CiteULike: cactus's kinetics

The lattice as allosteric effector: Structural studies of alphabeta- and gamma-tubulin clarify the role of GTP in microtubule assembly

Luke Rice — 2008-05-02T10:24:15-00:00

Proceedings of the National Academy of Sciences, Vol. 105, No. 14. (8 April 2008), pp. 5378-5383.

GTP-dependent microtubule polymerization dynamics are required for cell division and are accompanied by domain rearrangements in the polymerizing subunit, alpha-tubulin. Two opposing models describe the role of GTP and its relationship to conformational change in alpha-tubulin. The allosteric model posits that unpolymerized alpha-tubulin adopts a more polymerization-competent conformation upon GTP binding. The lattice model posits that conformational changes occur only upon recruitment into the growing lattice. Published data support a lattice model, but are largely indirect and so the allosteric model has prevailed. We present two independent solution probes of the conformation of alpha-tubulin, the 2.3 A crystal structure of gamma-tubulin bound to GDP, and kinetic simulations to interpret the functional consequences of the structural data. These results (with our previous gamma-tubulin:GTPgammaS structure) support the lattice model by demonstrating that major domain rearrangements do not occur in eukaryotic tubulins in response to GTP binding, and that the unpolymerized conformation of alpha-tubulin differs significantly from the polymerized one. Thus, geometric constraints of lateral self-assembly must drive alpha-tubulin conformational changes, whereas GTP plays a secondary role to tune the strength of longitudinal contacts within the microtubule lattice. alpha-Tubulin behaves like a bent spring, resisting straightening until forced to do so by GTP-mediated interactions with the growing microtubule. Kinetic simulations demonstrate that resistance to straightening opposes microtubule initiation by specifically destabilizing early assembly intermediates that are especially sensitive to the strength of lateral interactions. These data provide new insights into the molecular origins of dynamic microtubule behavior. 10.1073/pnas.0801155105

Kinetic Analysis of the Slow Skeletal Myosin MHC-1 Isoform from Bovine Masseter Muscle

MJ Bloemink — 2008-04-17T10:41:17-00:00

Journal of Molecular Biology, Vol. 373, No. 5. (9 November 2007), pp. 1184-1197.

Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as [beta]-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin-ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant KD) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (KAD/KD ~ 5 instead of KAD/KD ~ 50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s-1) than reported previously (138 s-1). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.

The kinetic mechanism of kinesin.

RA Cross — 2005-03-03T06:35:47-00:00

Trends Biochem Sci, Vol. 29, No. 6. (June 2004), pp. 301-309.

The chemical kinetic mechanism of kinesin (K) is considered by using a consensus scheme incorporating biochemically defined open, closed and trapped states. In the absence of microtubules, the dominant species is a trapped K*ADP state, which is defined by its ultra-slow release of ADP (off rate, k(off) approximately 0.002 s(-1)) and weak microtubule binding (dissociation constant, K(d) approximately 10-20 microM). Once bound, this trapped state equilibrates with a strongly binding open state that rapidly releases ADP (k(off) approximately 300 s(-1)). After ADP release, Mg*ATP binds (on rate, k(on) approximately 2 microM(-1)s(-1)) driving formation of a closed state that is defined by hydrolysis competence and by strong binding to microtubules. Hydrolysis (k(hyd) approximately 100-300 s(-1)) and phosphate release (k(off)>100 s(-1)) both occur in this microtubule-bound closed state. Phosphate release acts as a gate that controls reversion to the trapped K*ADP state, which detaches from the microtubule, completing the cycle.

Weak and Strong States of Kinesin and ncd

Isabelle Crevel — 2008-03-26T08:32:30-00:00

Journal of Molecular Biology, Vol. 257, No. 1. (22 March 1996), pp. 66-76.

Kinesin superfamily molecular motors step along microtubules (MTs)viaa cycle of conformational changes which is coupled to ATP turnover. To probe the coupling mechanism, we titrated the effects of various nucleotides on MT binding by two superfamily members; MT plus-end-directed kinesin and MT minus-end-directed non claret disjunctional (ncd). For both motors, the nucleotide-free state induced by apyrase was the strongest binding (Kkind~0.003 [mu]M,Kncdd~0.24 [mu]M), whilst the ADP state was the weakest binding (Kkind~11.32 [mu]M,Kncdd~12.02 [mu]M). In ATP, the motor.ADP state dominates and the binding is accordingly ADP-like, but in the presence of the slowly hydrolysed analogue adenosine 5'-O-(3-thiotriphosphate) there is a shift towards tighter binding (Kkind~4.23 [mu]M,Kncdd~2.34 [mu]M), consistent with a tight-binding motor.ATP-like state being enriched. In the presence of non-hydrolysable analogue [beta],[gamma]- imidoadenosine 5'-triphosphate the binding is still tighter (Kkind~<0.27 [mu]M,Kncdd~0.21 [mu]M), close to the values obtained with apyrase. For both kinesin and ncd, ADP has the unique quality that it traps the motor in a weak binding state. MT tight binding catalyses escape from this state, changing the active site conformation such that both ADP release and ADP binding are accelerated. The data are consistent with a simple two-state scheme in which both kinesin and ncd switch from weak to strong bindingviaADP release, and back againviaADP trapping. In a two-state model, the transition from weak to strong binding is force-generating.

Kinetic Characterization of the Function of Myosin Loop 4 in the Actin-Myosin Interaction

M Gyimesi — 2008-02-28T09:48:50-00:00

Biochemistry, Vol. 47, No. 1. (8 January 2008), pp. 283-291.

Abstract: Myosin interacts with actin during its enzymatic cycle, and actin stimulates myosin's ATPase activity. There are extensive interaction surfaces on both actin and myosin. Several surface loops of myosin play different roles in actomyosin interaction. However, the functional role of loop 4 in actin binding is still ambiguous. We explored the role of loop 4 by either mutating its conserved acidic group, Glu-365, to Gln (E365Q), or by replacing the entire loop with three glycines (AL) in a Dictyostelium discoideum myosin II motor domain (MD) containing a single tryptophan residue. This native tryptophan (Trp-501) is located in the relay loop and is sensitive to nucleotide binding and lever-arm movement. Fluorescence and fast kinetic measurements showed that the mutations in loop 4 do not alter the enzymatic steps of the ATPase cycle in the absence of actin. By contrast, actin binding was significantly weakened in the absence and presence of ADP and ATP in both mutants. Because the strength of actin-myosin interaction increases in the order of rigor, ADP, and ATP complex, we conclude that loop 4 is a functional actin-binding region that stabilizes actomyosin complex, particularly in weak actin-binding states.

The role of three-state docking of myosin S1 with actin in force generation.

MA Geeves — 2007-11-20T09:26:17-00:00

Biophys J, Vol. 68, No. 4 Suppl. (April 1995)

It has been shown that in solution myosin subfragment 1 binds to actin in three principal steps: [formula: see text] The nucleotide bound to myosin has a major influence on the equilibrium constant of the third of these steps but little effect on the other two. The third step is thought to be coupled to the force-generating event. Three-step binding and structure: The formation of the collision complex is strongly ionic strength dependent but independent of temperature. The isomerization to the A state is not strongly dependent on ionic strength but is affected by organic solvent and temperature. In contrast the isomerization to the R state-is affected by both ionic strength and organic solvent but little affected by temperature. The recent docking of the three-dimensional structures of actin and S1 suggest possible structural correlates of these events. These studies lead to predictions for the docking process, which may be tested using site-directed mutagenesis or peptide inhibitors. Three-step binding and head-head interactions: Studies of HMM binding to actin compared with S1 binding show that binding of two heads in the A state are unlikely presumably because of strain effects. However, binding of two heads as one A and one R state shows little evidence of strain while the isomerization of the second head to give two R states is fivefold weaker than for an isolated S1 head. These results suggest that in a rapidly shortening muscle only one head is likely to be attached at a time.(ABSTRACT TRUNCATED AT 250 WORDS)

Coupling of Rotation and Catalysis in F1-ATPase Revealed by Single-Molecule Imaging and Manipulation

Kengo Adachi — 2007-08-16T16:54:15-00:00

Cell, Vol. 130, No. 2. (27 July 2007), pp. 309-321.

Summary F1-ATPase is a rotary molecular motor that proceeds in 120[degree sign] steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F1 that phosphate release drives the last 40[degree sign] of the 120[degree sign] step, and that the 40[degree sign] rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F1 is forced to rotate slowly, that release of Cy3-ADP occurs at ~240[degree sign] after it is bound as Cy3-ATP at 0[degree sign]. This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete.

Kinetic Characterization of a Cytoplasmic Myosin Motor Domain Expressed in Dictyostelium discoideum

Md Ritchie — 2007-06-07T05:49:15-00:00

PNAS, Vol. 90, No. 18. (15 September 1993), pp. 8619-8623.

10.1073/pnas.90.18.8619

Role of the salt-bridge between switch-1 and switch-2 of Dictyostelium myosin

Marcus Furch — 2007-06-06T16:00:12-00:00

Journal of Molecular Biology, Vol. 290, No. 3. (1 January 1999), pp. 797-809.

Motifs N2 and N3, also referred to as switch-1 and switch-2, form part of the active site of molecular motors such as myosins and kinesins. In the case of myosin, N3 is thought to act as a [gamma]-phosphate sensor and moves almost 6 A relative to N2 during the catalysed turnover of ATP, opening and closing the active site surrounding the [gamma]-phosphate. The closed form seems to be necessary for hydrolysis and is stabilised by the formation of a salt-bridge between an arginine residue in N2 and a glutamate residue in N3. We examined the role of this salt-bridge in Dictyostelium discoideum myosin. Myosin motor domains with mutations E459R or R238E, that block salt-bridge formation, show defects in nucleotide-binding, reduced rates of ATP hydrolysis and a tenfold reduction in actin affinity. Inversion of the salt-bridge in double-mutant M765-IS eliminates most of the defects observed for the single mutants. With the exception of a 2,500-fold higher KM value for ATP, the double-mutant displayed enzymatic and functional properties very similar to those of the wild-type protein. Our results reveal that, independent of its orientation, the salt-bridge is required to support efficient ATP hydrolysis, normal communication between different functional regions of the myosin head, and motor function.

Coupling between phosphate release and force generation in muscle actomyosin

Y Takagi — 2005-01-14T07:34:48-00:00

Philosophical Transactions: Biological Sciences, Vol. 359, No. 1452., 1913.

Limping of Homodimeric Kinesin Motors

Ping Xie — 2007-05-07T08:28:21-00:00

Journal of Molecular Biology, Vol. 366, No. 3. (23 February 2007), pp. 976-985.

Conventional kinesin, a homodimeric motor protein that transports cargo in various cells, walks limpingly along microtubule. Here, based on our previously proposed partially coordinated hand-over-hand model, we present a new mechanism for the limping behaviors of both wild-type and mutant kinesin homodimers. The limping is caused by different vertical forces acting on the heads in two successive steps during the processive movement of the dimer. From the model, various theoretical results, such as the dependences of the mean dwell time and dwell time ratio on the coiled-coil length and on the external load as well as the effect of vertical force on velocity, are in good agreement with previous experimental results. We predict that a high degree of limping will correlate strongly with a high sensitivity of ATP turnover rate to upwards force.