The Way Things Move: Looking Under the Hood of Molecular Motor Proteins

Vale, Ronald D.; Milligan, Ronald A.

doi:10.1126/science.288.5463.88

Cited by 1,384 publications

(1,197 citation statements)

References 60 publications

Supporting

Mentioning

1,158

Contrasting

Unclassified

Order By: Relevance

“…In Figure 2, the rate of ATP turnover was plotted as a function of MgATP concentration, and the data were fit to the following quadratic equation: (1) where rate is the concentration of product formed per second per Eg5 site, k cat is the maximum rate constant of product formation at saturating substrate, E 0 is the total Eg5 site concentration, and K m,ATP is the MgATP concentration needed to provide one-half the maximal velocity.…”

Section: Steady-state Atpase Kineticsmentioning

confidence: 99%

ATPase Mechanism of Eg5 in the Absence of Microtubules: Insight into Microtubule Activation and Allosteric Inhibition by Monastrol

Cochran

Gilbert

2005

Biochemistry

View full text Add to dashboard Cite

The ATPase mechanism of kinesin superfamily members in the absence of microtubules remains largely uncharacterized. We have adopted a strategy to purify monomeric human Eg5 (HsKSP/ Kinesin-5) in the nucleotide-free state (apoEg5) in order to perform a detailed transient state kinetic analysis. We have used steady-state and presteady-state kinetics to define the minimal ATPase mechanism for apoEg5 in the absence and presence of the Eg5-specific inhibitor, monastrol. ATP and ADP binding both occur via a two-step process with the isomerization of the collision complex limiting each forward reaction. ATP hydrolysis and phosphate product release are rapid steps in the mechanism, and the observed rate of these steps is limited by the relatively slow isomerization of the Eg5-ATP collision complex. A conformational change coupled to ADP release is the rate-limiting step in the pathway. We propose that the microtubule amplifies and accelerates the structural transitions needed to form the ATP hydrolysis competent state and for rapid ADP release, thus stimulating ATP turnover and increasing enzymatic efficiency. Monastrol appears to bind weakly to the Eg5-ATP collision complex, but after tight ATP binding, the affinity for monastrol increases, thus inhibiting the conformational change required for ADP product release. Taken together, we hypothesize that loop L5 of Eg5 undergoes an "open" to "closed" structural transition that correlates with the rearrangements of the switch-1 and switch-2 regions at the active site during the ATPase cycle.Motor proteins from the myosin, kinesin, and dynein superfamilies are important molecular machines that utilize the energy of ATP turnover to generate force and perform various functions in eukaryotic cells. These enzymes coordinate movements of conserved structural elements located at the nucleotide binding site (P-loop, switch-1, switch-2) with structural elements that interact with the filament surface (actin-or microtubule-binding interface) (1)(2)(3)(4)(5)(6)(7)(8). The ATPase activity and enzymatic efficiency of these molecular motors are activated in the presence of their filament partner, which is thought to be mediated through acceleration of the rate of product release (reviewed in ref 9). However, the structural basis for this phenomenon is not well understood.The ATPase mechanisms of several different monomeric kinesins have been extensively studied in the presence of microtubules: conventional kinesin/Kinesin-1 (10-12), Eg5/ Kinesin-5 (13), and Ncd/Kar3/ . On the other hand, very little is known about the ATPase mechanism of kinesins in the absence of microtubules (17). Historically, kinesins have been purified with ADP bound to the nucleotide binding site (18), and attempts † This work was supported by Grant GM54141 from NIGMS, National Institutes of Health (NIH), and through Career Development Award K02-AR47841 from NIAMS, NIH, Department of Health and Human Services (to S.P.G.). to isolate a homogeneous, nucleotide-free population have been difficult due to the in...

show abstract

Section: Steady-state Atpase Kineticsmentioning

confidence: 99%

ATPase Mechanism of Eg5 in the Absence of Microtubules: Insight into Microtubule Activation and Allosteric Inhibition by Monastrol

Cochran

Gilbert

2005

Biochemistry

View full text Add to dashboard Cite

show abstract

“…10 The regulation of this contraction is intimately linked to Ca 21 signals that are primarily transmitted by Tn and Tm. On calcium influx into the sarcomere, troponin binds Ca 21 , which induces a conformational change in the protein that causes Tm to move on the thin filament to a position that opens myosin S1 binding sites on actin.…”

Section: Introductionmentioning

confidence: 99%

Invited review: Probing the structures of muscle regulatory proteins using small‐angle solution scattering

Jeffries

Trewhella

2011

Biopolymers

View full text Add to dashboard Cite

Small‐angle X‐ray and neutron scattering with contrast variation have made important contributions in advancing our understanding of muscle regulatory protein structures in the context of the dynamic molecular processes governing muscle action. The contributions of the scattering investigations have depended upon the results of key crystallographic, NMR, and electron microscopy experiments that have provided detailed structural information that has aided in the interpretation of the scattering data. This review will cover the advances made using small‐angle scattering techniques, in combination with the results from these complementary techniques, in probing the structures of troponin and myosin binding protein C. A focus of the troponin work has been to understand the isoform differences between the skeletal and cardiac isoforms of this major calcium receptor in muscle. In the case of myosin binding protein C, significant data are accumulating, indicating that this protein may act to modulate the primary calcium signals from troponin, and interest in its biological role has grown because of linkages between gene mutations in the cardiac isoform and serious heart disease. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 505–516, 2011.

show abstract

“…These fall into two classes, the first of which consists of rotary motors, such as the F 1 ATPase 22 and the bacterial flagellar motor 23 , which turn a spindle by a rotary conformational change mechanism. The second class consists of linear motors, like those required for muscle contraction 24 , where conformational fluctuations undergo a translation with each step. The ϕ29 DNA packaging motor as we have described it represents a third class, where rotary motion is converted into translational movement.…”

mentioning

confidence: 99%

Structure of the bacteriophage φ29 DNA packaging motor

Simpson

Tao

Leiman

et al. 2000

Nature

493

658

View full text Add to dashboard Cite

Motors generating mechanical force, powered by the hydrolysis of ATP, translocate doublestranded DNA into preformed capsids (proheads) of bacterial viruses 1,2 and certain animal viruses 3 . Here we describe the motor that packages the double-stranded DNA of the Bacillus subtilis bacteriophage ϕ29 into a precursor capsid. We determined the structure of the head-tail connector-the central component of the ϕ29 DNA packaging motor-to 3.2Å resolution by means of X-ray crystallography. We then fitted the connector into the electron densities of the prohead and of the partially packaged prohead as determined using cryo-electron microscopy and image reconstruction analysis. Our results suggest that the prohead plus dodecameric connector, prohead RNA, viral ATPase and DNA comprise a rotary motor with the head-prohead RNAATPase complex acting as a stator, the DNA acting as a spindle, and the connector as a ball-race. The helical nature of the DNA converts the rotary action of the connector into translation of the DNA.The bacteriophage ϕ29 (Fig. 1) is a 19-kilobase (19-kb) double-stranded DNA (dsDNA) virus with a prolate head and complex structure 4 . The prohead (Fig. 1), into which the DNA is packaged, is about 540Å long and 450Å wide 5 . The ϕ29 connector, a cone-shaped dodecamer of gene product 10 (gp10), occupies the pentagonal vertex at the base of the prohead 5 and is the portal for DNA entry during packaging and DNA ejection during infection 6 . The connector, in association with the oligomeric, ϕ29-encoded prohead RNA (pRNA) and a viral ATPase (gp16), is required for DNA packaging [7][8][9] . However, only the first 120 bases of the 174-base pRNA are essential for packaging 7 the genomic dsDNA with gp3 (DNA-gp3) can be packaged into proheads in about three minutes in vitro (P.J.J., unpublished results). The connector proteins of tailed phages 6 vary in relative molecular mass (M r ) from 36,000 (36K) in ϕ29 to 83K in phage P22, and assemble into oligomers with a central channel. The structure of the isolated ϕ29 connector has been studied by atomic force microscopy 10 and cryo-electron microscopy (cryo-EM) of two-dimensional arrays 11 , immuno-electron microscopy 12 and X-ray crystallography 13,14 .The connector structure, as now determined by X-ray crystallography, can be divided into three, approximately cylindrical regions: the narrow end, the central part, and the wide end, having external radii (Å ) of 33, 47 and 69, respectively (Fig. 2). These regions are respectively 25, 28 and 22Å in height, making the total connector 75Å long. The internal channel has a diameter of about 36Å at the narrow end, increasing to 60Å at the wide end.Comparison with electron microscopy reconstructions 5,11 shows that the narrow end protrudes from the portal vertex of the phage head, is associated with the multimeric pRNA, and binds the lower collar in the mature virus.The electron density of the connector was interpreted in terms of the amino-acid sequence 15 and was confirmed by the two Hg sites (see Methods section)...

show abstract

The Way Things Move: Looking Under the Hood of Molecular Motor Proteins

Cited by 1,384 publications

References 60 publications

ATPase Mechanism of Eg5 in the Absence of Microtubules: Insight into Microtubule Activation and Allosteric Inhibition by Monastrol

ATPase Mechanism of Eg5 in the Absence of Microtubules: Insight into Microtubule Activation and Allosteric Inhibition by Monastrol

Invited review: Probing the structures of muscle regulatory proteins using small‐angle solution scattering

Structure of the bacteriophage φ29 DNA packaging motor

Contact Info

Product

Resources

About