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 kinesin⅐AMPPPNP 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 ␥-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.

“Snapshots” of Ispinesib-induced Conformational Changes in the Mitotic Kinesin Eg5

“…The first of these contains a single cysteine in L5 (Eg5 W127C ). Residue Trp 127 makes a -stacking aromatic interaction with a tyrosine in ␣3 (Tyr 211 ) in the AMPPNP and monastrol ϩ ADP crystal structures (11,19). Like tryptophan, the cysteine-reactive fluorescent probe monobromobimane (mBBr) is also composed of two rings.…”

Prime movers: the mechanochemistry of mitotic kinesins

“…During ATP hydrolysis, a critical salt bridge links these two elements, forming the phosphate tube. This salt bridge positions two water molecules that are proposed to split the beta-gamma phosphate bond of ATP by extracting a proton 17 . The Mg 2+ ion of Mg•ATP is essential for catalytic activity of the kinesin ATPase.…”

“…The Mg 2+ ion of Mg•ATP is essential for catalytic activity of the kinesin ATPase. The Mg 2+ ion bridges the beta and gamma phosphates of ATP and, once docked into the active site, is coordinated by residues from the P-loop and (both directly and via water molecules) from the two switch elements 17 (FIG. 2b).…”

Molecular dynamics simulation of GTPase activity in polymers of the cell division protein FtsZ

“…1, inset): first the distance ''d'' from the oxygen atom of the water molecule (O wat ) to the phosphorus atom of the gamma phosphate (P c ) of GTP and also the angle ''a'' between the axis defined by the same O wat and P c atoms, and the axis defined by P c and the oxygen 3 atom of the beta phosphate (O3 b ) of GTP. Only water molecules located at a distance of 3.0-3.8 Å from P c and at an angle greater than 110°with respect to the P c -O3 b axis, can hydrolyze the GTP molecule [20][21][22]. Those FtsZ polymer interfaces containing water molecules that met these two conditions in a stable way were considered as ''active interfaces'' in terms of GTPase activity.…”