AMX – the highly automated macromolecular crystallography (17-ID-1) beamline at the NSLS-II

Schneider, Dieter K.; Soares, Alexei S.; Lazo, Edwin; Kreitler, D.F.; Qian, Kun; Fuchs, Martin R.; Bhogadi, Dileep; Antonelli, Steve; Myers, Stuart S.; Martins, Bruno; Skinner, John M.; Aishima, Jun; Bernstein, H. J.; Langdon, Thomas; Lara, John; Petkus, Robert; Cowan, Matt; Flaks, Leonid; Smith, Thomas J.; Shea-McCarthy, Grace; Idir, Mourad; Huang, Lei; Chubar, Oleg; Sweet, Robert M.; Berman, L. E.; McSweeney, Seán; Jakoncic, Jean

doi:10.1107/s1600577522009377

Cited by 13 publications

(7 citation statements)

References 38 publications

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“…X-ray diffraction data were collected on the NSLS-II AMX beamline at Brookhaven National Laboratory . Data were indexed and scaled on the AMX automated fast-dp pipeline.…”

Section: Methodsmentioning

confidence: 99%

“…X-ray diffraction data were collected on the NSLS-II AMX beamline at Brookhaven National Laboratory. 70 Data were indexed and scaled on the AMX automated fast-dp pipeline. The electron density map was phased from these data by molecular replacement using the program Phaser and the structure of unliganded HDAC6 CD2 less water molecules (PDB: 5EEM).…”

Section: Lc-ms To Determine H 2 18mentioning

confidence: 99%

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Difluoromethyl-1,3,4-oxadiazoles Are Selective, Mechanism-Based, and Essentially Irreversible Inhibitors of Histone Deacetylase 6

König,

Watson,

Reßing

et al. 2023

J. Med. Chem.

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Histone deacetylase 6 (HDAC6) is an important drug target in oncological and non-oncological diseases. Most available HDAC6 inhibitors (HDAC6i) utilize hydroxamic acids as a zinc-binding group, which limits therapeutic opportunities due to its genotoxic potential. Recently, difluoromethyl-1,3,4-oxadiazoles (DFMOs) were reported as potent and selective HDAC6i but their mode of inhibition remained enigmatic. Herein, we report that DFMOs act as mechanism-based and essentially irreversible HDAC6i. Biochemical data confirm that DFMO 6 is a tight-binding HDAC6i capable of inhibiting HDAC6 via a two-step slow-binding mechanism. Crystallographic and mechanistic experiments suggest that the attack of 6 by the zinc-bound water at the sp2 carbon closest to the difluoromethyl moiety followed by a subsequent ring opening of the oxadiazole yields deprotonated difluoroacetylhydrazide 13 as active species. The strong anionic zinc coordination of 13 and the binding of the difluoromethyl moiety in the P571 pocket finally result in an essentially irreversible inhibition of HDAC6.

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“…X-ray diffraction data were collected on the NSLS-II AMX beamline at Brookhaven National Laboratory . Data were indexed and scaled on the AMX automated fast-dp pipeline.…”

Section: Methodsmentioning

confidence: 99%

Section: Lc-ms To Determine H 2 18mentioning

confidence: 99%

Difluoromethyl-1,3,4-oxadiazoles Are Selective, Mechanism-Based, and Essentially Irreversible Inhibitors of Histone Deacetylase 6

König,

Watson,

Reßing

et al. 2023

J. Med. Chem.

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show abstract

“…Single crystals were harvested and flash frozen in liquid nitrogen with 20% glycerol in well solution as a cryoprotectant. Diffraction data were collected at the National Synchrotron Light Source II (NSLS II) Highly Automated Macromolecular Crystallography (AMX) (67) beamline 17-ID-1 at the Brookhaven National Laboratory on an Eiger 9M Pixel detector at 100 K and a wavelength of 0.920219 Å. Diffraction data were indexed, integrated, and scaled using XDS. The M pro -TRMT1 structure was solved in space group P2 1 2 1 2 1 using the Phaser package in the CCP4 suite and a modified PDB 7MGS as search model.…”

Section: Methodsmentioning

confidence: 99%

Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS-CoV-2 Main Protease

D'Oliviera

Dai

Mottaghinia

et al. 2023

Preprint

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The SARS-CoV-2 main protease (Mpro) plays a crucial role in the production of functional viral proteins during infection and, like many viral proteases, can also target and cleave host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 can be recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification at the G26 position of mammalian tRNA, which promotes global protein synthesis, cellular redox homeostasis, and has links to neurological disability. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain that is required for tRNA modification activity in cells. Evolutionary analysis shows that the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 may be resistant to cleavage. In primates, regions outside of the cleavage site with rapid evolution could indicate possible adaptation to ancient viral pathogens. To visualize how Mpro recognizes the TRMT1 cleavage sequence, we determined the structure of a TRMT1 peptide in complex with Mpro, which reveals a substrate binding conformation distinct from the majority of available SARS-CoV-2 Mpro-peptide complexes. Kinetic parameters for peptide cleavage showed that while TRMT1(526-536) is cleaved much slower than the Mpro nsp4/5 autoprocessing sequence, it is proteolyzed with comparable efficiency to the Mpro-targeted nsp8/9 viral cleavage site. Mutagenesis studies and molecular dynamics simulations together indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis that follows substrate binding. Our results provide new information about the structural basis for Mpro substrate recognition and cleavage that could help inform future therapeutic design and raise the possibility that proteolysis of human TRMT1 during SARS-CoV-2 infection may impact protein translation or oxidative stress response and contribute to viral pathogenesis.

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“…The final SeMet and native datasets of CifR TR :dsDNA 26 used for structure determination were collected at NSLS-II beamline 17-ID-1. Diffraction data were collected on an Eiger 9M detector through 360° of total rotation using the NSLS-II AMX beamline’s vector collection mode (22) in 0.2° frames at a detector distance of 200 mm. For the SAD experiment using the SeMet crystals, the optimal peak energies were determined from X-ray absorption scans.…”

Section: Methodsmentioning

confidence: 99%

Molecular basis for the transcriptional regulation of an epoxide-based virulence circuit inPseudomonas aeruginosa

He,

Taher,

Hvorecny

et al. 2024

Preprint

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The opportunistic pathogenPseudomonas aeruginosainfects cystic fibrosis (CF) patient airways and produces a virulence factor Cif that is associated with worse outcomes. Cif is an epoxide hydrolase that reduces cell-surface abundance of the cystic fibrosis transmembrane conductance regulator (CFTR) and sabotages pro-resolving signals. Its expression is regulated by a divergently transcribed TetR family transcriptional repressor. CifR represents the first reported epoxide-sensing bacterial transcriptional regulator, but neither its interaction with cognate operator sequences nor the mechanism of activation has been investigated. Using biochemical and structural approaches, we uncovered the molecular mechanisms controlling this complex virulence operon. We present here the first molecular structures of CifR alone and in complex with operator DNA, resolved in a single crystal lattice. Significant conformational changes between these two structures suggest how CifR regulates the expression of the virulence gene cif. Interactions between the N-terminal extension of CifR with the DNA minor groove of the operator play a significant role in the operator recognition of CifR. We also determined that cysteine residue Cys107 is critical for epoxide sensing and DNA release. These results offer new insights into the stereochemical regulation of an epoxide-based virulence circuit in a critically important clinical pathogen.

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AMX – the highly automated macromolecular crystallography (17-ID-1) beamline at the NSLS-II

Cited by 13 publications

References 38 publications

Difluoromethyl-1,3,4-oxadiazoles Are Selective, Mechanism-Based, and Essentially Irreversible Inhibitors of Histone Deacetylase 6

Difluoromethyl-1,3,4-oxadiazoles Are Selective, Mechanism-Based, and Essentially Irreversible Inhibitors of Histone Deacetylase 6

Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS-CoV-2 Main Protease

Molecular basis for the transcriptional regulation of an epoxide-based virulence circuit inPseudomonas aeruginosa

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