HAX1 stimulates CLPB disaggregase and ATPase activities by enhancing oligomerization.

(A) Reactivation of firefly luciferase aggregates by 0.25 μM CLPB, ± 0.25 μM HAX1 or 0.25 μM model substrate FITC-casein, n = 2. (B) ATP hydrolysis rate of 1 μM CLPB in the presence of 0-5 μM HAX1, FITC-casein, or aggregated luciferase (FFLppts), fit to a quadratic velocity equation, n = 2. 95% confidence interval of fit in parentheses. Fit to FFLppts data was not statistically meaningful. The p value for Kapp differing between HAX1 and casein <0.0001. (C) Oligomerization of CLPB, monitored using ATP hydrolysis as a proxy, fit to allosteric sigmoidal equation. HAX1, when present, is included at a 2:1 molar ratio with CLPB, n = 20 -HAX1, 10 +HAX1. p for K1/2 < 0.0001. (D) Reactivation of luciferase aggregates by 0.25 μM CLPB in the presence of 0-2 μM HAX1 (n = 6) or HAX1 alone (n = 3). For all plots, error = SEM; 95% confidence interval indicated for parameters extracted from fitted equations. Statistical significance denoted by p < 0.05 (*), p < 0.005 (**), p < 0.0005 (***).

A short helix of HAX1 is necessary for CLPB binding but is insufficient to stimulate CLPB activities.

(A) Prediction of the CLPB-HAX1 interaction site, using AlphaFold3. HAX1 is depicted in salmon and the CLPB ankyrin repeat domain is depicted in blue, with the linked subdomain in light blue. (B) Map of HAX1 and truncated variants, with predicted secondary structures indicated. (C) Relative reactivation of aggregated firefly luciferase of 0.25 μM CLPB ± 0.25 μM HAX1 (WT or variants in predicted CLPB-interaction region), n = 10 for wildtype and 6 for variants. (D) ATP hydrolysis rate of 1 μM CLPB in presence of 0-5 μM HAX1 variants, n = 2. p < 0.0001 for each variant differing from wildtype HAX1. (E) Reactivation of firefly luciferase aggregates by 0.25 μM CLPB in the presence of HAX1 variants lacking several elements of predicted secondary structure, n = 10 for wildtype and 6 for variants. (F) Oligomerization of CLPB, monitored using ATP hydrolysis as a proxy. HAX1 variants, when present, are included at a 2:1 molar ratio with CLPB, n = 22 for CLPB alone, n = 10 for CLPB with HAX1, n = 7 for CLPB with HAX15Δss. The p value for K1/2 between HAX1 and HAX15Δss = 0.0033; the p value for kcat = 0.032. For all plots, error = SEM; 95% confidence interval indicated for parameters extracted from fitted equations. Statistical significance denoted by p < 0.05 (*), p < 0.005 (**), p < 0.0005 (***).

HAX1 specifically stimulates the mature form of CLPB isoform 2.

(A) Domain maps of CLPB splice forms (B) Comparison of the ankyrin domain conformation in CLPB (PDB: 8FDS) and CLPBL (PDB: 8DEH). (C) Ratio of expression of exon 1, which is contained in both CLPB and CLPBL, and exon 5, which is unique to CLPBL, in several representative tissues. Exon expression values were obtained from GTEx Analysis Release V10. (D) Relative reactivation of aggregated firefly luciferase by 0.25 μM CLPB isoforms and cleavage variant ± 0.25 μM HAX1 WT, n = 10. (E) ATP hydrolysis rate of 1 μM CLPB isoforms and cleavage variant in presence of 0-5μM HAX1 WT, n for CLPB = 12, for CLPBL = 6. (F) Oligomerization of CLPBL, monitored using ATP hydrolysis rate as a proxy. HAX1, when present, is included at a 2:1 molar ratio with CLPB, n = 18 without HAX1 and n = 14 with HAX1. Data were fit to an allosteric sigmoidal equation. The p value for K1/2 = 0.13. For all plots, error = SEM; 95% confidence interval indicated for parameters extracted from fitted equations. Statistical significance denoted by p < 0.05 (*), p < 0.005 (**), p < 0.0005 (***).

CLPB-HAX1 complexes have enhanced refoldase activity compared to CLPB homo-oligomers.

(A) Refolding of soluble denatured firefly luciferase, monitored by luciferase activity over time, with 0.5 μM CLPB ± 0.5 μM HAX1 or HAX1 alone as indicated, n ≥ 4. P-values compare -CLPB vs +CLPB and CLPB vs CLPB+HAX1 refolding after 60 min. (B, C) The effect of 0.5 μM HAX1 variants with mutations in (B) the predicted CLPB interaction site or (C) the predicted secondary structures on 0.5 μM CLPB refoldase activity compared to HAX1 WT, n ≥ 4 (D) Refoldase activity of 0.5 μM CLPB isoforms ± 0.5 μM HAX1 WT, n ≥ 7. (E) The effect of increasing HAX1 concentration (0-4 μM) on the refoldase activity of 0.5 μM CLPB, n ≥ 5. (F) The effect of equimolar HAX1 on increasing CLPB concentration (0.125-3.0 μM) on the fraction of luciferase refolded after 15 minutes, n ≥ 6. Denatured luciferase was diluted to 100 nM in assay buffer. For all plots, error = SEM. Statistical significance denoted by p < 0.05 (*), p < 0.005 (**), p < 0.0005 (***).

HAX1 induces formation of a smaller oligomer of CLPB.

The size of CLPB, HAX1, and CLPB-HAX1 complexes were assessed using size exclusion-chromatography. Samples were prepared with an ATP-hydrolysis-blocked Walker B variant of CLPB (E425Q for isoform 2 or E455Q for isoform 1) (32 μM) and/or HAX1 (37.5 μM, in slight excess) in the presence of 2 mM ATP and separated on a Superose 6 Increase 10/300 column, equilibrated in the same buffer with 2 mM ATP. Elution fractions highlighted between vertical dashed lines were analyzed by SDS-PAGE and Sypro Red or Orange staining. At the top of each chromatogram, the molecular weight (in kDa) of three protein standards is noted at their elution volumes. (A) CLPBE425Q and HAX151-251; (B) CLPBE425Q and HAX1114-143. (C) CLPBL-E455Q and HAX151-251; (D) CLPBL-E455Q and HAX1114-143. (E) The ratios of HAX1:CLPB molecules were by quantifying the fluorescence intensity of protein bands in SDS-PAGE gels from size exclusion eluate fractions in comparison with concentration standards of these proteins. The average HAX1:CLPB across the peak of interest highlighted in orange on each chromatogram is plotted for each CLPB-HAX1 combination. (F) Molecular weights of possible CLPB homo-oligomer and CLPB-HAX1 stoichiometric oligomer complex formations. For all chromatograms n = 2.

(A) Disaggregase activity of 0.25 μM CLPB ± 0.25 μM MBP-HAX1, untagged HAX1 isolated from inclusion bodies and renatured, and MBP, n = 2. (B) ATPase rate of 1 μM CLPB in the presence of 0-5 μM MBP-HAX1, untagged HAX1, or MBP, n = 2. (C) ATP hydrolysis rate of 0.25 μM CLPB ± 0.25 μM MBP-HAX1 in the presence of 50 nM aggregated firefly luciferase. Assay becomes nonlinear below ∼A340 = 0.5 due to depletion of its components. n = 2. (D) Disaggregase activity of 0.125-2.0 μM CLPB in the presence of 0 or equimolar MBP-HAX1, n = 5. For all plots, error = SEM. Statistical significance denoted by p < 0.05 (*), p < 0.005 (**), p < 0.0005 (***).

(A) Co-isolation of CLPB with His6-tagged HAX1, n = 6. (B) Diagram of HAX1 predicted secondary structure and truncations. (C) Relative reactivation of aggregated firefly luciferase of 0.25 μM CLPB ± 0.25 μM HAX1 variants as indicated, n = 8 for wt and Δ209, n = 6 for Δ81, and n = 4 for Δ187Δ209. (D) kcat for ATP of CLPB with HAX1 variants as indicated, n = 2. (E) ATP hydrolysis activity of 1 μM CLPB in presence of 0-5 μM HAX1 variants, fit to a quadratic velocity equation, n = 2. (F) HAX1 variants were analyzed using a Superose 6 increase 10/300 column, equilibrated in buffer SD (250 mM NaCl, 5% glycerol, 25 mM Tris pH 7.8, 1 mM DTT). For all plots, error = SEM; fitted parameters represented with 95% confidence interval. Statistical significance denoted by p < 0.05 (*), p < 0.005 (**), p < 0.0005 (***).

(A) Disaggregation of FFLagg by 0.5 μM CLPB variants ± equimolar HAX1 after 90 min, n = 5 for CLPB variants and 10 for CLPB. (B) Disaggregation of FFLagg by 5 μM CLPB variants after 90 min; n = 2 for CLPB and point variants; n = 3 for CLPBL. P values denote difference from wildtype CLPB. (C) ATP hydrolysis rate of 1 μM CLPB variants over HAX1 concentration (0-5 μM), n = 2 for variants and 12 for wildtype CLPB. (D, E) Pulldown results of HAX1 (bait) with different CLPB variants, n = 3. For all plots, error = SEM. Statistical significance denoted by p < 0.05 (*), p < 0.005 (**), p < 0.0005 (***).

(A) The effect of 0.5 μM HAX1 on 0.5 μM CLPB refoldase activity compared to 0.5 μM HAX1 variants or protein controls, n ≥ 4. (B) The effect of 0.5 μM HAX1 on the refoldase activities of 0.5 μM CLPB variants, n ≥ 3. (C) Basal refoldase activity of CLPB variants at 5 μM, n = 2 for CLPBT238M and CLPBA239T, 4 for CLPBE425Q, 6 for CLPB, and 8 for CLPBL. (D) HAX1 (0-4 μM) effect on the refoldase activity of CLPB over several concentrations (0.25-2.0 μM CLPB) after 90 min; n = 2. Error = SEM for all plots. Statistical significance denoted by p < 0.05 (*), p < 0.005 (**), p < 0.0005 (***).

Size exclusion chromatography to assess CLPB, HAX1, and CLPB-HAX1 complex formation with (A) CLPBE425Q and HAX1 51-251 Δ125-130; (B) CLPBL-E455Q and HAX1 51-251 Δ125-130.

Elution fractions of CLPB+HAX1 highlighted between the vertical dashed lines were analyzed by SDS-PAGE and Sypro Orange staining and are displayed below each chromatogram. For all chromatograms, n=1.