John Tainer

MD Anderson Cancer Center, UT Texas, Houston, Department of Molecular and Cellular Oncology, Faculty Member

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I study structures to gain a far more complete understanding of life. I focus on connecting structures of nano to micro-scale macromolecular machines to seeing, predicting, and controlling biological outcomes for cancer, disease, and biotechnology. Research includes integrating x-ray scattering (SAXS) in solution, crystallography, EM, and NMR with super-resolution imaging with computation to define predictive biological structures and chemical inhibitors that control outcomes in cells - see sibyls.als.lbl.gov

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Structure of double mutant of human iNOS oxygenase domain with bound immidazole

Structure of murine iNOS oxygenase domain with inhibitor AR-C102222

Structure of inhibited murine iNOS oxygenase domain

Murine Inosoxy Dimer with Isothiourea Bound in the Active Site

Super resolution for X-ray scattering and biological insights from its applications to dynamic DNA replication and repair complexes

Acta Crystallographica, May 26, 2017

Biological small angle X-ray scattering (BioSAXS) is an under appreciated and under utilized meth... more Biological small angle X-ray scattering (BioSAXS) is an under appreciated and under utilized method for structural analyses that accuractely defines solution conformations and assemblies that are directly relevant to connecting structures to biological insights. Bio-SAXS is the only true high-throughput structural biology method. It furthermore provides information on the entire complex including both ordered and flexible regions when interpreted with appropriate metrics and methods. We will present data on dynamic complexes acting in DNA replication and repair. The results will show that SAXS can probe at resolutions sufficient to distinguish different conformational states, characterize flexible macromolecules, and screen in high-throughput under most solution conditions. Methods with exemplary experimental results will be considered with a focus on interpretation tools. Results will be presented to show how SAXS can provide accurate shapes, assembly states, and comprehensive conformations in solution that inform biology in fundamental ways 1-9. The approaches for measuring flexibility, conformational changes, and assembly processes discussed here are relevant for accurate understanding, simulation, and prediction of many mechanisms in cell biology and nanotechnology.

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Direct interaction of DNA repair protein tyrosyl DNA phosphodiesterase 1 and the DNA ligase III catalytic domain is regulated by phosphorylation of its flexible N-terminus

Journal of Biological Chemistry, Aug 1, 2021

Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-st... more Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3′-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped by either DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA-binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII170–755, as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small-angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in nonmalignant and cancer cells.

Structure of human INOSOX with inhibitor AR-C95791

A condensate forming tether for lariat debranching enzyme is defective in non-photosensitive trichothiodystrophy

SummaryThe pre-mRNA life cycle requires intron processing; yet, how intron processing defects inf... more SummaryThe pre-mRNA life cycle requires intron processing; yet, how intron processing defects influence splicing and gene expression is unclear. Here, we find TTDN1, which is frequently mutated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to the spliceosome. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, while its N-terminal intrinsically disordered region (IDR) binds the intron binding complex (IBC). The IDR forms condensatesin vitroand is needed for IBC interaction. TTDN1 loss causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells.Ttdn1Δ/Δmice recapitulate intron processing defects and neurodevelopmental phenotypes seen in NP-TTD. A DBR1-IDR fusion recruits DBR1 to the IBC and circumvents the requirement for TTDN1, indicating this tethering role as its major molecular function. Collectively, our find...

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Incorporating HT-SAXS into drug-discovery pipelines

Acta Crystallographica Section A Foundations and Advances

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Using Supercomputing Resources in Genomic Research

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XRCC1 promotes replication restart, nascent fork degradation and mutagenic DNA repair in BRCA2-deficient cells

NAR Cancer, 2020

Homologous recombination/end joining (HR/HEJ)-deficient cancers with BRCA mutations utilize alter... more Homologous recombination/end joining (HR/HEJ)-deficient cancers with BRCA mutations utilize alternative DNA double-strand break repair pathways, particularly alternative non-homologous end joining or microhomology-mediated end joining (alt-EJ/MMEJ) during S and G2 cell cycle phases. Depletion of alt-EJ factors, including XRCC1, PARP1 and POLQ, is synthetically lethal with BRCA2 deficiency; yet, XRCC1 roles in HR-deficient cancers and replication stress are enigmatic. Here, we show that after replication stress, XRCC1 forms an active repair complex with POLQ and MRE11 that supports alt-EJ activity in vitro. BRCA2 limits XRCC1 recruitment and repair complex formation to suppress alt-EJ at stalled forks. Without BRCA2 fork protection, XRCC1 enables cells to complete DNA replication at the expense of increased genome instability by promoting MRE11-dependent fork resection and restart. High XRCC1 and MRE11 gene expression negatively impacts Kaplan–Meier survival curves and hazard ratios ...

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Uncovering DNA-PKcs ancient phylogeny, unique sequence motifs and insights for human disease

Progress in Biophysics and Molecular Biology, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

format_quoteDNA-PKcs autophosphorylation surprisingly decreases its kinase activity, affecting DNA repair processes.format_quote

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Visualizing functional dynamicity in the DNA-dependent protein kinase holoenzyme DNA-PK complex by integrating SAXS with cryo-EM

Progress in Biophysics and Molecular Biology, 2020

Assembly of KU and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at DNA double strand... more Assembly of KU and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at DNA double strand breaks (DSBs) forms DNA-PK holoenzyme as a critical initiating step for non-homologous end joining (NHEJ) repair of DSBs produced by radiation and chemotherapies. Advanced cryo-electron microscopy (cryo-EM) imaging together with breakthrough macromolecular X-ray crystal (MX) structures of KU and DNA-PKcs recently enabled visualization of the~600 kDa DNA-PK assembly at near atomic resolution. These important static structures provide the foundation for definition and interpretation of functional movements crucial to mechanistic understanding that can be tested through solution state structure analysis. We herein therefore leverage Cryo-EM and MX structures for the interpretation of synchrotron small-angle X-ray scattering (SAXS) data on DNA-PK conformations in solution to inform the structural mechanism for NHEJ initiation. SAXS, which measures thermodynamic solution-state conformational states and assemblies outside of cryo-and solid-state conditions, unveils the inherent flexibility of KU, DNA-PKcs and DNA-PK. The combined structural measurements reveal mobility of KU80 C-terminal region (KU80CTR), motion/plasticity of HEAT (DNA-PKcs Huntingtin, Elongation Factor 3, PP2 A, and TOR1) regions, allosteric switching upon DNA-PKcs autophosphorylation, and dimeric arrangements of DNA-PK assembly. Importantly, the results uncover displacement of the N-terminal HEAT domain during autophosphorylation as suitable for a regulated release mechanism of DNA-PKcs from DNA-PK to control unproductive access to toxic and mutagenic DNA repair intermediates. These integrated analyses show that the marriage of SAXS with cryo-EM leverages the strengths of both techniques to enable assessment of functional conformations and flexibility defining atomic-resolution molecular mechanisms for DSB repair.

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63PClinical dysregulation of DNA repair by the polynucleotide kinase/phosphatase-XRCC4-DNA ligase IV in neurological disease

Annals of Oncology, 2017

Inducible Nitric Oxide Synthase with 3-Bromo-7-Nitroindazole bound

Human Endothelial Nitric Oxide Synthase with 6-NITROINDAZOLE Bound

human endothelial nitric oxide synthase with 3-Bromo-7-Nitroindazole bound

Inducible Nitric Oxide Synthase with 5-NITROINDAZOLE Bound

Inducible Nitric Oxide Synthase with 7-NITROINDAZOLE Bound

Structural Control of Nonnative Ligand Binding in Engineered Mutants of Phosphoenolpyruvate Carboxykinase

Biochemistry, 2018

Protein engineering to alter recognition underlying ligand binding and activity has enormous pote... more Protein engineering to alter recognition underlying ligand binding and activity has enormous potential. Here, ligand binding for Escherichia coli phosphoenolpyruvate carboxykinase (PEPCK), which converts oxaloacetate into CO 2 and phosphoenolpyruvate as the first committed step in gluconeogenesis, was engineered to accommodate alternative ligands as an exemplary system with structural information. From our identification of bicarbonate binding in the PEPCK active site at the supposed CO 2 binding site, we probed binding of nonnative ligands with three oxygen atoms arranged to resemble the bicarbonate geometry. Crystal structures of PEPCK and point mutants with bound nonnative ligands thiosulfate and methanesulfonate along with strained ATP and reoriented oxaloacetate intermediates and unexpected bicarbonate were determined and analyzed. The mutations successfully altered the bound ligand position and orientation and its specificity: mutated PEPCKs bound either thiosulfate or methanesulfonate but never both. Computational calculations predicted a methanesulfonate binding mutant and revealed that release of the active site ordered solvent exerts a strong influence on ligand binding. Besides nonnative ligand binding, one mutant altered the Mn 2+ coordination sphere: instead of the canonical octahedral ligand arrangement, the mutant in question had an only five-coordinate arrangement. From this work, *

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