Rasia, R. M. | bioRxiv Science

bioRxiv2025

The WRC domain of GRF transcription factors. Structure and DNA recognition

Growth-regulating factors (GRFs) belong to a plant-specific family of transcription factors which display important roles in plant growth and development. GRF transcriptional activity is finely tuned by regulatory processes involving post-transcriptional silencing exerted by miRNA396, and protein-protein interactions involving a family of co-transcriptional regulators known as GRF-interacting factors (GIFs). In this way, expression of GRF target genes is modulated by a highly complex interplay between GRF/GIF isoform diversity and expression patterns along with miR396 and GIF gradients throughout plant tissues. At the protein level, GRFs are composed of two highly conserved domains known as QLQ and WRC and a less conserved C-terminal trans-activation domain. Whereas QLQ mediates GRF-GIF interaction by forming a complex with SNH domains found in GIFs, the WRC has been proposed as a putative zinc finger domain responsible for target DNA recognition and nuclear import. However, the structural aspects governing GRF transcriptional activity and target recognition remain unknown. In this work, we applied bioinformatic and biophysical analysis to comprehensively characterize the structural features that modulate the biological function of this protein family with a focus on the WRC domain. We provide insights into the structure of the WRC domain in GRFs and explore the WRC features driving GRFs:DNA complex formation. These findings offer new insights into how WRC domains modulate the biological functions of GRFs, laying the groundwork for future studies on their structure-function relationship in gene regulation and development of plants.

biophysics↗

bioRxiv2024

Tertiary and quaternary structure remodeling by occupancy of the substrate binding pocket in a large glutamate dehydrogenase

Glutamate dehydrogenases (GDHs) catalyze the oxidative deamination of L-glutamate to 2-oxoglutarate using NAD(P)+ as a cofactor. The large type of GDHs (L-GDHs) displays a dynamic homotetrameric architecture that alternates between open and closed states. However, the catalytic mechanism and the functional relevance of the large conformational changes in L-GDHs remain poorly understood. Here, we use cryo-EM to investigate the structure and the conformational landscape of the mycobacterial L-GDH composed of 180 kDa subunits (mL-GDH180) when incubated with L-glutamate and NAD+. Classification of the heterogeneous population of tetramers reveals opening-closing motions and sorting of individual subunits resolves the occupancy of the cofactor and substrate binding pockets. Cryo-EM maps show that ligand binding to the glutamate binding pocket is accompanied by structural changes in a region approximately two nanometers away from the active site, leading to the formation of a previously undetected interaction between the catalytic domains of neighboring subunits in mL-GDH180 closed tetrameric states. Our findings indicate that the occupancy of the substrate binding site of mL-GDH180 is linked to a remodeling of both the tertiary and quaternary structure of the enzyme. STATEMENT FOR A BROADER AUDIENCEThis work reveals how the binding of L-glutamate and NAD+ reshapes the architecture of a large glutamate dehydrogenase, linking active site occupancy to long-range structural remodeling. By capturing previously unseen conformational transitions with cryo-electron microscopy, we provide insights into the molecular logic of enzyme function in mycobacteria. These findings establish a framework to understand how structural plasticity supports metabolic control.

molecular biology↗