Bortezomib Inhibits Open Configurations of the 20S Proteasome.

Bortezomib, a small dipeptide-like molecule, is a proteasome inhibitor used widely in the treatment of myeloma and lymphoma. This molecule reacts with threonine side chains near the center of the 20S proteasome and disrupts proteostasis by blocking enzymatic sites that are responsible for protein degradation. In this work, we use novel mass-spectrometry-based techniques to examine the influence of bortezomib on the structures and stabilities of the 20S core particle. These studies indicate that bortezomib binding dramatically favors compact 20S structures (in which the axial gate is closed) over larger structures (in which the axial gate is open)─suppressing gate opening by factors of at least ∼400 to 1300 over the temperature range that is studied. Thus, bortezomib may also restrict degradation in the 20S proteasome by preventing substrates from entering the catalytic pore. That bortezomib influences structures at the entrance region of the pore at such a long distance (∼65 to 75 Å) from its binding sites raises a number of interesting biophysical issues.

Increasing Isolation Efficiency Using a Segmented Quadrupole Mass Filter Operated with Rectangular Waveforms.

The performance of a segmented quadrupole mass filter operated with rectangular waveforms and capacitively coupled rectangular waveforms applied to the prefilters was examined on a home-built quadrupole-Orbitrap platform. For peak widths of 50 m/z, 100% isolation efficiency was achieved, which fell to approximately 20% for 5 m/z peak width for a rectangular waveform of 150 V0-p. Due to a small exit aperture following the mass filter, peak structure was observed in both experimental peak shapes and those simulated using SIMION. A larger radius quadrupole was examined and achieved similar performance. While the segmented quadrupole does remove the defocusing effects of the fringing fields, the ion beam is only slightly refocused due to the low RF voltage which limits achievable gains in isolation efficiency.

Nonlinear Frequency Modulation for Fourier Transform Ion Mobility Mass Spectrometry Improves Experimental Efficiency.

Through optimization of terminal frequencies and effective sampling rates, we have developed nonlinear sawtooth-shaped frequency sweeps for efficient Fourier transform ion mobility mass spectrometry (FT-IM-MS) experiments. This is in contrast to conventional FT-IM-MS experiments where ion gates are modulated according to a linear frequency sweep. Linear frequency sweeps are effective but can be hindered by the amount of useful signal obtained using a single sweep over a large frequency range imposed by ion gating inefficiencies, particularly small ion packets, and gate depletion. These negative factors are direct consequences of the inherently low gate pulse widths of high-frequency ion gating events, placing an upper bound on FT-IM-MS performance. Here, we report alternative ion modulation strategies. Sawtooth frequency sweeps may be constructed for the purpose of either extending high-SNR transients or conducting efficient signal-averaging experiments for low-SNR transients. The data obtained using this approach show high-SNR signals for a set of low-mass tetraalkylammonium salts (<1000 m/z) where resolving powers in excess of 500 are achieved. Data for low-SNR obtained for multimeric protein complexes streptavidin (53 kDa) and GroEL (800 kDa) also reveal large increases in the signal-to-noise ratio for reconstructed arrival time distributions.

BRD4354 Is a Potent Covalent Inhibitor against the SARS-CoV-2 Main Protease.

Numerous organic molecules are known to inhibit the main protease (MPro) of SARS-CoV-2, the pathogen of Coronavirus Disease 2019 (COVID-19). Guided by previous research on zinc-ligand inhibitors of MPro and zinc-dependent histone deacetylases (HDACs), we identified BRD4354 as a potent inhibitor of MPro. The in vitro protease activity assays show that BRD4354 displays time-dependent inhibition against MPro with an IC50 (concentration that inhibits activity by 50%) of 0.72 ± 0.04 µM after 60 min of incubation. Inactivation follows a two-step process with an initial rapid binding step with a KI of 1.9 ± 0.5 µM followed by a second slow inactivation step, kinact,max of 0.040 ± 0.002 min-1. Native mass spectrometry studies indicate that a covalent intermediate is formed where the ortho-quinone methide fragment of BRD4354 forms a covalent bond with the catalytic cysteine C145 of MPro. Based on these data, a Michael-addition reaction mechanism between MPro C145 and BRD4354 was proposed. These results suggest that both preclinical testing of BRD4354 and structure-activity relationship studies based on BRD4354 are warranted to develop more effective anti-COVID therapeutics.

Expanding Native Mass Spectrometry to the Masses.

At the 33rd ASMS Sanibel Meeting, on Membrane Proteins and Their Complexes, a morning roundtable discussion was held discussing the current challenges facing the field of native mass spectrometry and approaches to expanding the field to nonexperts. This Commentary summarizes the discussion and current initiatives to address these challenges.

Double and triple thermodynamic mutant cycles reveal the basis for specific MsbA-lipid interactions.

Structural and functional studies of the ATP-binding cassette transporter MsbA have revealed two distinct lipopolysaccharide (LPS) binding sites: one located in the central cavity and the other at a membrane-facing, exterior site. Although these binding sites are known to be important for MsbA function, the thermodynamic basis for these specific MsbA-LPS interactions is not well understood. Here, we use native mass spectrometry to determine the thermodynamics of MsbA interacting with the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL). The binding of KDL is solely driven by entropy, despite the transporter adopting an inward-facing conformation or trapped in an outward-facing conformation with adenosine 5'-diphosphate and vanadate. An extension of the mutant cycle approach is employed to probe basic residues that interact with KDL. We find the molecular recognition of KDL is driven by a positive coupling entropy (as large as -100 kJ/mol at 298 K) that outweighs unfavorable coupling enthalpy. These findings indicate that alterations in solvent reorganization and conformational entropy can contribute significantly to the free energy of protein-lipid association. The results presented herein showcase the advantage of native MS to obtain thermodynamic insight into protein-lipid interactions that would otherwise be intractable using traditional approaches, and this enabling technology will be instrumental in the life sciences and drug discovery.

Variation of CI-2 Conformers upon Addition of Methanol to Water: An IMS-MS-Based Thermodynamic Analysis.

Chymotrypsin inhibitor 2 (CI-2) is a well-studied, textbook example of a cooperative, two-state, native ↔ denatured folding transition. A recent hybrid ion mobility spectrometry (IMS)/mass spectrometry (MS) thermal denaturation study of CI-2 (the well-studied truncated 64-residue model) in water reported evidence that this two-state transition involves numerous (∼41) unique native and non-native (denatured) solution conformations. The characterization of so many, often low-abundance, states is possible because of the very high dynamic range of IMS-MS measurements of ionic species that are produced upon electrospraying CI-2 solutions from a variable temperature electrospray ionization source. A thermodynamic analysis of these states revealed large changes in enthalpy (ΔH) and entropy (ΔS) at different temperatures, and it was suggested that such variation might arise because of temperature-dependent conformational changes of the protein in response to changes in the conformational entropy and the dielectric permeability of water, which drops from a value of ε ∼ 79 at 24 °C to ∼ 60 at 82 °C. Herein, we examine how adding methanol to water influences the distributions of CI-2 conformers and their ensuing stabilities. The dielectric constant of a 60:40 water:methanol (MeOH) drops from ε ∼ 60 at 24 °C to ∼ 51 at 64 °C. Although the same set of conformers observed in water appears to be present in 60:40 water:MeOH, the abundance of each is substantially altered by the presence of methanol. Relative free energy values (ΔG) and thermodynamic values [ΔH and ΔS and heat capacities (ΔCp)] are derived from a Gibbs-Helmholtz analysis. A comparison of these data from water and water:MeOH systems allows rare insight into how variations in solvation and temperature affect many-state protein equilibria. While these studies confirm that variations in solvent dielectric constant with temperature affect the distributions of conformers that are observed, our findings suggest that other solvent differences may also affect abundances.

Polyamine detergents tailored for native mass spectrometry studies of membrane proteins.

Native mass spectrometry (MS) is a powerful technique for interrogating membrane protein complexes and their interactions with other molecules. A key aspect of the technique is the ability to preserve native-like structures and noncovalent interactions, which can be challenging depending on the choice of detergent. Different strategies have been employed to reduce charge on protein complexes to minimize activation and preserve non-covalent interactions. Here, we report the synthesis of a class of polyamine detergents tailored for native MS studies of membrane proteins. These detergents, a series of spermine covalently attached to various alkyl tails, are exceptional charge-reducing molecules, exhibiting a ten-fold enhanced potency over spermine. Addition of polyamine detergents to proteins solubilized in maltoside detergents results in improved, charge-reduced native mass spectra and reduced dissociation of subunits. Polyamine detergents open new opportunities to investigate membrane proteins in different detergent environments that have thwarted previous native MS studies.

Double and triple thermodynamic mutant cycles reveal the basis for specific MsbA-lipid interactions.

Structural and functional studies of the ATP-binding cassette transporter MsbA have revealed two distinct lipopolysaccharide (LPS) binding sites: one located in the central cavity and the other at a membrane-facing, exterior site. Although these binding sites are known to be important for MsbA function, the thermodynamic basis for these specific MsbA-LPS interactions is not well understood. Here, we use native mass spectrometry to determine the thermodynamics of MsbA interacting with the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL). The binding of KDL is solely driven by entropy, despite the transporter adopting an inward-facing conformation or trapped in an outward-facing conformation with adenosine 5'-diphosphate and vanadate. An extension of the mutant cycle approach is employed to probe basic residues that interact with KDL. We find the molecular recognition of KDL is driven by a positive coupling entropy (as large as -100 kJ/mol at 298K) that outweighs unfavorable coupling enthalpy. These findings indicate that alterations in solvent reorganization and conformational entropy can contribute significantly to the free energy of protein-lipid association. The results presented herein showcase the advantage of native MS to obtain thermodynamic insight into protein-lipid interactions that would otherwise be intractable using traditional approaches, and this enabling technology will be instrumental in the life sciences and drug discovery.

Digital Quadrupole Isolation and Electron Capture Dissociation on an Extended Mass Range Q-TOF Provides Sequence and Structure Information on Proteins and Protein Complexes.

Electron capture dissociation (ECD) is now a well-established method for sequencing peptides and performing top-down analysis on proteins of less than 30 kDa, and there is growing interest in using this approach for studies of larger proteins and protein complexes. Although much progress on ECD has been made over the past few decades, establishing methods for obtaining informative spectra still poses a significant challenge. Here we describe how digital quadrupole (DigiQ) ion isolation can be used for the mass selection of single charge states of proteins and protein complexes prior to undergoing ECD and/or charge reduction. First, we demonstrate that the DigiQ can isolate single charge states of monomeric proteins such as ubiquitin (8.6 kDa) and charge states of large protein complexes such as pyruvate kinase (234 kDa) using a hybrid quadrupole-TOF-MS (Agilent extended m/z range 6545XT). Next, we demonstrate that fragment ions resulting from ECD can be utilized to provide information about the sequence and structure of the cytochrome c/heme complex and the ubiquitin monomer. Lastly, an especially interesting result for DigiQ isolation and electron capture (EC) was noted; namely, the 16+ charge state of the streptavidin/biotin complex reveals different electron capture patterns for the biotinylated proteoforms of streptavidin. This result is consistent with previous reports that apo streptavidin exists in multiple conformations and that biotin binding shifts the conformational dynamics of the complex (Quintyn, R. Chem. Biol. 2015, 22 (55), 583-592).

Stability of 20S Proteasome Configurations: Preopening the Axial Gate.

Mass spectrometry studies of the stability of the S. cerevisiae 20S proteasome from 11 to 55 °C reveal a series of related configurations and coupled transitions that appear to be associated with opening of the proteolytic core. We find no evidence for dissociation, and all transitions are reversible. A thermodynamic analysis indicates that configurations fall into three general types of structures: enthalpically stabilized, tightly closed (observed as the +54 to +58 charge states) configurations; high-entropy (+60 to +66) states that are proposed as precursors to pore opening; and larger (+70 to +79) partially and fully open pore structures. In the absence of the 19S regulatory unit, the mechanism for opening the 20S pore appears to involve a charge-priming process that loosens the closed-pore configuration. Only a small fraction (≤2%) of these 20S precursor configurations appear to open and thus expose the catalytic cavity.

Modified Ion Source for the Improved Collisional Activation of Protein Complexes.

The analysis of large molecules is challenging, as they often have salts and adducts retained through the electrospray process, which increase the observed mass and compromise the achievable mass resolution. Mild collisional activation has been shown to be very effective for the removal of adducts and increases both measurement accuracy and mass resolution of large (>100 kDa) protein complexes. Collisionally activated protein ions are more completely desolvated due to the increased number of collisions when trapped following activation. A short square quadrupole maintained at 300 mTorr by a mechanical pump was added between the ion funnel and transmission quadrupole. This configuration and operation effectively removed adducts from the 800 kDa tetradecamer GroEL as well as fragmented smaller protein complexes like C-reactive protein. Due to the gas high pressure, ions of low size-to-charge ratio, such as those in charge reducing buffers, had low ejection efficiency. We show that segmenting the quadrupole rods greatly improves signal intensity for charge reduced GroEL D398A mutant compared to nonsegmented rods when operating at high pressure.

Optimization of a Digital Mass Filter for the Isolation of Intact Protein Complexes in Stability Zone 1,1.

Digital mass filters are advantageous for the analysis of large molecules due to the ability to perform ion isolation of high-m/z ions without the generation of very high radio frequency (RF) and DC voltages. Experimentally determined Mathieu stability diagrams of stability zone 1,1 for capacitively coupled digital waveforms show a voltage offset between the quadrupole rod pairs is introduced by the capacitors which is dependent on the voltage magnitude of the waveform and the duty cycle. This changes the ion's a value from a = 0 to a < 0. These effects are illustrated for isolation for single-charge states for various protein complexes up to 800 kDa (GroEL) for stability zone 1,1. Isolation resolving power (m/Δm) of approximately 280 was achieved for an ion of m/z 12,315 (z = 65+ for 800.5 kDa GroEL D398A), which corresponds to an m/z window of 44.

A Novel Y-Shaped, S-O-N-O-S-Bridged Cross-Link between Three Residues C22, C44, and K61 Is Frequently Observed in the SARS-CoV-2 Main Protease.

As the COVID-19 pathogen, SARS-CoV-2 relies on its main protease (MPro) for pathogenesis and replication. During crystallographic analyses of MPro crystals that were exposed to the air, a uniquely Y-shaped, S-O-N-O-S-bridged post-translational cross-link that connects three residues C22, C44, and K61 at their side chains was frequently observed. As a novel covalent modification, this cross-link serves potentially as a redox switch to regulate the catalytic activity of MPro, a demonstrated drug target of COVID-19. The formation of this linkage leads to a much more open active site that can potentially be targeted for the development of novel SARS-CoV-2 antivirals. The structural rearrangement of MPro by this cross-link indicates that small molecules that lock MPro in the cross-linked form can potentially be used with other active-site-targeting molecules such as paxlovid for synergistic effects in inhibiting SARS-CoV-2 viral replication.

An ion mobility-mass spectrometry study of copper-metallothionein-2A: binding sites and stabilities of Cu-MT and mixed metal Cu-Ag and Cu-Cd complexes.

The presence of Cu, a highly redox active metal, is known to damage DNA as well as other cellular components, but the adverse effects of cellular Cu can be mitigated by metallothioneins (MT), small cysteine rich proteins that are known to bind to a broad range of metal ions. While metal ion binding has been shown to involve the cysteine thiol groups, the specific ion binding sites are controversial as are the overall structure and stability of the Cu-MT complexes. Here, we report results obtained using nano-electrospray ionization mass spectrometry and ion mobility-mass spectrometry for several Cu-MT complexes and compare our results with those previously reported for Ag-MT complexes. The data include determination of the stoichiometries of the complex (Cui-MT, i = 1-19), and Cu+ ion binding sites for complexes where i = 4, 6, and 10 using bottom-up and top-down proteomics. The results show that Cu+ ions first bind to the ß-domain to form Cu4MT then Cu6MT, followed by addition of four Cu+ ions to the α-domain to form a Cu10-MT complex. Stabilities of the Cui-MT (i = 4, 6 and 10) obtained using collision-induced unfolding (CIU) are reported and compared with previously reported CIU data for Ag-MT complexes. We also compare CIU data for mixed metal complexes (CuiAgj-MT, where i + j = 4 and 6 and CuiCdj, where i + j = 4 and 7). Lastly, higher order Cui-MT complexes, where i = 11-19, were also detected at higher concentrations of Cu+ ions, and the metalated product distributions observed are compared to previously reported results for Cu-MT-1A (Scheller et al., Metallomics, 2017, 9, 447-462).

Performance evaluation of in-source ion activation hardware for collision-induced unfolding of proteins and protein complexes on a drift tube ion mobility-mass spectrometer.

Native ion mobility-mass spectrometry (IM-MS) has emerged as an information-rich technique for gas phase protein structure characterization; however, IM resolution is currently insufficient for the detection of subtle structural differences in large biomolecules. This challenge has spurred the development of collision-induced unfolding (CIU) which utilizes incremental gas phase activation to unfold a protein in order to expand the number of measurable descriptors available for native protein ions. Although CIU is now routinely used in native mass spectrometry studies, the interlaboratory reproducibility of CIU has not been established. Here we evaluate the reproducibility of the CIU data produced across three laboratories (University of Michigan, Texas A&M University, and Vanderbilt University). CIU data were collected for a variety of protein ions ranging from 8.6-66 kDa. Within the same laboratory, the CIU fingerprints were found to be repeatable with root mean square deviation (RMSD) values of less than 5%. Collision cross section (CCS) values of the CIU intermediates were consistent across the laboratories, with most features exhibiting an interlaboratory reproducibility of better than 1%. In contrast, the activation potentials required to induce protein CIU transitions varied between the three laboratories. To address these differences, three source assemblies were constructed with an updated ion activation hardware design utilizing higher mechanical tolerance specifications. The production-grade assemblies were found to produce highly consistent CIU data for intact antibodies, exhibiting high precision ion CCS and CIU transition values, thus opening the door to establishing databases of CIU fingerprints to support future biomolecular classification efforts.

Mechanistic Insights into IscU Conformation Regulation for Fe-S Cluster Biogenesis Revealed by Variable Temperature Electrospray Ionization Native Ion Mobility Mass Spectrometry.

Iron-sulfur (Fe-S) cluster (ISC) cofactors are required for the function of many critical cellular processes. In the ISC Fe-S cluster biosynthetic pathway, IscU assembles Fe-S cluster intermediates from iron, electrons, and inorganic sulfur, which is provided by the cysteine desulfurase enzyme IscS. IscU also binds to Zn, which mimics and competes for binding with the Fe-S cluster. Crystallographic and nuclear magnetic resonance spectroscopic studies reveal that IscU is a metamorphic protein that exists in multiple conformational states, which include at least a structured form and a disordered form. The structured form of IscU is favored by metal binding and is stable in a narrow temperature range, undergoing both cold and hot denaturation. Interestingly, the form of IscU that binds IscS and functions in Fe-S cluster assembly remains controversial. Here, results from variable temperature electrospray ionization (vT-ESI) native ion mobility mass spectrometry (nIM-MS) establish that IscU exists in structured, intermediate, and disordered forms that rearrange to more extended conformations at higher temperatures. A comparison of Zn-IscU and apo-IscU reveals that Zn(II) binding attenuates the cold/heat denaturation of IscU, promotes refolding of IscU, favors the structured and intermediate conformations, and inhibits the disordered high charge states. Overall, these findings provide a structural rationalization for the role of Zn(II) in stabilizing IscU conformations and IscS in altering the IscU active site to prepare for Zn(II) release and cluster synthesis. This work highlights how vT-ESI-nIM-MS can be applied as a powerful tool in mechanistic enzymology by providing details of relationships among temperature, protein conformations, and ligand/protein binding.

Repurposing Halicin as a potent covalent inhibitor for the SARS-CoV-2 main protease.

The rapid spread of COVID-19 has caused a worldwide public health crisis. For prompt and effective development of antivirals for SARS-CoV-2, the pathogen of COVID-19, drug repurposing has been broadly conducted by targeting the main protease (MPro), a key enzyme responsible for the replication of virus inside the host. In this study, we evaluate the inhibition potency of a nitrothiazole-containing drug, halicin, and reveal its reaction and interaction mechanism with MPro. The in vitro potency test shows that halicin inhibits the activity of MPro an IC50 of 181.7 â€‹nM. Native mass spectrometry and X-ray crystallography studies clearly indicate that the nitrothiazole fragment of halicin covalently binds to the catalytic cysteine C145 of MPro. Interaction and conformational changes inside the active site of MPro suggest a favorable nucleophilic aromatic substitution reaction mechanism between MPro C145 and halicin, explaining the high inhibition potency of halicin towards MPro.

Surface Activity of Amines Provides Evidence for the Combined ESI Mechanism of Charge Reduction for Protein Complexes.

Charge reduction reactions are important for native mass spectrometry (nMS) because lower charge states help retain native-like conformations and preserve noncovalent interactions of protein complexes. While mechanisms of charge reduction reactions are not well understood, they are generally achieved through the addition of small molecules, such as polyamines, to traditional nMS buffers. Here, we present new evidence that surface-active, charge reducing reagents carry away excess charge from the droplet after being emitted due to Coulombic repulsion, thereby reducing the overall charge of the droplet. Furthermore, these processes are directly linked to two mechanisms for electrospray ionization, specifically the charge residue and ion evaporation models (CRM and IEM). Selected protein complexes were analyzed in solutions containing ammonium acetate and selected trialkylamines or diaminoalkanes of increasing alkyl chain lengths. Results show that amines with higher surface activity have increased propensities for promoting charge reduction of the protein ions. The electrospray ionization (ESI) emitter potential was also found to be a major contributing parameter to the prevalence of charge reduction; higher emitter potentials consistently coincided with lower average charge states among all protein complexes analyzed. These results offer experimental evidence for the mechanism of charge reduction in ESI and also provide insight into the final stages of the ESI and their impact on biological ions.

Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP), and Stoichiometry of GroEL-GroES Complexes.

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, a chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL-GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL-GroES complexes. The results show clear evidence for destabilization of both GroEL14 and GroES7 at temperatures of 50 and 45 °C, respectively, substantially below the previously reported melting temperature (Tm ∼ 70 °C). This destabilization is accompanied by temperature-dependent reaction products that have previously unreported stoichiometries, viz. GroEL14-GroESy-ATPn, where y = 1, 2, 8 and n = 0, 1, 2, 8, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry reveals new insights about the stability of GroEL in response to temperature effects: (i) temperature-dependent ATP binding to GroEL; (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiometry of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and (iii) a change in the temperature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 and 40 °C. The similarities between results obtained by using native MS and cryo-EM [Clare et al. An expanded protein folding cage in the GroEL-gp31 complex. J. Mol. Biol. 2006, 358, 905-911; Ranson et al. Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes.Nat. Struct. Mol. Biol. 2006, 13, 147-152] underscore the utility of native MS for investigations of molecular machines as well as identification of key intermediates involved in the chaperonin-assisted protein folding cycle.