Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology.

We present a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS2) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-curation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS1-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS2 straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-curation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells.

The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA.

GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with tRNA aminoacylation, led to the hypothesis that eIF2α kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost histidine and ATP binding but retains tRNA binding abilities. Hydrogen deuterium exchange mass spectrometry, site-directed mutagenesis and computational docking experiments support a tRNA binding model that is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function.

Novobiocin blocks nucleic acid binding to Polθ and inhibits stimulation of its ATPase activity.

Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.

Utilization of Fast Photochemical Oxidation of Proteins and Both Bottom-up and Top-down Mass Spectrometry for Structural Characterization of a Transcription Factor-dsDNA Complex.

A combination of covalent labeling techniques and mass spectrometry (MS) is currently a progressive approach for deriving insights related to the mapping of protein surfaces or protein-ligand interactions. In this study, we mapped an interaction interface between the DNA binding domain (DBD) of FOXO4 protein and the DNA binding element (DAF16) using fast photochemical oxidation of proteins (FPOP). Residues involved in protein-DNA interaction were identified using the bottom-up approach. To confirm the findings and avoid a misinterpretation of the obtained data, caused by possible multiple radical oxidations leading to the protein surface alteration and oxidation of deeply buried amino acid residues, a top-down approach was employed for the first time in FPOP analysis. An isolation of singly oxidized ions enabled their gas-phase separation from multiply oxidized species followed by CID and ECD fragmentation. Application of both fragmentation techniques allowed generation of complementary fragment sets, out of which the regions shielded in the presence of DNA were deduced. The findings obtained by bottom-up and top-down approaches were highly consistent. Finally, FPOP results were compared with those of the HDX study of the FOXO4-DBD·DAF16 complex. No contradictions were found between the methods. Moreover, their combination provides complementary information related to the structure and dynamics of the protein-DNA complex. Data are available via ProteomeXchange with identifier PXD027624.

Chimeric Cellobiose Dehydrogenases Reveal the Function of Cytochrome Domain Mobility for the Electron Transfer to Lytic Polysaccharide Monooxygenase.

The natural function of cellobiose dehydrogenase (CDH) to donate electrons from its catalytic flavodehydrogenase (DH) domain via its cytochrome (CYT) domain to lytic polysaccharide monooxygenase (LPMO) is an example of a highly efficient extracellular electron transfer chain. To investigate the function of the CYT domain movement in the two occurring electron transfer steps, two CDHs from the ascomycete Neurospora crassa (NcCDHIIA and NcCDHIIB) and five chimeric CDH enzymes created by domain swapping were studied in combination with the fungus' own LPMOs (NcLPMO9C and NcLPMO9F). Kinetic and electrochemical methods and hydrogen/deuterium exchange mass spectrometry were used to study the domain movement, interaction, and electron transfer kinetics. Molecular docking provided insights into the protein-protein interface, the orientation of domains, and binding energies. We find that the first, interdomain electron transfer step from the catalytic site in the DH domain to the CYT domain depends on steric and electrostatic interface complementarity and the length of the protein linker between both domains but not on the redox potential difference between the FAD and heme b cofactors. After CYT reduction, a conformational change of CDH from its closed state to an open state allows the second, interprotein electron transfer (IPET) step from CYT to LPMO to occur by direct interaction of the b-type heme and the type-2 copper center. Chimeric CDH enzymes favor the open state and achieve higher IPET rates by exposing the heme b cofactor to LPMO. The IPET, which is influenced by interface complementarity and the heme b redox potential, is very efficient with bimolecular rates between 2.9 × 105 and 1.1 × 106 M-1 s-1.

The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses. Although LPMOs receive ample interest in industry and academia, their reaction mechanism is not yet fully understood. Recent studies showed that H2O2 is a more efficient cosubstrate for the enzyme than O2, which could greatly affect the utilization of LPMOs in industrial settings. RESULTS: We probe the reactivity of LPMO9C from the cellulose-degrading fungus Neurospora crassa with a turbidimetric assay using phosphoric acid-swollen cellulose (PASC) as substrate and H2O2 as a cosubstrate. The measurements were also followed by continuous electrochemical H2O2 detection and LPMO reaction products were analysed by mass spectrometry. Different systems for the in situ generation of H2O2 and for the reduction of LPMO's active-site copper were employed, including glucose oxidase, cellobiose dehydrogenase, and the routinely used reductant ascorbate. CONCLUSIONS: We found for all systems that the supply of H2O2 limited LPMO's cellulose depolymerization activity, which supports the function of H2O2 as the relevant cosubstrate. The turbidimetric assay allowed rapid determination of LPMO activity on a cellulosic substrate without the need for time-consuming and instrumentally elaborate analysis methods.

Photoinduced damage of AsLOV2 domain is accompanied by increased singlet oxygen production due to flavin dissociation.

Flavin mononucleotide (FMN) belongs to the group of very efficient endogenous photosensitizers producing singlet oxygen, 1O2, but with limited ability to be targeted. On the other hand, in genetically-encoded photosensitizers, which can be targeted by means of various tags, the efficiency of FMN to produce 1O2 is significantly diminished due to its interactions with surrounding amino acid residues. Recently, an increase of 1O2 production yield by FMN buried in a protein matrix was achieved by a decrease of quenching of the cofactor excited states by weakening of the protein-FMN interactions while still forming a complex. Here, we suggest an alternative approach which relies on the blue light irradiation-induced dissociation of FMN to solvent. This dissociation unlocks the full capacity of FMN as 1O2 producer. Our suggestion is based on the study of an irradiation effect on two variants of the LOV2 domain from Avena sativa; wild type, AsLOV2 wt, and the variant with a replaced cysteine residue, AsLOV2 C450A. We detected irradiation-induced conformational changes as well as oxidation of several amino acids in both AsLOV2 variants. Detailed analysis of these observations indicates that irradiation-induced increase in 1O2 production is caused by a release of FMN from the protein. Moreover, an increased FMN dissociation from AsLOV2 wt in comparison with AsLOV2 C450A points to a role of C450 oxidation in repelling the cofactor from the protein.

Structural Dynamics of Lytic Polysaccharide Monooxygenase during Catalysis.

Lytic polysaccharide monooxygenases (LPMOs) are industrially important oxidoreductases employed in lignocellulose saccharification. Using advanced time-resolved mass spectrometric techniques, we elucidated the structural determinants for substrate-mediated stabilization of the fungal LPMO9C from Neurosporacrassa during catalysis. LPMOs require a reduction in the active-site copper for catalytic activity. We show that copper reduction in NcLPMO9C leads to structural rearrangements and compaction around the active site. However, longer exposure to the reducing agent ascorbic acid also initiated an uncoupling reaction of the bound oxygen species, leading to oxidative damage, partial unfolding, and even fragmentation of NcLPMO9C. Interestingly, no changes in the hydrogen/deuterium exchange rate were detected upon incubation of oxidized or reduced LPMO with crystalline cellulose, indicating that the LPMO-substrate interactions are mainly side-chain mediated and neither affect intraprotein hydrogen bonding nor induce significant shielding of the protein surface. On the other hand, we observed a protective effect of the substrate, which slowed down the autooxidative damage induced by the uncoupling reaction. These observations further complement the picture of structural changes during LPMO catalysis.

14-3-3 protein binding blocks the dimerization interface of caspase-2.

Among all species, caspase-2 (C2) is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser139 and Ser164 , within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14-3-3, which maintains C2 in its immature form procaspase (proC2). However, the mechanism of 14-3-3-dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14-3-3 by hydrogen/deuterium mass spectrometry and protein crystallography to determine the molecular basis for 14-3-3-mediated inhibition of C2 activation. Our data reveal that the 14-3-3 dimer interacts with proC2 not only through ligand-binding grooves but also through other regions outside the central channel, thus explaining the isoform-dependent specificity of 14-3-3 protein binding to proC2 and the substantially higher binding affinity of 14-3-3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14-3-3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14-3-3 protein interacts with and masks both the nuclear localization sequence and the C-terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14-3-3. This masked region of p12 domain is involved in C2 dimerization. Therefore, 14-3-3 protein likely inhibits proC2 activation by blocking its dimerization surface. DATABASES: Structural data are available in the Protein Data Bank under the accession numbers 6SAD and 6S9K.