Edwin W. Taylor

A structural change in the kinesin motor protein that drives motility

Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of a ∼15-amino-acid region (the neck linker) in kinesin using electron paramagnetic resonance, fluorescence resonance energy transfer, pre-steady state kinetics and cryo-electron microscopy. This region becomes immobilized and extended towards the microtubule ‘plus’ end when kinesin binds microtubules and ATP, and reverts to a more mobile conformation when γ-phosphate is released after nucleotide hydrolysis. This conformational change explains both the direction of kinesin motion and processive movement by the kinesin dimer.

Interacting Head Mechanism of Microtubule-Kinesin ATPase

Kinetic and equilibrium properties are compared for a monomeric kinesin construct (K332) and a dimeric construct (K379). MtK379 has a low affinity (5 × 104 M−1) and a high affinity (5 × 106 M−1) binding site for mant ADP while MtK332 has a single low affinity site (5 × 104 M−1). Rate constants of dissociation of mant ADP are <1 s−1 for the high affinity site and 75-100 s−1 for the low affinity site for MtK379. For MtK332, the effective rate constant is 200-300 s−1. It is proposed that the two heads of the dimer are different through the interaction with the microtubule, a strongly bound head with low affinity for 2′-(3′)-O-(N-methylanthraniloyl) adenosine 5′-diphosphate (mant ADP), similar to the single strongly bound head of the monomer and a weakly bound or detached head with high affinity for mant ADP. Rate of binding of mant ADP gave an “S“-shaped dependence on concentration for MtK379 and a hyperbolic dependence for MtK332. Binding of K379·mant ADP dimer to microtubules releases only one mant ADP at a rate of 50 s−1. The second strongly bound mant ADP is released by binding of nucleotides to the other head. Rates are 100 s−1 for ATP, 30 s−1 for AMPPNP or ATPγS, and 2 s−1 for ADP. The rate of binding of mant ATP to MtK379 showed an “S“-shaped concentration dependence and limiting rate at zero concentration is <1 s−1 while MtK332 gave a hyperbolic dependence and limiting rate of 100 s−1. The limiting rate is determined by the rate of dissociation of mant ADP in the hydrolysis cycle. The evidence is consistent with an interacting site model in which binding of ATP to one head is required for release of ADP from the other head in the hydrolysis cycle. This model, in which the cycles are maintained partly out of phase, is an extension of the alternating site model of Hackney (Hackney, D. D. (1994) Proc. Nat. Acad. Sci. U. S. A. 91, 6865-6869). It provides a basis for a processive mechanism.

Kinetic Mechanism of a Monomeric Kinesin Construct

The kinetic mechanism is analyzed for a monomeric human kinesin construct K332. In the absence of microtubules, the rate constants of the ATPase cycle are very similar to dimeric human kinesin K379 and whole kinesin from bovine brain. The microtubule-activated ATPase is 60 s−1 at 20°C; Km(Mt) is 5 μM; dissociation constants in the presence of ATP and ADP are 9 μM and 16 μM, respectively. The values of dissociation constants are 5 times larger than for K379. Binding of K332 to microtubules increased the rate of the hydrolysis step from 7 s−1 to greater than 200 s−1 and the 2′-(3′)-O-(N-methylanthraniloyl) (mant) ADP dissociation step from 0.02 s−1 to greater than 100 s−1. At higher ionic strength, more than one ATP is hydrolyzed before dissociation of MtK332 (small processivity). Data are fitted to the kinetic scheme.

Kinetic Mechanism of Monomeric Non-claret Disjunctional Protein (Ncd) ATPase

The non-claret disjunctional protein (Ncd) is a kinesin-related microtubule motor that moves toward the negative end of microtubules. The kinetic mechanism of the monomer motor domain, residues 335–700, satisfied a simple scheme for the binding of 2′-3′-O-(N-methylanthraniloyl) (MANT) ATP, the hydrolysis step, and the binding and release of MANT ADP, Equation 1 MtN + T   ⇌ k 1 > 300   s − 1   MtN · T   ⇌ k 2 = 15   s − 1   MtN · D · P i   ⇌ k 3 ≥ 10   s − 1   ( − P ) MtN · D       ⇌ k − 4 = 220   s − 1 k 4 = 4   s − 1   MtN + D   where T, D, and Pi refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively, and MtN is the complex of an Ncd motor domain with a microtubule site. Rate constantsk 1 and k −4 are the rates of a first order step, an isomerization induced by nucleotide binding. The apparent second order rate constants for the binding steps are 1.5 × 106 m −1s−1 for MANT ATP and 3.5 × 106 m −1 s−1 for MANT ADP (conditions, 50 mm NaCl, pH 6.9, 21 °C). The rate constant of the hydrolysis step (k 2) was obtained from quench flow measurements of the phosphate burst phase corrected for the contribution of the rate of product release to the transient rate constant. The rate of phosphate dissociation was not measured; the value was assigned to account for a steady state rate of 3 s−1. The MtN complex is dissociated by ATP at a rate of 10 s−1 based on light scattering measurements. Dissociation constants of Ncd-nucleotide complexes from microtubules increased in the order adenosine 5′-O-(thiotriphosphate) (ATPγS) < ADP-AlF4 < ATP < ADP < ADP-vanadate. Comparison of the properties of Ncd with a monomeric kinesin K332 (Ma and Taylor (1997) J. Biol. Chem. 272, 717–723) showed a close similarity, except that the rate constants for the hydrolysis and ADP release steps and the steady state rate are approximately 15–20 times smaller for Ncd. There are two differences that may affect the reaction pathway. The rate of dissociation of MtN by ATP is comparable to the rate of the hydrolysis step, and N·T may dissociate in the cycle, whereas for kinesin, dissociation occurs after hydrolysis. The rate of dissociation of MtN by ADP is larger than the rate of ADP release from MtN·D, whereas for the microtubule-kinesin complex, the rate of dissociation by ADP is smaller than the rate of ADP release. The monomeric Mt·Ncd complex is not processive.

Kinetic studies of normal and modified heavy meromyosin and subfragment-1

SummaryThe kinetic behaviour of heavy meromyosin (HMM) and subfragment-1 (S-1) has been compared for a range of pH and temperature for normal and SH-1 modified proteins. No significant differences were found between S-1 and HMM and the evidence is consistent with an identical independent site mechanism of the form first proposed by Bagshaw and Trentham:

Structural mechanisms in muscle. Jean Hanson’s legacy: to celebrate the 50th anniversary of the sliding filament theory of muscle contraction

\begin{gathered} M + ATP\mathop { \leftharpoondown \rightharpoonup }\limits^{K_1 } M.ATP \mathop { \leftharpoondown \rightharpoonup }\limits^{k_2 } M.ATP^* \mathop { \leftharpoondown \rightharpoonup }\limits^{k_3 } M.Pr^{**} \mathop {\mathop { \leftharpoondown \rightharpoonup }\limits_{H^ + ,P_i } }\limits^{k_4 } \hfill \\ M.ADP^* \mathop { \leftharpoondown \rightharpoonup }\limits^{k_5 } M.ADP \mathop { \leftharpoondown \rightharpoonup }\limits^{K_6 } M + ADP \hfill \\ \end{gathered}

PROPERTIES OF THE PROTEIN SUBUNIT OF CENTRAL-PAIR AND OUTER-DOUBLET MICROTUBULES OF SEA URCHIN FLAGELLA

where asterisks refer to states of enhanced tryptophan fluorescence and a M.Pr is a state in which both products are bound.k3 andk5 had a large temperature dependence (Arrhenius activation energy approximately 100 kJ mol−1).k3 andk5 increased with increasing pH and the variation fitted a titration curve with pK of 7.4. The rate of step 4 was measured by decay of tryptophan fluorescence, proton release and phosphate release measured as an increase in conductance. The temperature dependence curves fork4 andk5 cross over at 11–13° C for Mn2+-ATPase, 3–5° C for SH-1 modified Mg2+-ATPase and below 0° C for normal Mg2+-ATPase. A change of the rate-determining step fromk4 tok5 accounts for the nonlinear Arrhenius plots of Mn2+-ATPase and SH-1 modified Mg2+-ATPase. Modification also reduced the rate of the hydrolytic stepk3 measured by the maximum rate of the phosphate early burst. The two fluorescence transitions (steps 2 and 3) are easily resolved for the modified enzyme andk3 measured by fluorescence was equal to the rate of hydrolysis obtained from phosphate measurements.

Mechanism of microtubule kinesin ATPase.

Muscle cells have an extremely efficient regulation system which reduces actomyosin ATPase activity by more than one thousand fold in relaxed versus active muscle. A surprising fact is that two quite different regulation systems are used in striated and smooth muscles. The actomyosin ATPase of smooth muscles as well as non muscle cells is activated by a calmodulin dependent protein kinase which specifically phosphorylates a myosin light chain (LC-2), Actomyosin is inhibited by dephosphorylation by a specific phosphatase.1 Activation of enzymes by phosphorylation is a very common control mechanism and its use in muscle regulation is not surprising. The second mechanism apparently evolved to meet the need for faster switching on and off in striated muscles. Although a phosphorylatable light chain has been retained by striated muscle myosin and the level of phosphorylation can be altered by stimulation, phosphorylation no longer activates the actomyosin ATPase. Regulation is obtained by a structural change of the thin filament. The thin filaments in striated muscle contain troponin which is not present in smooth muscle or non-muscle cells.