Kinetics characterization of ASXL1/2-mediated allosteric regulation of BAP1 deubiquitinase

BAP1 is a ubiquitin hydrolase whose deubiquitinase activity is mediated by polycomb group-like protein ASXL2. Cancer-related mutations/deletions of BAP1 lead to loss-of-function either by directly targeting the catalytic (UCH) or ULD domains of BAP1, the latter disrupts binding to ASXL2, an obligate partner for BAP1 enzymatic activity. However, the biochemical and biophysical properties of the domains involved in forming the enzymatically active complex are unknown. Here we investigate the molecular dynamics, kinetics and stoichiometry of these interactions. We demonstrate that the BAP1 and ASXL2 domain/proteins or protein complexes produced in either bacteria or baculovirus are structurally and functionally active. The interaction between BAP1 and ASXL2 is direct, specific, and stable to in vitro biochemical and biophysical manipulations as detected by isothermal titration calorimetry, GST association, and optical biosensor assays. Association of the ASXL2-AB box greatly stimulates BAP1 deubiquitinase activity. A stable ternary complex can be formed comprised of the BAP1-UCH, BAP1-ULD, and ASXL2-AB domains. Binding of the BAP1-ULD domain to the ASXL2-AB box is rapid, with fast association and slow dissociation rates. Stoichiometric analysis revealed that one molecule of the ULD domain directly interacts with one molecule of the AB Box. Real-time kinetics analysis of ULD/AB protein complex to the UCH domain of BAP1, based on SPR, indicated that formation of the ULD/AB complex with the UCH domain is a single-step event with fast association and slow dissociation rates. These structural and dynamic parameters implicate the possibility for future small-molecule approaches to reactivate latent wild-type UCH activity in BAP-mutant malignancies.


171
Interactions between the ASXL-AB and BAP1-ULD domains were studied by SPR using a 172 Biacore T200 instrument. GST-antibody (Abcam ab9085) was coupled to all flow cells of a CM5 sensor 173 chip using standard amine coupling procedures in a HEPES-buffered saline running buffer. After 174 coupling of the GST antibody, the running buffer was changed to 25 mM HEPES, pH 7.4, 150 mM NaCl, 175 5 mM DTT and 0.05% Tween20. GST-ULD was immobilized onto the chip surface at a ligand density of 176 400 RU, followed by a 120-s stabilization period. A single concentration His-AB was then injected over 177 both the reference cell, with GST antibody alone, and the flow cell covered with GST-ULD at 30 uL/min. 178 The binding reaction was monitored for 240 s followed by a 300-s dissociation time. Specific binding was 179 determined by subtracting the refractive index change in the reference cell from the flow cell containing 180 GST-ULD. After each concentration of His-AB, the GST-ULD was stripped from the surface using a 60-s 181 injection of 20 mM glycine, pH 2.0 at 30 uL/min, followed by another 120-s stabilization period. Fresh 182 GST-ULD was then immobilized as above. Experiments were done in triplicate.
212 Interestingly, cancer-related genes such as Fos were negatively correlated with Bap1. Among the 20 213 tissues, the majority of Bap1-expressing cells had none of the Asxl1-3 genes expressed (60.7%), with 214 21.1% of cells repressing Asxl2, 11.3% of cells with Asxl1, 5.9% of cells expressing both Asxl1 and 215 Asxl2, and 0.9% with AsxL3 (Suppl. Fig. S1C), suggesting ASXL2 kinetic interactions are of the highest 216 priority for ASXL proteins. The domain structure of BAP1 is unique from other UCH proteins (Fig. 1A). The N-terminus of 228 BAP1 has similarity to other mammalian UCHs (UCH-L1, UCH-L3, and UCH-L5); however, BAP1 also 229 has several additional conserved motifs and domains throughout the remainder of the protein including 230 the ULD found only in UCHL5. Alignments of the UCH domain of the four proteins and the ULD of 231 BAP1 and UCH-L5 identify many amino acids conserved throughout, especially at sites with cancer 232 (COSMIC) mutations within the UCH (Fig. 1B-C). Using the structure of Drosophila Calypso UCH/ULD 233 interaction with Asx (PDB 6HGC) converted into human BAP1 UCH/ULD and ASXL2 merged with our 234 previous models of interaction with H2A and Ubiquitin (16), we can pinpoint the human contact maps of 235 the ULD with ASXL2 with high confidence (Fig. 1D, E). The BAP1 ULD contact amino acids have 11 237 unique to BAP1, suggesting a lower kinetics of interaction between ASXL2 and UCH-L5 than with 238 BAP1. The conservation of ASXL1-3 identifies a shared highly conserved ASXH domain critical for 239 ULD interaction, with an additional conserved PHD-type domain being poorly defined (Fig. 1F). Of the 240 BAP1 contact amino acids within ASXL2, 20/29 sites are conserved between Drosophila ASX and 241 ASXL1-3 (Fig. 1G). A total of 23/29 BAP1 contact sites are conserved throughout ASXL1-3, suggesting 242 that contact between ASXL1-3 with BAP1 are maintained throughout all three proteins.  (Fig. 2B) and the proteins were functional (see below). The bacterial-expressed 293 GST-BAP1-UCH and GST-BAP1-ULD were soluble using GST-chromatography under native 294 purification conditions (Fig. 2B) and the proteins were functional (see below). The bacterial-expressed 295 His-BAP1-ULD and His-ASXL2-AB proteins were purified under denaturing conditions, followed by a 296 re-naturation protocol that yielded soluble, highly active proteins (Fig. 2B). However, the yield of re-297 folded proteins was not sufficient for structural studies. We thus used the pETDuet co-expression system 298 to co-express His-ULD and AB, or His-AB and ULD protein complexes in E. coli [Rosetta 2 (DE3) 299 pLysS]. The His-ULD/AB protein complex was successfully co-expressed and then purified using cobalt 300 beads (Talon) under native purification conditions. The protein complex was highly soluble and 301 functional (Fig. 2B).  312 Biophysical and biochemical characterization of BAP1-UCH, BAP1-ULD, ASXL2-AB, and the 520 ubiquitin hydrolase activity. In this study, we further quantified the AB box protein stimulation on either 521 full-length BAP1 or UCH domain deubiquitinase activity. We observed that ASXL2-AB dose-522 dependently increased the maximal velocity of BAP1 cleavage of Ub-AMC. Moreover, the ULD/AB 523 complex also increased the maximal velocity of BAP1 cleavage of Ub-AMC in a dose-dependent manner.
524 The data fit well into a one-site dose response equation. The AB box increases the maximal velocity of 525 BAP1-mediated cleavage of Ub-AMC rather than increasing the K m for this substrate. This is consistent 526 with our molecular modeling data suggesting that the AB box does not induce a conformational change in 527 the substrate's binding pocket, but rather binds to the ULD domain and stabilizes the UCH loop of BAP1.
528 The potency of the AB box for stimulating BAP1 mediated cleavage of Ub-AMC is similar to the 529 concentration of BAP1 in the enzyme assay, which suggests a 1:1 interaction. This is consistent with the 530 ITC results reported here.

531
Interestingly, the ULD/AB complex, but not the AB box alone, was able to bind the BAP1-UCH 532 domain, as determined by SPR, suggesting that interaction with the ULD domain is essential for 533 stabilizing the UCH domain of BAP1. As the ULD is also found in UCHL5, this makes sense. In 534 addition, most of the affinity for the AB box for BAP1 is through the ULD domain, as this interaction had 535 10-20 fold higher affinity compared to the affinity of the ULD/AB complex for the UCH domain. These 536 data suggest that the AB box binds the ULD domain first, and this complex then interacts with the BAP1-537 UCH domain to stimulate enzyme activity.

538
This is the first quantitative assessment of the inter-and intra-molecular interactions of the BAP1 539 tumor suppressor and its obligate partner for enzymatic activity, ASXL2, including the mode by which 540 the ASXL2-AB box mediates BAP1 deubiquitinase activity. The tripartite (UCH/ULD/AB) domain-541 domain interactions described here explain the loss of the BAP1 deubiquitinase activity when tumor-542 associated mutations in BAP1 occur outside of the catalytic UCH domain, each failing to productively 543 recruit the AB box to the wild-type BAP1 catalytic site via the ULD, resulting in loss of BAP1 544 deubiquitinase activity.