The mechanism of Torsin ATPase activation

Abstract

Torsins are membrane-associated ATPases whose activity is dependent on two activating cofactors, lamina-associated polypeptide 1 (LAP1) and luminal domain-like LAP1 (LULL1). The mechanism by which these cofactors regulate Torsin activity has so far remained elusive. In this study, we identify a conserved domain in these activators that is predicted to adopt a fold resembling an AAA+ (ATPase associated with a variety of cellular activities) domain. Within these domains, a strictly conserved Arg residue present in both activating cofactors, but notably missing in Torsins, aligns with a key catalytic Arg found in AAA+ proteins. We demonstrate that cofactors and Torsins associate to form heterooligomeric assemblies with a defined Torsin-activator interface. In this arrangement, the highly conserved Arg residue present in either cofactor comes into close proximity with the nucleotide bound in the neighboring Torsin subunit. Because this invariant Arg is strictly required to stimulate Torsin ATPase activity but is dispensable for Torsin binding, we propose that LAP1 and LULL1 regulate Torsin ATPase activity through an active site complementation mechanism.

Keywords: ATPase; DYT1 dystonia; Torsin; nuclear envelope.

Fig. 1.

LAP1 and LULL1 luminal domains are predicted to adopt an AAA+-like fold. (A) Predicted structural model of LAP1’s luminal domain using Phyre2. (B) As in A, but for LULL1. (C) Oligomeric model of TorA (green and yellow) using a least-squares superposition of alpha-carbons (Coot) onto ClpC’s hexameric structure (Protein Data Bank ID code 3PXI) bound to adenylylimidodiphosphate (AMPPNP). A least-squares superposition of LULL1^LD (magenta) onto a TorA monomer is shown. AMPPNP is colored by element. The arrowhead indicates TorA’s C terminus. (D, Left) Proposed mixed-ring assembly of LAP1/LULL1 (blue) with TorA (green). CNDs, cytoplasmic/nuclear domains; PNS, perinuclear space; LDs, luminal domains. (D, Right) Space-filling structural model of the LAP1/LULL1-TorA heterodimer. Regions not modeled are shown as dashed lines, membrane domains are shown as blocks, and the CND is shown as an ellipse.

Fig. 2.

TorA and LAP1 or LULL1 form a 1:1 stoichiometric complex. (A) Complex of MBP-tagged TorA Δ49EQ and LAP1^LD or LULL1^LD was analyzed by size exclusion chromatography. Elution positions of size markers are indicated by arrows. Elution fractions were subjected to SDS/PAGE, followed by colloidal blue staining. EQ, TorA E171Q Walker B mutant. (B) Size exclusion chromatography-purified complexes were incubated at room temperature for 10 min in the absence or presence of 0.1% (wt/vol) glutaraldehyde (GA) and resolved by gradient SDS/PAGE (6–9%), followed by silver staining.

Fig. 3.

Site-specific cross-linking between TorA and LAP1/LULL1. (A) Predicted interface between LAP1 (blue) and TorA (green). Cross-linker pBPA (orange) was installed on TorA at three sites (D327BPA, Y328BPA, and Y329BPA) at the predicted interface with LAP1. (B) Zoomed-in view of TorA and LAP1 interface. TorA D327BPA, Y328BPA, and Y329BPA are shown in orange. (C) HIS-LAP1^LD and TorA-Δ51 constructs were copurified from E. coli in 1 mM ATP. Copurified complexes were incubated at 30 °C for 5 min, cooled to 20 °C, and UV-irradiated for 15 min. Cross-linked species were immunoprecipitated with a TorA antibody, resolved by SDS/PAGE, and analyzed via immunoblotting using HIS antibody to detect LAP1. Arrows indicate the Ig heavy chain, and arrowheads indicate the TorA-LAP1 cross-linked species. Note that the order of loading (±UV) was inadvertently reversed for Y329BPA. IP, immunoprecipitate. (D) Copurified HIS-LAP1^LD and TorA-Δ51 constructs were treated with CIP overnight at 4 °C to remove nucleotide. ATP or ADP (10 mM) was added back to the cross-linking reaction. Cross-linked species were analyzed as described in C. (E) As in C, but for HIS-LULL1^LD.

Fig. 4.

Conserved Arg residue in LAP1 and LULL1 aligns with the Arg finger of other AAA+ proteins. (A) Alignment of LAP1 and LULL1 amino acid sequences to ClpB and ClpC using predicted structural homology. LAP1 and LULL1 sequences from Homo sapiens (NP_056417 and NP_001186189), Danio rerio (NP_001017552 and XP_001339602), and Drosophila melanogaster (NP_649149) were aligned to each other using ClustalW and formatted using BoxShade; black boxes indicate strict amino acid agreement, and gray boxes indicate similar amino acid agreement. Predicted secondary structures were generated and aligned to Thermus thermophilus ClpB and Bacillus subtilis ClpC using HHPred; boxes represent helices, arrows indicate sheets, and lines indicate random coils. A conserved Arg from LAP1 (H. sapiens R563) and LULL1 (H. sapiens R449) aligns with ClpB’s and ClpC’s AAA-2 Arg finger (R747 and R704, respectively) and is indicated with a red arrow and a red star. (B) Zoomed-in view of LAP1 and LULL1’s Arg fingers in proximity to AMPPNP-bound TorA (green). LAP1 R563 (orange) and LULL1 R449 (magenta) are shown.

Fig. 5.

Mutations of LAP1 and LULL1 Arg residues do not affect Torsin binding. (A) LAP1 and LULL1 Arg mutants interact with TorA in vivo. The 293T cells were transfected with TorA E171Q and the indicated LD constructs, lysed with Nonidet P-40 (Roche), and immunoprecipitated using an HA antibody. Input controls and immunoprecipitates were resolved by SDS/PAGE and blotted with the indicated antibodies. IB, immunoblot. (B) LDs of LAP1 or LULL1 were incubated at 30 °C in the absence or presence of equal molar TorA E171Q and 2 mM ATP. Protein complex formation was analyzed by size exclusion chromatography. Elution positions of size markers are indicated by arrows. Elution fractions were subjected to immunoblotting using the indicated antibodies. (C) Identical analysis for LAP1^LD R563A or LULL1^LD R449A. (D) Identical analysis for LAP1^LD R563E or LULL1^LD R449E.

Fig. 6.

LAP1 and LULL1 Arg mutants fail to stimulate TorA’s ATPase activity. (A and B) TorA ATP hydrolysis rates in the presence of WT or Arg mutant cofactors. ATP hydrolysis rates were measured as a function of Pi-production over time. TorA (3 μM) was incubated with ATP (2 mM) at 37 °C, either alone or with 3 μM indicated WT or mutant cofactor, and Pi-production was measured at various time points using a malachite green assay. Data were fit with a linear regression in Prism (GraphPad) to yield the ATP hydrolysis rate. (C and D) Rate constants were obtained by dividing the ATP hydrolysis rate by the TorA concentration. (E and F) ATP single-turnover kinetics of TorA in the presence of WT or Arg mutant cofactor. Each data point represents the mean of three independent experiments. Error bars indicate SD. (G) Rate constants were obtained by fitting the data from E and F with a single exponential decay function in Prism. TorA basal ATP hydrolysis could not be fit to a single exponential decay function in a statistically significant manner. (H) LAP1 R563 and LULL1 R449 are required to stimulate TorB’s ATPase activity. TorB (3 μM) was incubated with ATP (2 mM), either alone or with 3 μM WT or Arg mutant cofactor. ATP hydrolysis was measured as a function of Pi-production after 60 min using a malachite green assay.