Biology subjects

Golovenko, D.

Publications and source records attributed to Golovenko, D..

3 recordsLinked to original sources

bioRxiv2025

Structural mechanism of mRNA decoding by mammalian GTPase GTPBP1

GTP-binding protein 1 (GTPBP1) is a widespread translational GTPase closely related to elongation factor eEF1A. The loss of GTPBP1 leads to errors in neuronal development in animals and is associated with neurodegenerative disorders in humans. Although linked to translation and quality control mechanisms, GTPBP1 functions remain largely obscure. Similarly to eEF1A, GTPBP1 delivers cognate aminoacyl-tRNA to the ribosomal A site in a GTP-dependent manner, but GTP hydrolysis is not followed by rapid peptide bond formation, and GTPBP1-mediated elongation is slow. To establish the basis for GTPBP1 function, we determined cryo-EM structures of 80S ribosomal complexes bound to GTPBP1*aa-tRNA with GTP or the non-hydrolysable analog GDPCP. They revealed that the unique GTPBP1 architecture, including the additional eIF1/IF3-like N-terminal domain and the shoulder-interacting H-loop in place of the 2 helix of canonical GTPases, is responsible for establishing GTPBP1-specific interactions with tRNA and the ribosome, leading to slow GTPBP1 dissociation after GTP hydrolysis and thus delayed tRNA accommodation. Slow dissociation correlates with an extended proofreading stage resulting in higher accuracy of GTPBP1-mediated decoding and potentially allows GTPBP1 to elicit its putative quality control functions. GTPBP1 visualization provides the foundation for mapping and elucidating GTPBP1 mutations associated with human diseases.

biochemistry↗

bioRxiv2024

Cryo-EM Structure of HRSL Domain Reveals Activating Crossed Helices at the Core of GCN2

GCN2 is a conserved receptor kinase activating the Integrated Stress Response (ISR) in eukaryotic cells. The ISR kinases detect accumulation of stress molecules and reprogram translation from basal tasks to preferred production of cytoprotective proteins. GCN2 stands out evolutionarily among all protein kinases due to the presence of a histidyl tRNA synthetase-like (HRSL) domain, which arises only in GCN2 and is located next to the kinase domain. How HRSL contributes to GCN2 signaling remains unknown. Here we report a 3.2 [A] cryo-EM structure of HRSL from thermotolerant yeast Kluyveromyces marxianus. This structure shows a constitutive symmetrical homodimer featuring a compact helical-bundle structure at the junction between HRSL and kinase domains, in the core of the receptor. Mutagenesis demonstrates that this junction structure activates GCN2 and indicates that our cryo-EM structure captures the active signaling state of HRSL. Based on these results, we put forward a GCN2 regulation mechanism, where HRSL drives the formation of activated kinase dimers. Remaining domains of GCN2 have the opposite role and in the absence of stress they help keep GCN2 basally inactive. This autoinhibitory activity is relieved upon stress ligand binding. We propose that the opposing action of HRSL and additional GCN2 domains thus yields a regulated ISR receptor. Significance statementRegulation of protein synthesis (translation) is a central mechanism by which eukaryotic cells adapt to stressful conditions. In starving cells, this translational adaptation is achieved via the receptor kinase GCN2, which stays inactive under normal conditions, but is switched on under stress. The molecular mechanism of GCN2 switching is not well understood due to the presence of a structurally and biochemically uncharacterized histidyl tRNA synthetase-like domain (HRSL) at the core of GCN2. Here we use single-particle cryo-EM and biochemistry to elucidate the structure and function of HRSL. We identify a structure at the kinase/HRSL interface, which forms crossed helices and helps position GCN2 kinase domains for activation. These data clarify the molecular mechanism of GCN2 regulation.

biochemistry↗

bioRxiv2022

Structural Basis of MicroRNA Biogenesis by Dicer-1 and Its Partner Protein Loqs-PB

In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 and its partner Loqs-PB. The structures show Dicer-1*Loqs-PB (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer-1*Loqs-PB heterodimer, enabling conformational proofreading. The Dicer-1 dsRBD and three Loqs-PB dsRBD domains form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-mRNA cleavage shifts the RNA- binding domains and tightens Dicer-1, promoting product release. Our data suggest a model for how the Dicer-1*Loqs-PB complex effects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and sequential product release.

molecular biology↗