Satoko Akashi

Single-cell native mass spectrometry (MS) holds immense potential for highly sensitive in situ analysis of biological events within individual live cells. Herein, we present a significant advancement that enables the direct detection of protein-drug complexes in single human cells. We first established the robust and reproducible detection of enhanced green fluorescent protein (EGFP) expressed in HEK293T cells using our single-cell native MS approach. To investigate protein-inhibitor interactions with this approach, human Sirtuin 2 histone deacetylase domain (SIRT2 HDAC) was fused with EGFP and overexpressed in HEK293T cells. However, the expression level of this EGFP-SIRT2 HDAC fusion protein was too low for the detection of protein signals by single-cell native MS. To overcome this challenge, we employed a P2A peptide linker strategy to achieve coexpression of independent EGFP and SIRT2 HDAC at suitable levels within the same cell. This strategy enabled the clear identification of multiply charged ions for both proteins and, crucially, the observation of SIRT2 HDAC-drug complex ions within a single live cell. Our platform establishes a transformative approach for characterizing protein-drug interactions in their native cellular environment at the single-cell level, with potential applications in cell-cycle-resolved pharmacology and drug discovery.

Intramembrane metalloproteases are conserved in all three domains of life and regulate cellular signal transduction by cleaving membrane-anchored precursors or regulators of transcription factors. Bacterial intramembrane metalloproteases are promising targets for antimicrobial drugs as they are implicated in pathogenicity and drug resistance. Understanding the properties of intramembrane metalloproteases, particularly their interactions with zinc and inhibitors, is important for drug development. While native mass spectrometry (MS) is effective for studying intermolecular interactions, its application to membrane proteins, including intramembrane metalloproteases, presents a unique challenge for preserving noncovalent interactions in the gas phase due to their hydrophobic nature. In this study, we utilized native MS to investigate zinc and inhibitor binding to two bacterial intramembrane metalloproteases, Escherichia coli RseP (EcRseP) and its ortholog from Kangiella koreensis (KkRseP). Intact protein ions were successfully observed following optimized purification and buffer exchange protocols. Native MS revealed zinc binding to both orthologs, with EcRseP exhibiting higher affinity. In the presence of batimastat, a specific EcRseP inhibitor, both RseP orthologs formed stable complexes, demonstrating batimastat binds exclusively to zinc-bound RseP. These results demonstrate the ability of native MS for characterizing membrane protein interactions and highlight its potential as a platform for identifying specific binding events, thereby extending its established application with soluble proteins. Graphical Abstract

Misfolding of mutant Cu/Zn‐superoxide dismutase (SOD1) is a well‐established pathological feature of familial amyotrophic lateral sclerosis (ALS). While amino acid substitutions in mutant SOD1 destabilize its structure and promote misfolding, oxidation has also been implicated in the pathological alterations of wild‐type SOD1, particularly in neurodegenerative diseases including sporadic ALS. However, the impact of oxidation on SOD1 folding remains to be fully elucidated. Here, we demonstrate that Cys111 is primarily oxidized to sulfonic acid upon exposure of apo‐SOD1 to hydrogen peroxide, as confirmed by the quantitation of thiol groups and mass spectrometry. Molecular dynamics simulations showed that sulfonylation of Cys111 disrupts the dimer interface and promotes monomerization. This monomeric form then facilitates the subsequent oxidation of buried Cys6, leading to structural disruption, as evidenced by circular dichroism spectroscopy and loss of thiol groups. SOD1 denaturation triggered by Cys111 oxidation became evident when zinc binding was impaired due to pathological mutations and/or under zinc‐deficient conditions. Given that increased oxidative stress is frequently associated with many neurodegenerative diseases, modulating Cys111 oxidation may offer a potential strategy for maintaining SOD1 structural stability and preventing its pathological misfolding.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in glycolysis. Beyond this normal function, GAPDH acts as a moonlighting protein, interacting with nonglycolytic molecules to fulfill additional roles, such as apoptosis induction. However, the three-dimensional (3D) structural details underlying these interactions remain unclear, likely due to their dynamic and transient nature. To address this issue, we investigated the structural properties of human and porcine GAPDH using a combination of biophysical techniques, including nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, gel filtration chromatography, and thermal shift assays, with a particular focus on their 3D structures. Our results revealed that although GAPDH becomes unstable upon nicotinamide adenine dinucleotide (NAD⁺) depletion (apo state), its oligomeric structure as a tetramer remains preserved regardless of temperature. In contrast, the presence of adenosine triphosphate (ATP) promotes dimerization at low temperatures, as previously reported. Furthermore, our NMR data suggest that ATP binding exposes the dimer interface and increases the flexibility of side chains in this region. These findings indicate that GAPDH maintains a stable tetrameric structure in the presence of NAD⁺ but becomes structurally unstable and likely more susceptible to oxidation upon NAD⁺ depletion. Additionally, our analyses showed that partial nitrosylation of GAPDH subunits does not induce significant tertiary structural changes. However, significant structural alterations were observed when all four subunits were nitrosylated, although the possibility remains that residues other than the active site residue, Cys152, may have been oxidized. We propose that NAD⁺ depletion, along with oxidation or nitrosylation─most likely at Cys152─destabilizes the GAPDH conformation, and that subsequent ATP binding promotes dimerization. This subunit dissociation may serve as a structural basis for GAPDH's interactions with other molecules and its moonlighting functions.

Site-2 proteases (S2Ps), conserved intramembrane metalloproteases that maintain cellular homeostasis, are associated with chronic infection and persistence leading to multidrug resistance in bacterial pathogens. A structural model of how S2Ps discriminate and accommodate substrates could help us develop selective antimicrobial agents. We previously proposed that the Escherichia coli S2P RseP unwinds helical substrate segments before cleavage, but the mechanism for accommodating a full-length membrane-spanning substrate remained unclear. Our present cryo-EM analysis of Aquifex aeolicus RseP ( Aa RseP) revealed that a substrate-like membrane protein fragment from the expression host occupied the active site while spanning a transmembrane cavity that is inaccessible via lateral diffusion. Furthermore, in vivo photocrosslinking supported that this substrate accommodation mode is recapitulated on the cell membrane. Our results suggest that the substrate accommodation by threading through a conserved membrane-associated region stabilizes the substrate-complex and contributes to substrate discrimination on the membrane.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the sixth step of glycolysis. As a moonlighting protein, GAPDH interacts with other molecules, seemingly unrelated to glycolysis, to exert additional functions, such as inducing apoptosis. However, specific mechanisms underlying these interactions remain unclear. Therefore, in this study, we aimed to elucidate these mechanisms by analyzing human and porcine GAPDHs based on their three-dimensional (3D) structures using various biophysical techniques, including nuclear magnetic resonance spectroscopy, gel filtration chromatography, and thermal shift assay. Although GAPDH became unstable when nicotinamide adenine dinucleotide (NAD ⁺ ) was removed ( apo state), the 3D structure of the tetramer was maintained regardless of the temperature. However, in the presence of adenosine triphosphate (ATP), GAPDH split into dimers at low temperatures, exposing the interface between the dimers and increasing the flexibility of the side chains at the site. Moreover, subunit splitting also occurred upon exposure to oxidizing agents, such as hydrogen peroxide. These findings suggest that GAPDH maintains a stable tetramer in the presence of NAD ⁺ but becomes unstable and easily oxidized upon NAD ⁺ depletion. When multiple residues, including those other than the cysteine residue at the active site, are oxidized by reactive oxygen species or nitric oxide, or when it interacts with ATP, GAPDH splits into dimers. This subunit splitting may trigger interactions with other molecules.

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin‐resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin‐resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid‐selective unlabeling method. Because we found that the zinc ion‐coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

Sirtuin 2 (SIRT2) is a class III histone deacetylase that is highly conserved from bacteria to mammals. We prepared and characterized the wild‐type (WT) and mutant forms of the histone deacetylase (HDAC) domain of human SIRT2 ( h SIRT2) using various biophysical methods and evaluated their deacetylation activity. We found that WT h SIRT2 HDAC (residues 52–357) forms a homodimer in a concentration‐dependent manner with a dimer–monomer dissociation constant of 8.3 ± 0.5 μM, which was determined by mass spectrometry. The dimer was disrupted into two monomers by binding to the HDAC inhibitors SirReal1 and SirReal2. We also confirmed dimer formation of h SIRT2 HDAC in living cells using a NanoLuc complementation reporter system. Examination of the relationship between dimer formation and deacetylation activity using several mutants of h SIRT2 HDAC revealed that some non‐dimerizing mutants exhibited deacetylation activity for the N‐terminal peptide of histone H3, similar to the wild type. The h SIRT2 HDAC mutant Δ 292–306, which lacks a SIRT2‐specific disordered loop region, was identified to exist as a monomer with slightly reduced deacetylation activity; the X‐ray structure of the mutant Δ 292–306 was almost identical to that of the WT h SIRT2 HDAC bound to an inhibitor. These results indicate that h SIRT2 HDAC forms a dimer, but this is independent of deacetylation activity. Herein, we discuss insights into the dimer formation of h SIRT2 based on our biophysical experimental results.

We aimed to develop a platform to rapidly investigate the responses of agonists and antagonists to G-protein-coupled receptors (GPCRs) using native mass spectrometry (MS). We successfully observed the ligand-bound human β₂ adrenergic receptor (hβ₂AR); however, it was challenging to quantitatively discuss drug efficacy from MS data alone. Since ligand-bound GPCRs are stabilized by the Gα subunit of G proteins on the membrane, mini-G_s and nanobody80 (Nb80) that can mimic the Gα interface of the GPCR were utilized. Ternary complexes of hβ₂AR, ligand, and mini-G_s or Nb80 were prepared and subjected to native MS. We found a strong correlation between the hβ₂AR-mini-G_s or -Nb80 complex ratio observed in the mass spectra and agonist/antagonist efficacy obtained using a cell-based assay. This method does not require radioisotope labeling and would be applicable to the analysis of other GPCRs, facilitating the characterization of candidate compounds as GPCR agonists and antagonists.