Walker-A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Abstract

Hexameric AAA+ ATPases induce conformational changes in a variety of macromolecules. AAA+ structures contain the nucleotide-binding P-loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA+ sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA+ ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker-A Thr with Asn in ClpB, a bacterial AAA+ chaperone which reactivates aggregated proteins. We found that the T-to-N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP-bound conformation and the high-affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker-A Thr in sensing the nucleotide's gamma-phosphate and in maintaining an allosteric linkage between the P-loop and the aggregate binding site of ClpB. We postulate that AAA+ ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate-remodeling activity.

Figure 1

Gel-filtration analysis of ClpB(T213N/T612N). Elution profiles were measured in the absence of nucleotides (thick solid line), with 2 mM ATP (thin solid line), or with 2 mM ADP (broken line). Elution times of the molecular-weight standards (kDa) are indicated.

Figure 2

ATPase activity of ClpB. The rate of ATP hydrolysis was measured for wt ClpB or its variants in the absence of other polypeptides (A), in the presence of 0.1 mg/mL κ-casein (B, black bars), or with 0.04 mg/mL poly-l-lysine (B, white bars).

Figure 3

Nucleotide binding to ClpB. Shown are the heat effects of titrating 50 μM wt ClpB (A) and ClpB(T213N/T612N) (B) with ATPγS (•) or ADP (○). Solid lines show fits of an empirical model of multiple ligand-binding sites (MCS ITC software, MicroCal Inc.). The enthalpy of binding is negative which indicates an exothermic reaction.

Figure 4

Interactions of ClpB with aggregated substrates. Wt ClpB, ClpB(T213N), ClpB(T612N), or ClpB(T213N/T612N) (1.5 μM protein) were incubated without or with aggregated 3 μM glucose-6-phosphate dehydrogenase (A) or 3 μM malate dehydrogenase (B) without nucleotides or with 5 mM ATP, ATPγS, or ADP. The solutions were passed through a 0.1-μm filter. SDS-PAGE analysis with a Coomassie stain is shown for the fractions retained on the filter and subsequently solubilized with an SDS buffer. Representative results from two independent experiments are shown.

Figure 5

Reactivation of aggregated G6PDH in the presence of ClpB and the DnaK chaperone system. Aggregated 3 μM G6PDH was incubated without ClpB with 1 μM DnaK, 1 μM DnaJ, 0.5 μM GrpE (KJE) (+), KJE with 1.5 μM wt ClpB (•), KJE with 1.5 μM ClpB(T213N) (▵), KJE with 1.5 μM ClpB(T612N) (▴), or KJE with 1.5 μM ClpB(T213N/T612N) (♦). After incubation at 30°C for the indicated time, the G6PDH activity was measured as described in Materials and Methods. Representative results from two independent experiments are shown.

Figure 6

In vivo chaperone activity of ClpB. E. coli cells were grown at 50°C until the indicated time and their survival was determined as described in Materials and Methods. The data are shown for wt MC4100 E. coli (○), ClpB-null E. coli containing a control pGB2 plasmid (+), pGB2 with wt ClpB sequence (•), pGB2 with ClpB(T213N) (▵), pGB2 with ClpB(T612N) (▴), or pGB2 with ClpB(T213N/T612N) (♦). Inset shows immunodetection of ClpB in bacterial cultures after 6 h of heat shock. Purified ClpB is shown in Lane 1, empty pGB2 in Lane 2, wt E. coli in Lane 3, pGB2(wt ClpB) in Lane 4, pGB2(T213N) in Lane 5, pGB2(T612N) in Lane 6, and pGB2(T213N/T612N) in Lane 7.

Figure 7

Location of the Walker-A Thr in the nucleotide-binding site of ClpB. Shown is a fragment of the AMP-PNP-bound D1 from T. thermophilus ClpB. The nucleotide is shown using a stick representation. The Walker-A Lys (cyan) and Thr (red) are shown in space-fill representation. The rest of P-loop is shown in magenta.