by regulating the motility of kinesin and dynein. MAP7 (ensconsin, E-MAP-115) is a ubiquitous MAP that organizes the microtubule cytoskeleton in mitosis and neuronal branching. MAP7 also promotes the interaction of kinesin-1 with microtubules. We expressed and purified full-length kinesin-1 and MAP7 in Sf9 cells. In single-molecule motiity assays, MAP7 recruits kinesin-1 to microtubules, increasing the frequency of both diffusive and processive runs. Optical trapping assays on beads transported by single and teams of kinesin-1 motors indicate that MAP7 increases the relative binding rate of kinesin-1 and the number of motors simultaneously engaged in ensembles. To examine the role of MAP7 in regulating bidirectional transport, we isolated late phagosomes along with their native set of kinesin-1, kinesin-2, and dynein motors. Bidirectional cargoes exhibit a clear shift towards plus-end directed motility on MAP7-decorated microtubules due to increased forces exerted by kinesin teams. Collectively, our results indicate that MAP7 enhances kinesin-1 recruitment to microtubules and targets organelle transport to the plus end.

biophysics↗

bioRxiv2018

Recruitment of Two Dyneins to an mRNA-Dependent Bicaudal D Transport Complex

We investigated the role of binding partners of full-length Drosophila Bicaudal D (BicD) in the activation of dynein-dynactin motility for mRNA transport on microtubules. In single-molecule assays, full-length BicD robustly activated dynein-dynactin only when both the mRNA binding protein Egalitarian (Egl), and K10 mRNA cargo were present. Electron microscopy showed that both Egl and mRNA were needed to disrupt an auto-inhibited, looped BicD conformation that sterically prevents dynein-dynactin binding. In vitro reconstituted messenger ribonucleoprotein (mRNP) complexes with two Egl molecules showed faster speeds and longer run lengths than mRNPs with one Egl, suggesting that cargo binding enhances dynein recruitment. Labeled dynein showed that BicD can recruit two dimeric dyneins to the mRNP, resulting in faster speeds and longer run lengths than with one dynein. The fully reconstituted mRNP provides a model for understanding how adaptor proteins and cargo cooperate to confer optimal transport properties to a dynein-driven transport complex.

biophysics↗

bioRxiv2017

Binding Of A Newly Identified Essential Light Chain To Expressed Plasmodium falciparum Class XIV Myosin Enhances Actin Motility

Motility of the apicomplexan parasite Plasmodium falciparum, the causative agent of malaria, is enabled by the glideosome, a multi-protein complex containing the class XIV myosin motor, PfMyoA. Parasite motility is necessary for invasion into host cells and for virulence. Here we show that milligram quantities of functional PfMyoA can be expressed using the baculovirus/Sf9 cell expression system, provided that a UCS (UNC-45/CRO1/She4p) family myosin co-chaperone from Plasmodium spp. is co-expressed with the heavy chain. The homologous chaperone from the apicomplexan Toxoplasma gondii does not functionally substitute. We expressed a functional full-length PfMyoA with bound myosin tail interacting protein (MTIP), the only known light chain of PfMyoA. We then identified an additional \"essential\" light chain (PfELC) that co-purified with PfMyoA isolated from parasite lysates. PfMyoA expressed with both light chains moved actin at ~3.8 m/sec, more than twice that of PfMyoA-MTIP (~1.7 m/sec), consistent with the light chain binding domain acting as a lever arm to amplify nucleotide-dependent motions in the motor domain. Surprisingly, PfMyoA moved skeletal actin or expressed P. falciparum actin at the same speed. Duty ratio estimates suggest that PfMyoA may be able to move actin at maximal speed with as few as 6 motors. Under unloaded conditions, neither phosphorylation of Ser19 of the heavy chain, phosphorylation of several Ser residues in the N-terminal extension of MTIP, or calcium affected the speed of actin motion. These studies provide the essential framework for targeting the glideosome as a potential drug target to inhibit invasion by the malaria parasite.\n\nSignificanceMotility of the apicomplexan parasite Plasmodium falciparum, the causative agent of malaria, relies on a divergent actomyosin system powered by the class XIV myosin, PfMyoA. We show that functional PfMyoA can be expressed in Sf9 cells if a Plasmodium spp. myosin chaperone is co-expressed. We identified an \"essential\" light chain (PfELC) that binds to PfMyoA in parasites. In vitro expression of PfMyoA heavy chain with PfELC and the known light chain MTIP produced the fastest speeds of actin movement (~3.8 m/sec). Duty ratio estimates suggest that ~6 PfMyoA motors can move actin at maximal speed, a feature that may facilitate interaction with short, dynamic Plasmodium actin filaments. Our findings enable drug screening for myosin-based inhibitors of Plasmodium cellular invasion.

biochemistry↗

bioRxiv2017

Small teams of myosin Vc coordinate their stepping for efficient cargo transport on actin bundles

Myosin Vc (myoVc) is unique among vertebrate class V myosin isoforms in that it requires teams of motors to transport cargo. Single molecules of myoVc cannot take multiple steps on single actin filaments, in stark contrast to the well-studied myosin Va (myoVa) isoform. Consistent with in vivo studies (1), only teams of myoVc motors can move continuously on actin bundles at physiologic ionic strength (2), raising the question of how motor-motor interactions cause this preference. Here, using DNA nanostructures as synthetic cargos for linking defined numbers of myoVa or myoVc molecules, we compared the stepping behavior of myoVa versus myoVc teams, and myoVc stepping patterns on single actin filaments versus actin bundles. Run lengths of both myoVa and myoVc teams increased with motor number, but the run lengths of myoVc teams were longer on actin bundles than on filaments. By resolving the stepping behavior of individual myoVc motors with a Qdot bound to the motor domain, we found that coupling of two myoVc molecules significantly decreases futile back/side steps, which were frequently observed for single myoVc motors. Data showing how changes in the inter-motor distance of two coupled myoVc motors affected stepping dynamics suggested that mechanical tension coordinates the stepping behavior of two molecules for efficient directional motion. Our study thus provides a molecular basis to explain how teams of myoVc motors are suited to transport cargos such as zymogen granules on actin bundles.

biophysics↗