Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor

Zeymer, C.; Barends, T.R.M.; Werbeck, N.D.; Schlichting, I.; Reinstein, J.

doi:10.1107/S1399004713030629

Figure 5
The active-site mutation R621Q facilitates the incorporation of Mg²⁺ into ClpB NBD2. (a) Structure of ClpB NBD2 R621Q in complex with AMPPCP. The essential Mg²⁺ ion is coordinated by the β- and γ-phosphates of the bound nucleotide, the Walker A threonine Thr602 and three water molecules. Owing to the mutation and the high MgCl₂ concentration of 75 mM in the crystallization solution, a second Mg²⁺ ion with a complete hydration shell [d(Mg–O) = 2.0–2.3 Å] is present in the catalytic site at a distance of 4.5 Å from the γ-phosphate. The Walker A lysine Lys601 adopts the stretched conformation observed in the previous structures and does not contact the nucleotide. The Walker B aspartate Asp667 forms hydrogen bonds to water molecules of the hydration shell of the essential Mg²⁺ ion. However, there is no significant rearrangement of the catalytic site compared with the Mg-free structures. Residues 621 and 709 were modelled using alternative conformations, which are both shown in the figure. (b) Structure of ClpB NBD2 R621Q in complex with AMPPCP and GdmCl. Using a lower MgCl₂ concentration of 10 mM and additionally soaking with 100 mM GdmCl, the second Mg²⁺ ion was replaced by a Gdm⁺ ion, whereas the essential Mg²⁺ ion bound at the phosphates of the nucleotide remained. The Gdm⁺ ion represents a mobile arginine side-chain mimic, suggesting that the mutated Arg621 would usually occupy this position. Residue 709 was modelled using alternative conformations, which are both shown in the figure. (c) Superposition of the two crystal forms observed for ClpB NBD2 showing two different positions for the conserved active-site residue Arg621 (green, wild-type NBD2 + AMPPCP; blue, NBD2 K601Q + MANT-dADP). Additionally, the AMPPCP, the essential Mg²⁺ ion and the Gdm⁺ ion of the NBD2 R621Q structure shown in (b) are overlaid in yellow, illustrating the two possible positions of the Arg621 side chain. In position 1, Arg621 is located close to the nucleotide and impairs Mg²⁺ incorporation. In position 2, it is at a greater distance from the nucleotide, allowing Mg²⁺ binding.

BIOLOGICAL
CRYSTALLOGRAPHY

ISSN: 1399-0047

Volume 70| Part 2| January 2014| Pages 582-595

doi:10.1107/S1399004713030629

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