Biomedical subjects

Elena B Krementsova

Publications and source records attributed to Elena B Krementsova.

8 recordsLinked to original sources

PubMed2006

Regulation of myosin V processivity by calcium at the single molecule level.

Calcium can affect myosin V (myoV) function in at least two ways. The full-length molecule, which adopts a folded inhibited conformation in EGTA, becomes extended and active in the presence of calcium. Calcium also dissociates one or more calmodulin molecules from the extended neck. Here we investigated at the single molecule level how calcium regulates the processive run length of full-length myosin V (dFull) and a truncated dimeric construct (dHMM), which cannot adopt the folded conformation. The processivity of dFull and dHMM is tightly controlled by the calcium and calmodulin concentration, with shorter runs occurring at higher calcium concentration. The data indicate that a calcium-dependent dissociation of calmodulin from the neck region of myoV terminates its processive run. dFull showed unexpected processive movement in EGTA, suggesting that a small population of extended, active molecules are in equilibrium with the inhibited, folded form. Single turnover assays showed that the ATPase activity of the folded full-length molecule is inhibited by more than 50-fold compared with the extended molecule. The results imply that activation and termination of the processive runs of myoV can be accomplished by multiple mechanisms.

Actins↗

PubMed2006

Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography.

Unconventional myosin V (myoV) is an actin-based molecular motor that has a key function in organelle and mRNA transport, as well as in membrane trafficking. MyoV was the first member of the myosin superfamily shown to be processive, meaning that a single motor protein can 'walk' hand-over-hand along an actin filament for many steps before detaching. Full-length myoV has a low actin-activated MgATPase activity at low [Ca2+], whereas expressed constructs lacking the cargo-binding domain have a high activity regardless of [Ca2+] (refs 5-7). Hydrodynamic data and electron micrographs indicate that the active state is extended, whereas the inactive state is compact. Here we show the first three-dimensional structure of the myoV inactive state. Each myoV molecule consists of two heads that contain an amino-terminal motor domain followed by a lever arm that binds six calmodulins. The heads are followed by a coiled-coil dimerization domain (S2) and a carboxy-terminal globular cargo-binding domain. In the inactive structure, bending of myoV at the head-S2 junction places the cargo-binding domain near the motor domain's ATP-binding pocket, indicating that ATPase inhibition might occur through decreased rates of nucleotide exchange. The actin-binding interfaces are unobstructed, and the lever arm is oriented in a position typical of strong actin-binding states. This structure indicates that motor recycling after cargo delivery might occur through transport on actively treadmilling actin filaments rather than by diffusion.

Actins↗

PubMed2005

Processivity of chimeric class V myosins.

Unconventional myosin V takes many 36-nm steps along an actin filament before it dissociates, thus ensuring its ability to move cargo intracellularly over long distances. In the present study we assessed the structural features that affect processive run length by analyzing the properties of chimeras of mouse myosin V and a non-processive class V myosin from yeast (Myo4p) (Reck-Peterson, S. L., Tyska, M. J., Novick, P. J., and Mooseker, M. S. (2001) J. Cell Biol. 153, 1121-1126). Surprisingly a chimera containing the yeast motor domain on the neck and rod of mouse myosin V (Y-MD) showed longer run lengths than mouse wild type at low salt. Run lengths of mouse myosin V showed little salt dependence, whereas those of Y-MD decreased steeply with ionic strength, similar to a chimera containing yeast loop 2 in the mouse myosin V backbone. Loop 2 binds to acidic patches on actin in the weak binding states of the cycle (Volkmann, N., Liu, H., Hazelwood, L., Krementsova, E. B., Lowey, S., Trybus, K. M., and Hanein, D. (2005) Mol. Cell 19, 595-605). Constructs containing yeast loop 2, which has no net charge compared with +6 for wild type, showed a higher K(m) for actin in steady-state ATPase assays. The results imply that a positively charged loop 2 and a high affinity for actin are important to maintain processivity near physiologic ionic strength.

Actins↗

PubMed2005

The structural basis of myosin V processive movement as revealed by electron cryomicroscopy.

The processive motor myosin V has a relatively high affinity for actin in the presence of ATP and, thus, offers the unique opportunity to visualize some of the weaker, hitherto inaccessible, actin bound states of the ATPase cycle. Here, electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional (3D) reconstructions provide the atomic models of myosin V in both weak and strong actin bound states. One structure shows that ATP binding opens the long cleft dividing the actin binding region of the motor domain, thus destroying the strong binding actomyosin interface while rearranging loop 2 as a tether. Nucleotide analogs showed a second new state in which the lever arm points upward, in a prepower-stroke configuration (lever arm up) bound to actin before phosphate release. Our findings reveal how the structural elements of myosin V work together to allow myosin V to step along actin for multiple ATPase cycles without dissociating.

Actins↗

PubMed2005

Differential labeling of myosin V heads with quantum dots allows direct visualization of hand-over-hand processivity.

The double-headed myosin V molecular motor carries intracellular cargo processively along actin tracks in a hand-over-hand manner. To test this hypothesis at the molecular level, we observed single myosin V molecules that were differentially labeled with quantum dots having different emission spectra so that the position of each head could be identified with approximately 6-nm resolution in a total internal reflectance microscope. With this approach, the individual heads of a single myosin V molecule were observed taking 72-nm steps as they alternated positions on the actin filament during processive movement. In addition, the heads were separated by 36 nm during pauses in motion, suggesting attachment to actin along its helical repeat. The 36-nm interhead spacing, the 72-nm step size, and the observation that heads alternate between leading and trailing positions on actin are obvious predictions of the hand-over-hand model, thus confirming myosin V's mode of walking along an actin filament.

Actins↗

PubMed2004

Myosin V processivity: multiple kinetic pathways for head-to-head coordination.

Myosin V, a double-headed molecular motor, transports organelles within cells by walking processively along actin, a process that requires coordination between the heads. To understand the mechanism underlying this coordination, processive runs of single myosin V molecules were perturbed by varying nucleotide content. Contrary to current views, our results show that the two heads of a myosin V molecule communicate, not through any one mechanism but through an elaborate system of cooperative mechanisms involving multiple kinetic pathways. These mechanisms introduce redundancy and safeguards that ensure robust processivity under differing physiologic demands.

Adenosine Diphosphate↗

PubMed2004

Myosin V: regulation by calcium, calmodulin, and the tail domain.

Calcium activates the ATPase activity of tissue-purified myosin V, but not that of shorter expressed constructs. Here, we resolve this discrepancy by comparing an expressed full-length myosin V (dFull) to three shorter constructs. Only dFull has low ATPase activity in EGTA, and significantly higher activity in calcium. Based on hydrodynamic data and electron microscopic images, the inhibited state is due to a compact conformation that is possible only with the whole molecule. The paradoxical finding that dFull moved actin in EGTA suggests that binding of the molecule to the substratum turns it on, perhaps mimicking cargo activation. Calcium slows, but does not stop the rate of actin movement if excess calmodulin (CaM) is present. Without excess CaM, calcium binding to the high affinity sites dissociates CaM and stops motility. We propose that a folded-to-extended conformational change that is controlled by calcium and CaM, and probably by cargo binding itself, regulates myosin V's ability to transport cargo in the cell.

Actins↗

PubMed2004

Does the myosin V neck region act as a lever?

Recent research efforts from several groups have addressed the question of whether the amplitude of myosin's unitary step size is proportional to the length of the neck region. Unconventional myosin V, which has an extended neck region with 6IQ motifs, provides a natural template by which to test the lever arm model via mutational analysis. The most stringent test requires that a series of single-headed molecules from the same myosin class be analyzed. Here we characterized the unitary mechanics of three single-headed fragments of myosin V expressed in the baculovirus/insect cell system. Each construct consisted of the motor domain (MD) and a variable number of IQ motifs (MD2IQ, MD4IQ and MD6IQ) that bind calmodulin, followed by an epitope tag so that the molecule can be attached to the nitrocellulose surface via an antibody. The results show a correlation between the unitary step size and the number of IQ motifs, confirming that the myosin neck region acts as a lever. The step size of MD2IQ is twice that observed from single-headed subfragments of class II myosins with the same neck length. Our results are discussed in relation to data obtained concurrently from other laboratories with similar constructs.

Actins↗