Comparison of the E2-binding region among AREL1 and other HECT E3s

One intriguing aspect of the ubiquitination process is the interaction of the HECT domain with different E2 enzymes. The N-lobe small subdomain (~70-aa region) in the HECT domain is responsible for E2 binding, and this region varies considerably across the various HECT family members in terms of amino acid composition. However, within the NEDD4 subfamily, there is a 50–80% identity in this region, with ~10 amino acid residues identified as being involved in E2 interaction (Fig. S4A). In the AREL1 HECT domain, the equivalent region (aa 595–668) forms an N-lobe small subdomain, with only four common residues with that of the NEDD4 subfamily HECT E3s. Structural analysis of the AREL1 E2-interacting region shows that this region contains different structural elements, such as α-helices, β-sheets, and loops, in different orientations (Fig. 2, A–D) (29,–31). Notably, two “other” subfamily HECT domain structures (E6AP and HUWE1) have been determined, and their E2-binding regions have only 20–35% identity with the AREL1 E2-binding region.

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Figure 2.

Structural comparison of AREL1 E2 binding region (aa 595- 668) against the E2-binding region of different HECT E3 ligases. Superimposition of E2-binding regions between AREL1 (dark green) and WWP1 (warm pink) (A), NEDD4 (orange) (B), E6AP (yellow) (C), and HUWE1 (cyan) (D). The differences in the secondary structure elements in the E2 binding regions are highlighted by a dotted rectangular box.

The extended AREL1 HECT domain is catalytically active

Autoubiquitination is often used as a marker of E3 ligase activity, and studies have attributed to this feature to be involved in the regulation of the activity, stability, and downstream signaling functions of E3 ligases (30). We first conducted an in vitro ubiquitination assay of the extended AREL1 HECT domain with UBA1 (E1), UbcH7 (E2), ubiquitin (Ub), and ATP. SDS-PAGE and Western blot analysis, using immunoblotting with an anti-Ub antibody, showed higher-molecular weight bands than the AREL1 HECT domain alone (Fig. 3, A and B), indicative of AREL1 HECT-Ub polymers and autoubiquitination. TripleTOF 5600 MS analysis of these bands identified ~11 lysine residues all involved in autoubiquitination (Fig. S5 and Table S5).

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Figure 3.

Autoubiquitination activity of the extended AREL1 HECT domain and its E701A mutant. A, the various in vitro ubiquitination reactions were conducted in the presence (+) or absence (−) of E1 (UBA1), E2 (UbcH7), E3 (extended AREL1 HECT), Ub, and ATP, respectively. SDS-PAGE analysis shows the formation of polyubiquitin chains on the extended AREL1 HECT domain. B, Western blot analysis of A to highlight the catalytic activity of AREL1 and the activity of AREL1 without E2 enzyme. The AREL1 extended HECT domain autoubiquitination activity can be seen in the absence of E2 enzyme (lane 2). The products were immunoblotted with anti-ubiquitin antibody. C, SDS-PAGE analysis of in vitro ubiquitination reactions to compare the catalytic activities between WT extended AREL1 HECT and its E701A mutant. D, Western blot analysis of in vitro ubiquitination reactions in C to compare the catalytic activities between WT extended AREL1 HECT and E701A mutant. The products were immunoblotted with anti-ubiquitin antibody.

Notably, we have also observed weak autoubiquitination of the extended AREL1 HECT domain in the absence of UbcH7 (E2), as shown by the appearance of a higher-molecular weight band corresponding to monoubiquitinated extended AREL1 HECT (Fig. 3B, lane 2).

Our structural analysis of the extended AREL1 HECT domain showed that Glu⁷⁰¹ in the N-lobe forms hydrogen bonds with three residues of the C-lobe (Fig. 1C). To assess whether it is involved in ubiquitination, we mutated this Glu⁷⁰¹ to Ala and performed an autoubiquitination assay. Both SDS-PAGE and Western blot analysis highlighted that mutation of Glu⁷⁰¹ to Ala significantly increased the autopolyubiquitination activity as compared with the WT AREL1 HECT domain (Fig. 3, C and D). Therefore, Glu⁷⁰¹ is a critical residue in the AREL1 HECT domain and can alter autopolyubiquitination activity.

The extended AREL1 HECT domain ubiquitinates the substrate SMAC

Previous reports suggest that AREL1 can interact with its substrates in the absence of its N-terminal filamin domain (aa 52–158) (13). Therefore, we next characterized the activity of the extended AREL1 HECT domain in the presence of its substrate, SMAC, using an in vitro ubiquitination assay. Both SDS-PAGE and Western blot analysis reveal that the extended AREL1 HECT domain ubiquitinates SMAC via the conjugation of polyubiquitin chains, as noted by the presence of multiple higher-molecular weight bands corresponding to the sizes of mono- and multiubiquitinated SMAC (Fig. 4, A and B). Consistent with our observations (see above), we noted that the extended AREL1 HECT domain can ubiquitinate SMAC in the absence of E2, albeit at a lower efficiency (Fig. 4B, lane 2).

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Figure 4.

The extended AREL1 HECT ubiquitinates antagonist of inhibitor of apoptosis protein SMAC. A, the extended AREL1 HECT domain was incubated in the presence (+) or absence (−) of E1 (UBA1), E2 (UbcH7), WT SMAC, Ub, and ATP in an in vitro ubiquitination assay. SDS-PAGE analysis shows that the extended AREL1 HECT domain polyubiquitinates SMAC. The rightmost lane (lane 7) represents the control lane with all components required for the ubiquitination cascade present. B, Western blot analysis of A to further highlight AREL1-mediated SMAC ubiquitination. The extended AREL1 HECT domain is functionally active in the absence of E2 (lane 2) and ubiquitinates SMAC weakly. The products were immunoblotted with anti-SMAC antibody. C, SDS-PAGE analysis to compare the in vitro SMAC ubiquitination by WT extended AREL1 HECT domain and its E701A mutant. D, Western blot analysis of in vitro SMAC ubiquitination by WT extended AREL1 HECT domain and its E701A mutant. The products were immunoblotted with anti-SMAC antibody.

The AREL1 E701A mutant showed enhanced autoubiquitination activity (Fig. 3, C and D), and we were similarly interested in studying the effects of this mutation on SMAC ubiquitination. As such, we performed SMAC ubiquitination with the E701A mutant and compared its activity with the activity of the WT extended AREL1 HECT domain. Notably, we observed an increase in SMAC ubiquitination by the E701A mutant, which highlights that the mutation of the Glu⁷⁰¹ residue in the AREL1 HECT domain affects both its autoubiquitination and substrate ubiquitination activity (Fig. 4, C and D).

AREL1 HECT domain assembles Lys³³-, Lys⁴⁸-, and Lys⁶³-linked polyubiquitin chains

We next analyzed the specificity of the polyubiquitin chain linkages assembled by the extended AREL1 HECT domain in an in vitro autoubiquitination assay (Fig. 5A). This assay was performed with UBA1 (E1), UbcH7 (E2), extended AREL1 HECT domain (E3), and a panel of eight different Lys-to-Arg Ub mutants (K6, K11, K27, K29, K33, K48, and K63) that each retained only one lysine residue; K0 has all lysine residues mutated to arginine. Incubation of the extended AREL1 HECT domain with each of the eight different ubiquitination reactions resulted in the formation of mono and di-Ub chains with all mutants, whereas formation of polyubiquitin chains was only observed with K33, K48, and K63 ubiquitin single mutants. The Lys³³-, Lys⁴⁸-, and Lys⁶³-linked chains were significantly longer, and the bands in the Western blotting were of a higher intensity than the other single lysine Ub mutants. The Lys⁴⁸- and Lys⁶³-linked polyubiquitin chains might allow AREL1 to regulate its activity and stability in the cell; the biological significance of the Lys³³ linkage remains unknown.

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Figure 5.

Ubiquitin linkage assembly, sites of ubiquitination on SMAC, and its tetrameric crystal structure. A, in vitro autoubiquitination assay of the extended AREL1 HECT domain in the presence of WT ubiquitin and single-lysine Ub mutants. Each lane represents a Ub mutant with a single lysine residue conserved. A control reaction without ATP was conducted in the rightmost lane. The products were blotted with anti-ubiquitin antibodies. *, AREL1-Ub band. B, site-directed mutagenesis of SMAC followed by its in vitro ubiquitination assay to identify possible ubiquitination sites. The extended AREL1 HECT domain was incubated with E1 (UBA1), E2 (UbcH7), WT SMAC or its mutants, Ub, and ATP in an in vitro ubiquitination assay. WT SMAC and its various mutant constructs were used as labeled. The products were blotted with anti-SMAC antibodies. *, SMAC protein band. C, crystal structure of the tetrameric form of SMAC showing the arrangement of four SMAC monomers. D, Lys⁶² and Lys¹⁹¹ ubiquitination sites are highlighted in the SMAC monomers as red spheres. The Lys²⁰⁷ position is also labeled on the SMAC monomers as blue spheres.

Lys⁶¹ and Lys¹⁹¹ are two major sites on SMAC involved in ubiquitination

Next, we sought to identify the sites on SMAC that are subjected to ubiquitin conjugation by the extended AREL1 HECT domain. Ubiquitinated SMAC bands were analyzed by MS, which identified three lysine residues on SMAC modified with ubiquitin: Lys⁶², Lys¹⁹¹, and Lys²⁰⁷ (Table S5). We performed site-directed mutagenesis to mutate each lysine residue to arginine and generated single (K62R, K191R, and K207R) and double mutants (K62R/K191R, K62R/K207R, and K191R/K207R) (Fig. 5B). In vitro ubiquitination assays with these single and double mutants highlighted a significant reduction in the ubiquitination of K62R and K191R single mutants and almost no signal in the K62R/K191R double mutant (Fig. 5B). There was no significant change in the ubiquitination signal for the K207R mutant. Together, these studies suggest that Lys⁶² and Lys¹⁹¹ are the two major sites on SMAC targeted for ubiquitination by AREL1. The Lys²⁰⁷ residue might be involved in weak ubiquitination, which could be the reason that no changes were observed in the SMAC ubiquitination when this residue was mutated into arginine. However, in the crystal structure of SMAC (see “Crystal structure of tetrameric SMAC (aa 56–239)”), this residue is not buried in the tetrameric structure.

Crystal structure of tetrameric SMAC (aa 56–239)

To map the positions of Lys⁶² and Lys¹⁹¹ on the endogenously relevant tetrameric SMAC structure, we solved the structure of the tetrameric form of SMAC, which is not available in the literature. Previous studies have only captured the monomeric and dimeric forms of SMAC (32, 33). The molecular mass of monomeric SMAC is 20 kDa; however, it naturally exists in an active tetrameric form (~80 kDa) in solution to interact and inhibit IAPs (Fig. S6) (33). Here, we crystallized the tetrameric form of SMAC and determined its structure (Table 1). The final model was refined up to 2.8 Å resolution. There are four molecules in the asymmetric unit (Fig. 5, C and D). The tetrameric structure of SMAC shows the arrangement of four monomeric chains as a dimer of dimers (chain D and chain A:chain C and chain B). The residues Gln¹¹⁶, Glu¹²⁷, and Glu¹³¹ in chain A of one dimer form hydrogen bonds with the Glu¹⁹⁶, Gln⁹¹, and Gln¹⁹³ residues in chain C of the second dimer, respectively (Fig. S7 and Table S4). The buried surface area between the dimers of the tetramer (i.e. between chain A and chain C) is 508.7 Ų based on the PISA analysis (34).

As discussed in the previous section, AREL1 targets Lys⁶² and Lys¹⁹¹ on SMAC for its ubiquitination. Here, we show the arrangement of Lys¹⁹¹ on four different monomeric chains of the SMAC tetramer. Lys⁶² could not be mapped in the structure, as it is present at the beginning of the chains, and no electron density was obtained for the initial 16 amino acids.

Critical role of C-terminal residues of AREL1 HECT domain for ubiquitination

HECT E3s confer ubiquitin chain-type specificity, and it has been shown that the last 60 amino acids of the C-lobe are the determinants of linkage chain specificity (Fig. S4B) (35). Members of the NEDD4 subfamily have an invariable acidic residue as the last amino acid, which, if deleted or mutated, can result in WT transthiolation; however, it completely abrogates substrate ubiquitination (10). Notably, HECT E3s in the “other” subfamily do not have this acidic residue, and limited knowledge is available about the involvement of their C-terminal residues in the ubiquitination process. Previous work has shown that deletion of the last amino acid of E6AP (“other” subfamily member whose structure is known) does not affect its ubiquitination activity (36); however, substitution of the last four amino acids of NEDD4 with the last three amino acids of E6AP results in a substantial reduction in chain kinetic formation of the NEDD4 E3 ligase (10). Moreover, a conserved phenylalanine located at the fourth position from the C terminus is critical for HECT E3 substrate ubiquitination (36).

To assess the catalytic role of C-terminal residues of the extended AREL1 HECT domain in autoubiquitination and SMAC ubiquitination, we mutated the Phe residue located at the fourth position from the C terminus of the extended AREL1 HECT domain to alanine (F820A mutant) and generated another mutant without the last three amino acids (Δ3 CT mutant; aa 436–820). Notably, autoubiquitination was undetectable with both F820A and Δ3 CT mutants (Fig. 6, A and B). For SMAC ubiquitination, no ubiquitination was observed with the F820A mutant, whereas reduced SMAC ubiquitination was observed with the Δ3 CT mutant (Fig. 6, C and D). The last three amino acids of AREL1 are similar to the last three amino acids of E6AP, and deletion of these amino acids in E6AP did not affect E6AP-mediated p53 multiubiquitination (36). These differences between the AREL1 and E6AP E3 ligases suggest the possibility of differences in catalytic mechanisms.

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Figure 6.

Comprehensive analysis of the role of C-terminal residues of the extended AREL1 HECT domain in autoubiquitination and SMAC ubiquitination. A, involvement of C-terminal residues of AREL1 in the ubiquitination process. In vitro autoubiquitination assay to compare the catalytic activities of WT AREL1 extended HECT domain with its F820A and Δ3 CT mutants. SDS-PAGE analysis showed complete inhibition of autoubiquitination in both F820A and Δ3 CT mutants. B, Western blot analysis of WT extended AREL1 HECT domain and its F820A and Δ3 CT mutants to highlight the role of C-terminal residues on autoubiquitination activity. The products were blotted with anti-Ub antibodies. C, SDS-PAGE analysis to show the effects of F820A and Δ3 CT mutations on SMAC ubiquitination as compared with WT AREL1 extended HECT domain. D, Western blot analysis to further confirm the role of F820A and Δ3 CT mutations on SMAC ubiquitination. The products were blotted with anti-SMAC antibodies.

AREL1-specific Ub variant (UbV), KL.3, modulates AREL1 ubiquitination

A recent screening of selective UbV modulators for different NEDD4 HECT E3s uncovered distinct mechanisms of HECT E3 regulation (37). To assess the effects of ubiquitin variants on AREL1 activity, we expressed and purified a previously published ubiquitin variant of AREL1 (UbV KL.3) (37), selected based on its maximum affinity (EC₅₀ value) for AREL1. Based on our observation, KL.3 is a dimer in solution (Fig. S8). We incubated the extended AREL1 HECT domain with KL.3 in a 1:1 molar ratio and performed gel filtration using an S200 analytical column. We noted a shift in the profile of the extended AREL1 HECT:KL.3 sample, suggestive of a strong interaction (Fig. 7A). We next performed isothermal titration calorimetry (ITC) experiments to determine the binding affinities between the extended AREL1 HECT domain and UbV KL.3. The ITC experiments showed a dissociation coefficient of 427 nm for KL.3 binding to the extended AREL1 HECT domain (Fig. 7B). To confirm that KL.3 does not affect the functions of E1 (UBA1) and E2 (UbcH7) enzymes, we prepared a reaction mixture containing E1, E2, Ub, ATP, and KL.3 (10 and 20 μm) and kept the reaction at 37 °C for 1 h. SDS-PAGE analysis (nonreducing and reducing) of the reaction mixture showed that KL.3 does not inhibit the functions of E1 and E2 enzymes, as the formation of E2-Ub was unaffected in the presence of KL.3 (Fig. 7, C and D). We then performed an in vitro ubiquitination assay with 0.05 μm UBA1, 1 μm UbcH7, 3 μm extended AREL1 HECT, 20 μm WT Ub, and 10 μm SMAC. Two different concentrations (10 and 20 μm) of KL.3 were used in the ubiquitination assay. Interestingly, we observed that KL.3 hinders AREL1-mediated ubiquitination of SMAC, as noted by a decrease in the intensity of the ubiquitinated bands (Fig. 7, E and F). This suggests a possibility of modulating HECT E3 activity with ubiquitin variants.

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Figure 7.

Extended AREL1 HECT interaction with ubiquitin variant KL.3. A, analytical S200 profiles of the extended AREL1 HECT domain (blue), KL.3 (green), and AREL1 HECT:KL.3 mixture (orange). SDS-PAGE image analysis of the extended AREL1 HECT:KL.3 mixture to show that both proteins interact and elute together. B, isothermal titration calorimetry profile to show the interaction between the extended AREL1 HECT domain and ubiquitin variant KL.3. Nonreducing SDS-PAGE (C) and reducing SDS-PAGE (D) analysis showed that KL.3 has no effects on the activities of E1 and E2 enzymes. E, SDS-PAGE analysis showing extended AREL1- mediated SMAC in vitro ubiquitination assay in the presence of ubiquitin variant KL.3. F, Western blot analysis of E to highlight extended AREL1-mediated SMAC ubiquitination in the presence of KL.3. The products were immunoblotted with anti-SMAC antibody.

Reductive alkylation

Reductive alkylation was conducted with a commercially available reductive alkylation kit (Hampton Research) as described previously (46). Subsequently, gel filtration chromatography was performed in a buffer consisting of 10 mm Tris, pH 8.5, 150 mm NaCl, 5% glycerol, 1 mm DTT to separate the methylated protein sample.

Crystallization and structure determination of extended AREL1 HECT domain

The crystal screening of the extended AREL1 HECT domain was carried out using commercially available crystallization screens by incubating 1 μl of protein solution (10 mg/ml) with 1 μl of crystallization solution in a hanging-drop vapor-diffusion method. Crystals appeared after 3 days in 100 mm BisTris, pH 5.5, 200 mm ammonium sulfate at room temperature (23 °C) and were further optimized for obtaining diffraction quality crystals. The suitable crystals were immersed in mother liquor solution with 25% ethylene glycol as a cryoprotectant for synchrotron data collection. Complete native diffraction data were collected at 2.4 Å at the beamline MX2 Australian Synchrotron, part of ANSTO. Data were processed with the HKL-2000 program (47). The crystals belonged to space group C121 with three molecules in the asymmetric unit. CCP4 online program Balbes (48) generated an initial model with ITCH HECT (PDB code 3TUG) as the template (33% sequence identity), which was used in Phenix-Phaser for molecular replacement (49). The remaining model was built manually in COOT (50) and further refined using Phenix.Refine (49). The final model was refined at 2.4 Å resolution with an R-factor of 0.18 (R_free = 0.21) and has good stereochemical parameters evaluated with PROCHECK (51) (Table 1). PyMOL (52) was used to prepare all structure-related figures.

Crystallization and structure determination of SMAC

SMAC crystallization was carried out by incubating 1 μl of protein solution (4 mg/ml) with 1 μl of crystallization solution. The crystals were obtained with a condition consisting of 200 mm potassium phosphate monobasic, 20% PEG 3350, and complete native diffraction data were collected at 2.8 Å at the beamline 24-ID-E, APS, US. The structure was solved by molecular replacement in Phenix-Phaser using the monomeric form of SMAC protein (PDB code 1G73) as the search model. The remaining model was built manually in COOT and refined using Phenix.Refine. The current R-factor is 0.21 (R_free = 0.26) with good stereochemical parameters evaluated with PROCHECK (Table 1).

In vitro ubiquitination assays

Multiple in vitro ubiquitination assays were conducted to characterize the functional activity of the extended AREL1 HECT domain. The reaction mixtures contain 0.05 μm UBA1, 1 μm UbcH7, 3 μm WT extended AREL1 HECT or its mutants, 10 μm WT ubiquitin or its mutants, UbV KL.3 (10–20 μm), and 10 μm WT SMAC or its mutants. The ubiquitination mixtures were incubated at 37 °C for 120 min in 50 mm HEPES, pH 7.5, 100 mm NaCl, 5 mm ATP, 10 mm MgCl₂, 1 mm DTT. The ubiquitination reaction was terminated with the addition of SDS-PAGE loading buffer prior to 12.5% SDS-PAGE analysis.

Western blotting

SDS-polyacrylamide gels were transferred to polyvinylidene difluoride membranes (Sigma-Aldrich) followed by incubation with mouse monoclonal anti-SMAC antibody at 1:2000 dilutions or mouse monoclonal anti-ubiquitin antibody at 1:2000 dilutions (Santa Cruz Biotechnology, Inc.). After washing, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch) in a 1:10,000 dilution. The bands were visualized using ECL Western blotting substrate (Pierce) as instructed by the manufacturer. PageRuler Prestained protein ladder (catalogue no. 26616; lot 00355719; Thermo Scientific) was used in all of the gels to compare the protein bands.

Mass spectrometry

Following 12.5% SDS-PAGE analysis, bands corresponding to ubiquitinated species were excised and subjected to exhaustive trypsin digestion. Eksigent nanolC Ultra and ChiPLC-nanoflex (Eksigent, Dublin, CA) system was used in the Trap Elute configuration to separate peptides. The desalting of samples was performed with a Sep-Pak tC18 μElution Plate (Waters, Milford, MA) and subsequently reconstituted with 20 μl of diluent (98% water, 2% acetonitrile, 0.05% formic acid). The samples (2 μl) were loaded on a 200 μm × 0.5-mm trap column followed by elution on an analytical 75 μm × 150-mm column. Peptides were separated by a gradient formed by 2% acetonitrile, 0.1% formic acid (mobile phase A) and 98% acetonitrile, 0.1% formic acid (mobile phase B): 5–7% of mobile phase B in 0.1 min, 7–30% of mobile phase B in 10 min, 30–60% of mobile phase B in 4 min, 60–90% of mobile phase B in 5 min, 90 to 5% of mobile phase B in 0.1 min and 5 to 5% of mobile phase B in 10 min, at a flow rate of 300 nl/min. The TripleTOF 5600 system (AB SCIEX, Foster City, CA) was used to perform MS analysis in an information-dependent mode. MS spectra were acquired across the mass range of 400–1800 m/z in high-resolution mode (>30,000) using 250-ms accumulation time per spectrum. A maximum of 20 precursors per cycle were chosen for fragmentation from each MS spectrum with 100-ms minimum accumulation time for each precursor and dynamic exclusion for 15 s. Tandem mass spectra were recorded in high-sensitivity mode (resolution >15,000) with rolling collision energy on. Peptide identification and quantification were performed on the ProteinPilot^TM 5.0 software Revision 4679 (AB SCIEX) using the Paragon^TM database search algorithm (5.0.0.0, 4767) and the integrated false discovery rate analysis function. The data were searched against a database consisting of 2018_Juneuniprot_sprot (total 40,698 entries). The processing was specified as follows: digestion, trypsin; ID focus, biological modifications; special factors, ubiquitin/SUMO enrichment; search effort, thorough; detected protein threshold, 0.05 (10.0%). For false discovery rate determination, data were searched against a concatenated database with in silico on-the-fly reversal for decoy sequences automatically by the software. Peptides identified with confidence interval ≥95% were considered.

ITC

ITC experiments were performed using the MicroCal VP-ITC titration calorimeter (MicroCal, Northampton, MA). The extended AREL1 HECT domain and UbV KL.3 were purified separately in a buffer containing 20 mm Tris, pH 8.5, 100 mm NaCl, and 1 mm DTT. Both proteins were further concentrated to achieve a molar ratio of 1:10. All samples were spun and degassed before the experiment. A single-ligand-binding site approach was employed to analyze binding isotherms as implemented in Origin 7 (OriginLab).

CD spectrometry

Far-UV spectra (260–200 nm) measurements of the extended AREL1 HECT domain and its mutants were performed using a Jasco J-100 spectropolarimeter (Jasco Europe, Cremella, Italy) in 20 mm Tris, 150 mm NaCl, and 2 mm DTT (pH 8) at room temperature (23 °C) using a 0.1-cm path length and stoppered cuvettes. Ten scans were recorded and averaged, and the baseline was subtracted.

We acknowledge the beamline MX2 Australian Synchrotron, part of ANSTO, and NECAT beamline 24-ID-E at the Advanced Photon Source, Argonne National Laboratory. NECAT beamlines are funded by NIGMS, National Institutes of Health (NIH), Grant P41 GM103403. The Pilatus 6M detector on the 24-ID-E beamline is funded by NIH-ORIP HEI Grant S10 RR029205. The Advanced Photon Source is a United States Department of Energy User Facility operated by Argonne National Laboratory under Contract DE-AC02-06CH11357. We acknowledge the Protein and Proteomics center (NUS) for MS proteomics data acquisition and deposition.