Stoichiometry of Nucleotide Binding to Proteasome AAA+ ATPase Hexamer Established by Native Mass Spectrometry.

AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) six-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring. The number and type of nucleotides in an AAA+ ATPase hexamer is inherently linked to the mechanism that underlies cooperation among subunits and couples ATP hydrolysis with substrate translocation. We conducted a native mass spectrometry study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the wild-type protein. We utilized ADP and its analogues (TNP-ADP and mant-ADP), and a non-hydrolysable ATP analogue (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP- and ATP-binding states, respectively. Throughout all experiments we used a Walker A mutant (PANK217A) that is impaired in nucleotide binding as an internal standard to mitigate the effects of residual solvation on mass measurement accuracy and to serve as a reference protein to control for non-specific nucleotide binding. This approach led to the unambiguous finding that a wild-type PAN hexamer carried - from expression host - six tightly bound ADP molecules that could be exchanged for ADP and ATP analogues. While the Walker A mutant did not bind ADP analogues, it did bind AMP-PNP, albeit at multiple stoichiometries. We observed variable levels of hexamer dissociation and an appearance of multimeric species with the over-charged molecular ion distributions across repeated experiments. We posit that these phenomena originated during ESI process at the final stages of ESI droplet evolution.

its eukaryotic counterpart, archaeal PAN-20S proteasome assembly functions in a ubiquitinindependent fashion. For M jannaschii PAN the key amino acid residues within functional motifs were identified as K217 (Walker A), D270 (Walker B), T316 (Sensor I), and R328, R331 (Arginine finger) (3). Early studies on PAN demonstrated that its oligomeric form was highly stable in the absence of ATP. An interaction between PAN and its CP appeared to be transient (8) and a formation of PAN-20S assembly required the presence of ATP or its non-hydrolysable analogs (9). It was also demonstrated that while ATP hydrolysis was necessary for a degradation of globular proteins, it was not required for a translocation of the unfolded substrates through a central pore of the proteasome assembly (9). When expressed in E. coli, PAN was produced without the N-terminal methionine and was accompanied by its N-terminally truncated version, the latter resulting from the presence of an internal initiation site at M74 (8).
The early cryo-EM (9) and X-ray (10,11) analyses of archaeal AAA+ proteins, the latter employing truncated forms of PAN, demonstrated that the six AAA-ATPase subunits assemble in two stacked rings: the AAA ring formed by the AAA domains is of pseudo six fold symmetry and it is positioned proximal to the CP, whereas the distal N-terminal domains form the N ring with a pseudo threefold symmetry with coiled-coil pairs protruding from it. The underlying key to the asymmetry within the N ring is the main chain configuration of Pro91 (in M. jannaschii PAN), which alternates between the cis-and trans-type peptide bond across subunits within a hexameric ring (10). The N ring facilitates substrate unfolding whereas the AAA ring is believed to actively translocate substrates into the CP in an ATP-dependent manner.
For a subset of AAA+ ATPases that facilitate protein unfolding and degradation, e.g., PAN, it is generally held that the process of substrate translocation for further processing is enabled by a cooperation among six AAA+ subunits that cycle through the nucleotide binding-dependent conformational states. Numerous studies aimed at establishing a stoichiometry of nucleotide binding to AAA+ ATPases were performed over the years and surprisingly, they delivered for ClpX, an RP of the bacterial AAA+ protease while pointing to cooperativity in ATP binding to distinct classes of ATP-binding sites (13). Smith et al. (14) reported similar results for PAN using P 32 -labeled ATP. Likewise, incubation of PAN with P 32 -labeled ADP pointed to the presence of four P 32 -labeled ADP ligands bound to the PAN hexamer. They further suggested that out of six potential sites, two remained unoccupied while the other four formed two pairs of high-and lowaffinity ATP binding sites. The further study from the same laboratory utilized fluorescently labeled ATP analogs and confirmed their previous results (15). However, two independent studies on crystallization of E. coli HslU ATPase demonstrated the presence of 3, 4 or 6 nucleotides depending on the crystallization conditions, the type of nucleotide used and the presence or absence of its proteasomal partner CP, i.e., HslV (16,17). After completion of our studies, several high-resolution cryo-EM studies on human p97 (18) and human proteasome (12,19,20) were published, each demonstrating the presence of six nucleotides bound per hexamer. The latest cryo-EM study on archaeal (A. fulgidus) proteasome pointed to the coexisting nucleotide states with a hexamer occupied by five nucleotides and revealed "the structural basis for a sequential around-the ring-ATPase cycle" (21). To address an apparent variability of nucleotide:protein stoichiometry information reported to date, we employed native mass spectrometry (MS) to study nucleotide binding to the archaeal M. jannaschii proteasome AAA+ ATPase PAN homohexamer. Of note, native MS analysis of PAN from Methanosarcina mazei and M. jannaschii were published but the issue of proteinnucleotide occupancy was not addressed (22).
The term "native MS" was coined (23) to refer to MS assays performed under pseudophysiological conditions that were likely to preserve macromolecular interactions upon their transfer from solution to the gas phase. Most of the work in the native MS field to date has employed electrospray ionization (ESI) in its nanoflow (nanoESI) format. In comparison to other established techniques widely used for analysis of non-covalent assemblages, native MS offers unique advantages in terms of high throughput and the ability to handle mixtures, inclusive of differentiating among distinct folds of the same species. At the same time, native MS technology presents with challenges, of which an inherent heterogeneity of non-covalently bound molecular ion populations due to incomplete desolvation upon their transfer from the ESI droplet to the gas phase (24) needs to be addressed in studies on ligand binding stoichiometry. Nevertheless, as outlined in recent reviews (25)(26)(27)(28), the long-term stability of native-like structures in the gas phase enabled numerous informative native MS studies aimed at characterizing solution-phase properties, e.g., stoichiometry, topology, dynamics and binding affinities of interactions of diverse types of macromolecular assemblages, including nucleotide-binding complexes (listed in Supplemental material).
In view of successful studies referred to above, we utilized ADP and its analogues (TNP-ADP and mant-ADP), and a non-hydrolysable ATP analogue (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP-and ATP-binding states, respectively. The approach presented here can be applied to a broad range of other AAA+ ATPase machineries and non-AAA+ helicase hexamers as well, providing more accurate measurement of nucleotide by guest on September 15, 2020 https://www.mcponline.org Downloaded from AAA+ ATPase nucleotide binding 8 stoichiometry than is achievable using traditional biochemical methods. In addition to reporting on stoichiometry of PAN-nucleotide binding, we will also discuss the possibility that the variable level of hexamer dissociation and possibly, an appearance of multimer species of over-charged molecular ion distributions that were observed in our experiments originated during ESI process at the final stages of ESI droplet evolution.  mM Tris pH 8.0, 150 mM NaCl and 10 mM MgCl2. The PAN hexamer concentration in the mixture was kept above 5 µM to ensure reliable signal in nanoESI-MS. The nucleotide concentration in the mixtures ranged from 0.5 mM to 10 mM, i.e., at 100 to 2000-fold excess over the PAN hexamer concentration: typically, the nucleotide analog concentration was set to afford a ~80-fold molar excess over the number of nucleotide binding sites. The mixtures were left at 4˚C overnight (for ADP or its analogues) or for 15 min (for AMP-PNP) before MS experiments. After incubation, buffer was exchanged to 0.5 M ammonium acetate and free nucleotide was concomitantly removed from PAN WT by using the Micro Bio-Spin P-6 column (Bio-Rad), which according to the Manufacturer's instructions was expected to retain 98% of unincorporated nucleotides at 20 µL load volume. The procedures followed manufacturer instructions. Prior to nanoESI MS analysis, internal standard PAN KA mutant was buffer exchanged to 0.5 M ammonium acetate using the same type of a spin column and PAN WT and PAN KA were mixed at a 1:1 ratio. mm I.D., 2.5" length, Waters) at a potential of 3-3.5 kV. The instrument was mass calibrated using sodium iodide for m/z range 50-2,000. The mass spectrometer was operated in "high resolution" mode and spectra were acquired within 500-2,000 m/z range. MassLynx software (Version SCN781, Waters) was used for data analysis. Specifically, mass spectra were by guest on September 15, 2020 https://www.mcponline.org Downloaded from smoothed, background-subtracted and centroided for manual assignment of components.

ESI MS Analysis of PAN
Alternatively, raw data were submitted to MaxEnt-driven deconvolution of molecular ion envelopes to identify sample components and measure their molecular masses.
nanoESI MS Analysis of Native PAN Proteins: Samples were introduced into the QTOF mass spectrometer (SYNAPT G2 HDMS, Waters, Milford, MA) via homemade borosilicate glass nanoESI emitters that were prepared as described in (29). Typically, a 3 µL PAN sample (hexamer concentration 5-10 µM) was loaded into the emitter and nanospray was initiated by applying 0.6-1.0 kV potential via a platinum wire (0.127 mm diameter, Sigma-Aldrich, St. Louis, MO) that was inserted into a spray capillary and remained in contact with the solution. Typical instrumental settings were as follows: source temperature = 80-100˚C, desolvation temperature = 150˚C, sampling cone = 100-200 V, trap gas flow = 4 ml/min, backing pressure = 7 mbar, detector = 2,700-3,000 V, m/z range = 500-14,000, scan time 1 s, interscan time 0.024 s. The instrument was mass calibrated using cesium iodide for m/z range of 500-5,000. When required, data acquired in few separate MS experiments that were performed under the same conditions were combined -using MassLynx "Combine All Files" toolto increase signal-to-noise ratio.
Native MS data analysis utilized various modules of Massign software (30). Specifically, data were smoothed and linearized (settings: smooth curve 20, linearization setting one datapoint per Da), background-subtracted (step function 100, smooth curve 200), protein components were manually assigned and then simulated using a Levenberg-Marquardt algorithm; Lorentzian peak shape trailing edge settings were used. Detailed parameters generated for the measured mixture components using Massign simulations are listed in Tables S1c, S2c, S3c for analyses of mixtures of PAN KA with PAN WT before and after nucleotide exchange with mantADP, and liquid ethane that was kept slightly above -180˚C by using a Vitrobot IV (FEI Co., Hillsborough OR). The proteins in vitreous ice were inspected on a Tecnai TF20 transmission electron microscope (FEI Co., Hillsborough OR) equipped with a field emission gun operating at 200 kV.
Images were collected using a TemF816 8K x 8K CMOS camera (TVIPS GmbH, Gauting, Germany) at a nominal magnification of 62 kX (pixel size 1.204 Å). The defocus values of the micrographs were determined by using the software CTFFIND37. For each protein, ~4000 particles in the defocus range of 2.5-3.5 µm were classified into 64 class averages using the Kmean method.

Experimental Design and Statistical Rationale
All studies utilized the PAN sequence modified by M74A substitution, to prevent alternative protein translation starting from the M74 residue (7). For simplicity, we refer to this M74A variant of PAN as wild-type PAN (PAN WT ) while mutants are annotated with their secondary mutations, e.g., PAN K217A (PAN KA ) carrying KA mutation at position 217 in addition to the M74A mutation that was shared by all the constructs. . As all proteins in the mixture will be affected by residual solvation to the same extent, their respective residual solvation mass increments are expected to be the same. Hence, establishing residual solvation mass increment for one proteinwith a caveat that the residual solvation can reliably be expected to be solely responsible for the observed increase in the control protein experimental masswill allow to calculate the residual solvation related mass increases for all other proteins in the mixture. This way an increase in an experimental mass that exceeds the level established for the control protein will point to the presence of other factors responsible for the observed mass difference, e.g., the presence of ligands or errors in amino acid sequence used to calculate protein MW theor. Therefore, we included Walker A mutant PAN KA as an internal standard in all analyses of PAN WT samples. As PAN KA was expected to be devoid of bound nucleotides, all mass increase observed for this protein was due to residual solvation. Hence, after accounting for mass difference between the two proteins due to the K217A mutation, we were able to separate the measured [(MW exp -MW theor)] difference for PAN WT into a portion related to the residual solvation and a portion related to the presence of bound ligandssee details in p. 6 below. At the same time, PAN KA served as a control for non-specific ligand-protein interactions.
The following experimental scenario was utilized: 1. Express PAN WT and PAN mutants in E. coli and purify the proteins by a combination of anion exchange and size exclusion chromatography. multiple sets of nanoESI MS data were acquired and datasets with the highest resolution and S/N ratio were selected for analysis and are presented here.  Table ST3a.

Integrity of PAN Protein Preparations
The cryo-EM micrographs of frozen hydrated PAN proteins in TBS buffer showed a monodispersed population, whichas indicated by class averageswas composed of fully assembled hexamers, consistent with their (pseudo) 6-fold symmetry (10) Fig. 1B).
The UV spectra of PAN proteins carrying wild-type P-loop motif (PAN WT and arginine finger mutant PAN R328A ) showed higher absorbance at 260 nm than at 280 nm, pointing to the presence of nucleotides in those two preparations. In contrast, the UV profile of the PAN Walker species, nanoESI spectra contained varying levels of "satellite" narrow charge state distributions within m/z ranges of 3000-4200, 4200-5500, and 5500-7000, annotated with <M>, <D> and <T> ( Fig. 2A), whose molecular masses were consistent with the products of PAN dissociation, i.e., the monomeric (M), dimeric (D), and tetrameric (T) species, respectively. The simulated spectra of these species are shown in Fig. 2B and marked in yellow, blue, and green for the <M>, <D>, and <T> m/z ranges, respectively. Relative levels of hexamer (H) vs satellite native-like tended to vary, sometimes significantly, between experiments and in some cases, within the course of a by guest on September 15, 2020 single data acquisition. Fig. 3 and Fig. S2AB show some cases of significant changes in relative intensities of intact hexamer and products of its dissociation occurring during a single acquisition, generally accompanied by an abrupt alteration in TIC. We found no correlation between the extent of PAN dissociation and global instrumental parameters, e.g., cone and capillary voltages.
Charge states at the apices of molecular ion envelopes of the H as well as M, D, and T species correlated with their respective molecular masses in a manner characteristic for ESI spectra of folded globular proteins (31) (annotated with black dots in Fig. 2A insets) thus suggesting that the M, D and T species represented the native-like products of the solution-rather than gas phase-driven dissociation of PAN hexamer (see Discussion). Importantly, partially dissociated PAN species did maintain ligand binding. To this end, ligand loss that could be triggered by an increase in buffer concentration, was clearly detectable under the MS conditions used in the study. When using 1 M ammonium acetate buffer, the ADP concentration-dependent dissociation of PAN WT -bound ADP was well resolved at a monomer and dimer levels (Fig. 4AB) and resulting heterogeneity was clearly discerned at a tetramer level (data not shown). Under the same MS conditions, no nucleotide-protein dissociation was ever observed when using lower concentration of the same buffer (0.5 M), which ultimately was employed for all further studies. As PAN KA species carried a mutated Walker A binding site making it defective in ADP binding, no parallel appearance of a PAN KA -related peak at lower mass was observed in 1 M ammonium acetate buffer thus substantiating the reliability of using PAN KA as a ADP-free control in these studies.
The observed dissociation pattern of PAN hexamer (dimers and tetramers but not trimers or pentamers) is consistent with the known PAN architecture: PAN hexamer is built as a trimer of by guest on September 15, 2020 https://www.mcponline.org Downloaded from identical dimers (AB, CD and EF), each dimer being assembled from two monomers of the dissimilar Pro91 configuration (cis for monomers A, C, E vs. trans for monomers B, D, F) (10).
We note that species carrying higher number of charges than expected for the folded globular protein mass (annotated with "x" in Fig. 2A insets) were also detected at variable levels throughout the study. Specifically, over-charged hexamers spanning <D> and <T> m/z regions and over-charged dimers appearing within the <M> region, (annotated with "*" and "**" in Fig.   2B, respectively) were seen. As the over-charged species were not used to establish nucleotide-binding stoichiometry, their further description is provided in the Supplemental material.
In summary, PAN spectra show the presence of native hexamers (H), whichin most experimentsconstituted the major species accompanied by various levels of partially dissociated native-like as well as over-charged multimers. At the same time, our data point to the stability of protein-nucleotide interactions within all the observed species under MS conditions employed in the study. Therefore, we posit that notwithstanding the presence of the partially dissociated species in the spectra, not only did our analyses provide valid information on the stoichiometry of PAN-nucleotide binding but it benefited from the ability to analyze representative dimer species at the m/z region that afforded higher mass resolution than would otherwise be feasible to achieve for the higher m/z native hexamer region. To this end, whenever warranted by their good quality, we used the dimer-in addition to the hexamerderived data to calculate PAN WT nucleotide occupancy.

PAN WT hexamer binds six ADP ligands
The PAN WT  here. An example raw MS spectrum shown in Fig. 5 demonstrates that data generated for a PAN protein mixture has the same general characteristics as previously observed in MS analyses of individual protein components, i.e., an appearance of partially dissociated species (ranges <M>, <D> and <T>) in addition to folded hexamers (range <H>). Insets show the Massign software-processed spectra (grey lines) and mock spectra derived by summing up contributions of the simulated deconvoluted species for native-like monomers in m/z region <M>, native-like dimers and over-charged hexamers in m/z region <D>, and native hexamers in m/z region <H> (shown in yellow, blue and red, respectively).
We have analyzed the hexamer and dimer data by considering all potential forms of PAN WT hexamer fully and partially loaded with either nucleotide or their mixtures (result summary and detailed calculations are shown in tables ST1a and ST1d, respectively). All calculations were based on a premise that control protein PAN KA carried no nucleotides, as supported by the experiments performed in 1 M ammonium acetate buffer (Fig. 4), and that the residual solvation level measured for PAN KA could serve as a reliable approximation of the residual solvation of the WT protein after accounting for the presence of the KA mutation. Hence, we interpreted the experimental mass difference between the WT and KA proteins as representing bound nucleotides, as described in Experimental Design. Formulas in sections 6 and 7 therein were utilized to calculate the number of ADP and/or ATP for PAN WT carrying a single type or two types of nucleotides, respectively.
We considered the consistency between the residual solvation mass increments of KA and WT proteins as a measure of the reliability of the PAN WT composition assignment. To this end, the ratios between the KA and WT protein residual solvation mass incrementsnormalized to the same protein molecular masswere expected to be close to 1 Table 1 stood out, as they met the above criteria the best, with CV of PAN KA /PAN WT residual solvation mass increment ratios across all five experiments below 10% and 20% for hexamer and dimer results, respectively. ADP or with ATPcan conclusively be discerned (Fig. 6B). Taken together, these results strongly suggest that the presence of the PAN WT hexamer carrying only 5 nucleotides is highly unlikely and hence we conclude that the PAN WT is fully loaded with ADP.
The results of the experiment shown in Fig. 5  The results of analysis of PAN WT that was partially exchanged to TNP-ADP, described below  (Tables ST2ab and ST3ab). An incomplete ligand exchange was apparent in view of the significantly higher residual solvation observed for the KA mutant vs that calculated for the WT protein carrying full complement of ADP analogues of higher mass than ADP (Table   ST2ab and Fig. 7B). Importantly, the results of TNP-ADP exchange experiment (Fig. 7) support our conclusion that PAN WT carry 6 nucleotides since neither monomer and dimer apo forms nor a dimer carrying a single nucleotide that would be present for not-fully occupied PAN WT were detected.
In conclusion, the above experiments demonstrated that wild type PAN protein carried 6 ligands and that it was ADP rather than ATP that was bound to a nascent protein. It is noteworthy that across all experiments described above there was no indication of PAN KA protein carrying any ligands, neither from the expression host nor captured from a solution carrying excess of free ADP or its analogues (data not shown). Likewise, no species consistent with PAN WT protein carrying more than one ADP (or its analogue) per monomer were seen. Notably, no PAN WT -PAN K217A hybrid multimers were ever observed across all experiments performed in the study.

Both PAN WT and Walker A mutant bind AMP-PNP
We have utilized AMP-PNP, a non-hydrolysable ATP analogue, to establish stoichiometry of  representing PAN WT protein appears as a single species, its heterogeneity due to the presence of low level of unresolved contaminants, including potential PAN KA protein carrying 6 AMP-PNP, i.e., KA (0|6) cannot be ruled out. Of note, relative abundance distribution of PAN KA -associated peaks (P1-P4) suggests that small amount of KA (0|6) might be present (Fig. 9B)   carrying a total of 5 ATP analogs, i.e. WT (0|5), as suggested by the recent cryo-EM study (21) was way outside two standard deviations from the experimental average in both experiments.
Examination of the spectra at the monomer and dimer levels confirmed that PAN KA carried AMP-PNP, as the PAN KA dimer with 1 AMP-PNP (peak 2) was clearly discerned in addition to apo PAN KA monomer and dimer (peak 1) (see To confirm a phenomenon of ATP-analogue binding to PAN protein carrying Walker A mutation, we generated a double mutant PAN K217A/R328A . The double mutant interacted with nucleotides in a similar manner to that observed for PAN KA , i.e., it did not bind ADP and it did bind AMP-PNP ( Fig. S4 bottom and top spectrum, respectively). However, the degree of AMP-PNP ligand binding to the double mutant was much lower, with a maximum of three ligands per hexamer, vs five to six ligands seen in Fig. 9 for PAN K217A . We note that while distinct components of a double mutant-ATP analogue ladder were not resolved, the presence of multiple species was apparent upon comparing peak widths of the mutant vs wild type protein, as shown for two most by guest on September 15, 2020 https://www.mcponline.org Downloaded from abundant charge states (Fig. S4 inset a). A ratio between peak width (full width at half maximum, FWHM) calculated for the six most abundant charge states (33+ -38+) of a double mutant protein to its wild type counterpart was 1.0±0.14 and 1.5±0.15 in the absence and presence of AMP-PNP (T-test=1.49E-04), respectively, (Fig. S4 inset b).

Discussion
We divide the result discussion to two sections: the first one focused on the structural aspects of PAN and the second one addressing the issues regarding the methodological aspects of the study.

ATPase Structure
The characteristics of nucleotide binding to PAN revealed by the mass spectra provide information that is critical to unveiling the proteasome ATPase hexamer cooperation mechanisms. Proteasome is a large protein assembly that removes misfolded proteins and short-lived signaling proteins (2,4). The eukaryotic 26S proteasome consists of a 20S proteasome core particle (CP) and a 19S regulatory particle (RP). Six distinct Rpt subunits form the base of RP that interfaces with CP. PAN shares high homology with Rpt subunits and therefore it serves as a simpler model for the Rpt hexamer. Determining the precise number and type of nucleotides bound to proteasomal ATPases at a given state is important to understanding the details of the cooperation mechanism.
An earlier biochemical study of PAN and 26S proteasome suggested a pairwise model, in which a total of 4 nucleotides were bound to a hexameric proteasomal ATPase at any giving time, with two opposite subunits carrying ATP, another two opposite subunits carrying ADP and two remaining subunits in an apo state (14). Our results revealed that the purified wild-type PAN by guest on September 15, 2020 (PAN WT ) hexamer carried six tightly bound ADP molecules and that nascent ADP ligands could be replaced with ADP and ATP analogues. Of note, never did we observe an apo-form of PAN WT . Specifically, our study points to the full occupancy of all six nucleotide-binding sites within the PAN WT hexamer in both, the ADP-and ATP-binding states. We point out however, that we analyzed PAN in the absence of its functional partner, i.e., proteolytic core particle 20S and hence our results might not represent the PAN nucleotide loading in its active state in vivo.
Binding of ADP and its analogs is not likely to be affected by the exclusion of CP because the PAN-20S proteasome does not form in the absence of ATP (9). However, it is conceivable that PAN-20S interaction might alter ATP binding stoichiometry. Our results point to binding of a total of 6 nucleotides, either 6 AMP-PNP or 5 AMP-PNP and 1 ADP (or potentially 4 AMP-PNP and 2 ADP). According to our data, the presence of 5 AMP-PNP with one nucleotide-free site is unlikely (Fig. 6C). Taken together, our results support a sequential model of the AAA+ ATPase hexamer, where nucleotide can bind to all subunits simultaneously and the cooperation seems closely related to the synergy between neighboring subunits in binding ATP.
We also show that while the Walker A mutant PAN K217A hexamer carried no nascent ligands, it could be loaded with multiple copies of AMP-PNP. This unexpected AMP-PNP binding to Walker A mutant implies that the arginine finger might play a key role in ATP binding, which differs from the notion that arginine finger is not involved in ATP binding in a recent study of PAN (15). Indeed, introduction of an additional R328A mutation on one of the arginine finger residues reduced such binding, as shown by native MS of the protein mixture of PAN WT and PAN K217A/R328A treated with AMP-PNP (Fig. S4). jannaschii PAN that we have studied. However, three independent cryo-EM studies of human 26S proteasome at high resolution (12,19,20)  Our results are consistent with the hexameric structure of PAN, as demonstrated by cryo-EM data (9,21). Early reports suggested a dodecameric structure of PAN, in view of its elution volume in gel filtration chromatography that was consistent with a mass of a dodecamer (>600 kDa) rather than that of a hexamer (~300 kDa) (7). A notion of the possible weak association of PAN hexamers to form dodecameric structures was supported by an observation of PAN dodecamers in some of the cryo-EM studies on two species of archaeal PAN reported by Medalia et al (22). However, only hexameric structures were seen for the same proteins in the native MS analysis performed by the same authors. We note that on occasion we detected very weak signals that were consistent with PAN dodecamer structure (data not shown). However, we attribute their formation to an artefact of nonspecific protein-protein interactions in ESI droplets rather than to a representation of a native dodecamer arrangement. lived ESI droplets thus encouraging non-specific interactions at final stages of droplet evolution when final protein concentration gets very high (33).
The data pointing to the PAN (14,15) and ClpX (13) binding a maximum of 4 nucleotides are baffling in view of the results derived from cryo-EM and our native MS studies. The somewhat trivial explanation might be that biochemical methods that were used relied on two separate calibration routines: one for a protein and another one for a nucleotide, each relying on their own set of standards, thus compounding the likelihood of errors. It is well established that the typically used dye-based protein determination assays are highly dependent on sample composition, protein sequence and the relative abundance of specific amino acid residues within the sequence (34). To explore this possibility, we measured protein concentration in two PAN preparations by the three most often used protein assays (Fig. S5). Using the same vial of BSA solution as a standard, the concentration of the same PAN WT sample was determined to be 4.7 mg/ml by BCA assay, 8. If the PAN dissociation were to happen in the gas phase, it would most likely have proceeded in the ESI source and would have been driven by the collisionally induced dissociation (CID). Yet, the types of the observed dissociation products are inconsistent with the typical mechanism of the protein complex disassembly under CID conditions, which involves an asymmetric separation of charge and mass (37,38). Thus, the products of typical CID would have been an unfolded highly charged monomer and a charge-stripped pentamer rather than the observed folded tetramers, dimers, and monomers. While atypical symmetric CID driven fragmentation was observed, we note that a generation of monomers and pentamers under CID conditions was demonstrated in the previous native MS study on PAN (22). In addition, the fact that we observed no correlation between the extent of dissociation and cone voltage (data not shown), highly supports a notion that in-source CID was not responsible for the PAN complex disassembly.
As argued above, the characteristics charge state distributions of the partially dissociated "satellite" species points to their globular structures and solution origin ( Fig. 2A insets). droplet acidification contributes to the PAN hexamer dissociation or not, the significant variability in the extent of this process that we have observed across multiple experiments suggests that additional factors might also be at play, specifically the forces that affect evolution of a droplet during ESI (41). As compared to the bulk solution, an environment of a nanodroplet "in the throes of what is now known as a Rayleigh Instability, and sometimes referred to as a Coulomb explosion" (42) has distinct properties, e.g., high concentration of ions relative to that of the bulk solution; strong electric field due to the presence of charged macromolecules or complexes; a large surface to volume ratio (41). These unique nanodroplet characteristics impart significant changes onto chemical processes that occur in these small volume "reactors" (43) as opposed to the bulk solution. For example, chemical reactions, including unimolecular processes (44) occur much faster in nanodroplets; cases of acceleration up to the level of 10E6 were reported (45,46). By the same token, the rate of protein complex dissociation might also be greatly enhanced in the course of ESI-driven droplet evolution from its initial bulk solvent-like stage upon release from a capillary emitter to its final offspring phase that gives rise to the gas phase molecular ions. Consequently, it is likely that even small changes in the droplet evolution process that might affect the rate of the offspring droplet generation, their size, final pH or morphology might be accompanied by the large differences in the reaction rates within these droplets, thus in turn leading to distinct differences in substrate-to-product ratios. The absence of hybrid multimers that potentially could have been generated via a re-assembly of the released subunits suggests that the dissociation might occur at the late stages of the droplet transformation process when a nanodroplet carries a single or a few analytes. that in addition to the cone and capillary voltages and capillary-cone distance, the way in which the capillary was cut was also a crucial parameter in controlling the extent of complex dissociation, thus suggesting that minute changes to the capillary orifice, which potentially might occur during the spray might greatly affect ESI performance. Likewise, distinct differences in ESI spectra of non-covalent assemblages were seen in response to changes in a spraying mode (cone-jet vs pulsating mode), likely associated with the enhanced droplet acidification produced in the former mode (48) course of the spray) likely represent the major trigger of the observed variability in the extent of PAN dissociation in the native nanoESI mass spectra.

Conclusions
Our results demonstrate that native mass spectrometry can be utilized to determine the stoichiometry of nucleotide binding to an AAA+ ATPase hexamer. Its high precision and capability to maintain macromolecules in a native-like state allowed us to simultaneously measure the masses of protein and bound nucleotides whose molecular weights differ by ~ three orders of magnitude. Utilization of a Walker A mutant as an internal standard minimized the impact of residual solvation on mass measurement accuracy, as both PAN KA and PAN WT were subjected to identical ESI conditions thus allowing us to decouple the effects of a ballast of extraneous adducts, expected to be roughly the same for both proteins, from those of the PAN WT -specific binders. We propose that a generation of partially dissociated species observed in this study was driven by the chemistry of ESI droplets at the final stages of their evolution, rather than by the hexamer-dimer-tetramer equilibria in the bulk solution. While the triggers of the dissociation are presently unknown, we note that this process did not adversely affect the integrity of the results and it actually aided our investigation by supporting information gathered on a hexamer level with the data generated for monomers, dimers and tetramers.   Table 1, i.e., compositions with a total of 5 nucleotides, the presence of an apo form and a dimer carrying either single ADP (1) or ATP (2) is expected to be discernable in the spectrum none were detected.