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Griffin, Patrick R.

Publications and source records attributed to Griffin, Patrick R..

11 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Assembly of JAZ–JAZ and JAZ–NINJA complexes in jasmonate signaling

Jasmonates (JAs) are plant hormones with crucial roles in development and stress resilience. They activate MYC transcription factors by mediating the proteolysis of MYC inhibitors called JAZ proteins. In the absence of JA, JAZ proteins bind and inhibit MYC through the assembly of MYC–JAZ–Novel Interactor of JAZ (NINJA)–TPL repressor complexes. However, JAZ and NINJA are predicted to be largely intrinsically unstructured, which has precluded their experimental structure determination. Through a combination of biochemical, mutational, and biophysical analyses and AlphaFold-derived ColabFold modeling, we characterized JAZ–JAZ and JAZ–NINJA interactions and generated models with detailed, high-confidence domain interfaces. We demonstrate that JAZ, NINJA, and MYC interface domains are dynamic in isolation and become stabilized in a stepwise order upon complex assembly. By contrast, most JAZ and NINJA regions outside of the interfaces remain highly dynamic and cannot be modeled in a single conformation. Our data indicate that the small JAZ Zinc finger expressed in Inflorescence Meristem (ZIM) motif mediates JAZ–JAZ and JAZ–NINJA interactions through separate surfaces, and our data further suggest that NINJA modulates JAZ dimerization. This study advances our understanding of JA signaling by providing insights into the dynamics, interactions, and structure of the JAZ–NINJA core of the JA repressor complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro

SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic. Its genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-11 polyprotein, whose mature products include cofactors of the viral replicase, and identified the order of cleavages. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and x-ray scattering yielded a nsp7-11 structural ensemble, demonstrating shared secondary structural elements with individual nsps. The pattern of cross-links and HDX footprint of the C145A Mpro and nsp7-11 complex demonstrate preferential binding of the enzyme active site to the polyprotein junction sites and additional transient contacts to help orient the enzyme on its substrate for cleavage. Last, proteolysis assays were used to characterize the effect of inhibitors/binders on Mpro processing/inhibition using the nsp7-11 polyprotein as substrate.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Conformational Changes of RORγ During Response Element Recognition and Coregulator Engagement

The retinoic acid receptor-related orphan receptor γ (RORγ) is a ligand-dependent transcription factor of the nuclear receptor super family that underpins metabolic activity, immune function, and cancer progression. Despite being a valuable drug target in health and disease, our understanding of the ligand-dependent activities of RORγ is far from complete. Like most nuclear receptors, RORγ must recruit coregulatory protein to enact the RORγ target gene program. To date, a majority of structural studies have been focused exclusively on the RORγ ligand-binding domain and the ligand-dependent recruitment of small peptide segments of coregulators. Herein, we examine the ligand-dependent assembly of full length RORγ:coregulator complexes on cognate DNA response elements using structural proteomics and small angle x-ray scattering. The results from our studies suggest that RORγ becomes elongated upon DNA recognition, preventing long range interdomain crosstalk. We also determined that the DNA binding domain adopts a sequence-specific conformation, and that coregulatory protein may be able to ‘sense’ the ligand- and DNA-bound status of RORγ. We propose a model where ligand-dependent coregulator recruitment may be influenced by the sequence of the DNA to which RORγ is bound. Overall, the efforts described herein will illuminate important aspects of full length RORγ and monomeric orphan nuclear receptor target gene regulation through DNA-dependent conformational changes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), but how heme regulates REV-ERB activity remains unclear. Cellular studies indicate that heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription. However, fluorescence-based biochemical assays suggest that heme displaces NCoR; here, we show that this is due to a heme-dependent artifact. Using ITC and NMR spectroscopy, we show that heme binding remodels the thermodynamic interaction profile of NCoR receptor interaction domain (RID) binding to REV-ERBβ ligand-binding domain (LBD). We solved two crystal structures of REV-ERBβ LBD cobound to heme and NCoR peptides, revealing the heme-dependent NCoR binding mode. ITC and chemical cross-linking mass spectrometry reveals a 2:1 LBD:RID stoichiometry, consistent with cellular studies showing that NCoR-dependent repression of REV-ERB transcription occurs on dimeric DNA response elements. Our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A molecular switch regulating transcriptional repression and activation of PPARγ

Nuclear receptor (NR) transcription factors use a conserved activation function-2 (AF-2) helix 12 mechanism for agonist-induced coactivator interaction and NR transcriptional activation. In contrast, ligand-induced corepressor-dependent NR repression appears to occur through structurally diverse mechanisms. We report two crystal structures of peroxisome proliferator-activated receptor gamma (PPARγ) in an inverse agonist/corepressor-bound transcriptionally repressive conformation. Helix 12 is displaced from the solvent-exposed active conformation and occupies the orthosteric ligand-binding pocket enabled by a conformational change that doubles the pocket volume. Paramagnetic relaxation enhancement (PRE) NMR and chemical crosslinking mass spectrometry confirm the repressive helix 12 conformation. PRE NMR also defines the mechanism of action of the corepressor-selective inverse agonist T0070907, and reveals that apo-helix 12 exchanges between transcriptionally active and repressive conformations—supporting a fundamental hypothesis in the NR field that helix 12 exchanges between transcriptionally active and repressive conformations.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1987

Analysis of the fluid dynamics and heat transfer during micro-gravity Bridgman-Stockbarger growth of semiconductors in steady periodic gravitational fields

The effect of periodic variations in the magnitude and direction of gravitational acceleration on buoyancy-driven convection in the melt during microgravity growth of low Prandtl number semiconductor materials in Brigdman-Stockbarger configuration is investigated through the numerical solution of the transient Navier-Stokes and energy equations. Results are presented over a wide range of Rayleigh numbers and frequency spectrum of periodic g-level variations corresponding to conditions present on earth and on board spacecrafts. The frequency response of convection in the melt during growth is found to be similar to that of a first order dynamic system with a cutoff frequency determined by the diffusion of momentum in the system. Results indicate that during growth of small diameter Germanium crystals, convection in the melt is insensitive to the measured g-gitter on board the shuttle.

Griffin, Patrick R.↗