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Interacting head mechanism of microtubule-kinesin ATPase
1Department of Molecular Genetics and Cell Biology, The University of Chicago, Illinois 60637, USA.
Abstract:
Kinetic and equilibrium properties are compared for a monomeric kinesin construct (K332) and a dimeric construct (K379). MtK379 has a low affinity (5 x 10(4) M(-1)) and a high affinity (5 x 10(6) M(-1)) binding site for mant ADP while MtK332 has a single low affinity site (5 x 10(4) 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 x 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 ATPgammaS, 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.
Insights
Kinesin dimers (K379) exhibit distinct head affinities for mant ADP compared to monomers (K332), influencing nucleotide binding and release kinetics. This suggests an interacting site model for processive movement.
Area of Science:
- Biochemistry
- Molecular Biology
- Motor Proteins
Background:
- Kinesin motors are crucial for intracellular transport, utilizing ATP hydrolysis to generate force.
- Understanding the kinetic and equilibrium properties of kinesin constructs is essential for elucidating their mechanism of action.
- Previous models, like Hackney's alternating site model, provide a framework for kinesin function.
Purpose of the Study:
- To compare the kinetic and equilibrium properties of monomeric (K332) and dimeric (K379) kinesin constructs.
- To investigate the binding affinities and dissociation rates of 2'-(3')-O-(N-methylanthraniloyl) adenosine 5'-diphosphate (mant ADP) for different kinesin states.
- To explore the mechanism of nucleotide binding and release in dimeric kinesin and its implications for processivity.
Main Methods:
- Fluorescence-based assays to measure binding and dissociation kinetics of mant ADP.
- Comparison of kinetic parameters (affinity, rate constants) between monomeric and dimeric kinesin constructs.
- Analysis of nucleotide binding curves (mant ADP and mant ATP) to determine concentration dependence.
Main Results:
- Dimeric kinesin (K379) possesses distinct high and low affinity binding sites for mant ADP, unlike monomeric kinesin (K332) with a single low affinity site.
- Dissociation rates of mant ADP differ significantly between the high (<1 s⁻¹) and low (75-100 s⁻¹) affinity sites of K379, and the effective rate for K332 is 200-300 s⁻¹.
- Nucleotide binding curves show an 'S'-shaped dependence for K379 and hyperbolic for K332, indicating cooperative or independent binding, respectively.
Conclusions:
- The dimeric kinesin likely has two functionally distinct heads interacting differently with microtubules, one strongly bound with low ADP affinity and the other weakly bound with high ADP affinity.
- Binding of ATP to one head of the dimer is necessary for the release of ADP from the other head, supporting an interacting site model.
- This interacting site model, an extension of the alternating site model, provides a mechanistic basis for kinesin processivity.