ATP hydrolysis in Eg5 kinesin involves a catalytic two-water mechanism

Abstract

Motor proteins couple steps in ATP binding and hydrolysis to conformational switching both in and remote from the active site. In our kinesin.AMPPPNP crystal structure, closure of the active site results in structural transformations appropriate for microtubule binding and organizes an orthosteric two-water cluster. We conclude that a proton is shared between the lytic water, positioned for gamma-phosphate attack, and a second water that serves as a general base. To our knowledge, this is the first experimental detection of the catalytic base for any ATPase. Deprotonation of the second water by switch residues likely triggers subsequent large scale structural rearrangements. Therefore, the catalytic base is responsible for initiating nucleophilic attack of ATP and for relaying the positive charge over long distances to initiate mechanotransduction. Coordination of switch movements via sequential proton transfer along paired water clusters may be universal for nucleotide triphosphatases with conserved active sites, such as myosins and G-proteins.

FIGURE 1.

Structural features of Eg5·AMPPNP. a, F_o − F_c simulated annealing omit density map contoured at 3σ surrounds the AMPPNP, Mg²⁺, W1, and W2. b, highlighted in the Eg5·AMPPNP complex are L9 (yellow) containing switch I, L11 (green) containing switch II, the P-loop (pink), and the L5-loop (red), which is involved in binding allosteric inhibitors. AMPPNP and Mg²⁺ (dark sphere) are also shown. c, comparison of α6, α4, and the neck linker of Eg5·AMPPNP (red), Eg5·ADP (cyan), and Kif1A·AMPPNP (yellow) after superimposing the structures (see “Experimental Procedures”).

FIGURE 2.

Closed nucleotide-binding site. Comparisons of the α3 helix and switch I loop (a and b) and of the relay helix and switch II loops (c and d) from representative kinesin and myosin structures are shown. a, differences in α3 and L9 between Eg5·AMPPNP (red) with the equivalent sequences of Eg5·ADP (cyan) and Kif1A·AMPPNP (yellow). Disordered residues are shown as dashed lines. Note that L9 of Eg5·AMPPNP approaches the nucleotide in this closed conformation. b, Eg5·AMPPNP (red) and myosin·ADP·vanadate (gray) have similar switch I loop conformations. Conserved switch I arginines are shown. c, relay helices and switch II loops of Eg5·AMPPNP (red), Eg5·ADP (cyan), and Kif1A·AMPPNP (yellow). L11 of Eg5·AMPPNP is ordered, and the relay helix is in an ATP-like position. d, Eg5·AMPPNP (red) and myosin·ADP·vanadate (gray) have similar switch II loop conformations. Conserved switch II glutamates are shown.

FIGURE 3.

Nucleotide-binding pocket of Eg5·AMPPNP. a, waters W1 and W2 make extensive interactions with switch I residues Ser²³³ and Arg²³⁴ (yellow) and switch II residues Gly²⁶⁸ and Glu²⁷⁰ (green). Measured interatomic distances are shown. W1 is 3.3 Å from the γ-phosphorus atom. b, oxygen atoms of the nucleotide γ-phosphate are coordinated by Mg²⁺, switch I and switch II residues, and residues of the P-loop (pink).

FIGURE 4.

Coordination of the Mg²⁺ ion. The Mg²⁺ ion is tightly coordinated by oxygen atoms of the β- and γ-phosphate, Ser²³³ of switch I, Thr¹¹² of the P-loop, and two water molecules. The ion location is quite similar to that seen in the Eg5·ADP structure (18), in which the Mg²⁺ ion is coordinated by Thr¹¹², a β-phosphate oxygen atom, and four water molecules.

FIGURE 5.

Proposed model of ATP hydrolysis. Four different mechanistic steps are postulated for this human kinesin-5 motor domain: a, the prehydrolysis state, in which there is deprotonation of the nucleophilic water by W2; b, nucleophilic attack of the scissile phosphate of ATP; c, cleavage of the β-γ-phosphoanhydride bond; and d, deprotonation of W2 by Glu²⁷⁰. Shown are residues from switch I (tan), switch II (green), and the P-loop (pink) that coordinate W1, W2, and the β- and γ-phosphates of the substrate in the prehydrolysis state. Also shown are three of the interactions for the divalent cation (black sphere). Atomic details in b and c are speculative and are hypothetical interactions for residues shown in panel a. The atomic details in d reflect only a subset of the interactions reported for the Eg5·ADP structure (18).