Biology subjects

Valentin Gese, G.

Publications and source records attributed to Valentin Gese, G..

3 recordsLinked to original sources

bioRxiv2022

Structural inventory of cotranslational protein folding by the eukaryotic RAC complex

Folding of nascent chains emerging from the ribosome is a challenge in cellular protein homeostasis, which in eukaryotes is met by an Hsp70 chaperone triad directly binding at the ribosomal tunnel exit. The conserved ribosome-associated complex (RAC) consists of the non-canonical Hsp70 Ssz1 and the J-domain protein Zuotin (Zuo1), which in fungi acts together with the canonical Hsp70 protein Ssb. Here, we determined high-resolution cryo-electron microscopy structures of RAC bound to the 80S ribosome. RAC adopts two distinct conformations accommodating continuous ribosomal rotation by a flexible lever arm. The heterodimer is held together by a tight interaction between the Ssz1 substrate-binding domain (SBD) and the N-terminus of Zuo1, with additional contacts between the Ssz1 nucleotide-binding domain (NBD) and the Zuo1 J- and ZHD domains that form a rigid unit. The Zuo1 HPD-motif conserved in J-proteins is masked by the Ssz1 NBD, different from the canonical Hsp70 J-protein contact, however, allowing to position Ssb for activation by Zuo1. Our data provide the basis for understanding how RAC cooperates with Ssb at the ribosome in dynamic nascent chain interaction and protein folding.

biochemistry↗

bioRxiv2021

A dual allosteric pathway drives human mitochondrial Lon

The hexameric, barrel-forming, AAA+ protease Lon is critical for maintaining mitochondrial matrix protein homeostasis. Efficient substrate processing by Lon requires the coordinated action of six protomers. Despite Lons importance for human health, the molecular bases for Lons substrate recognition and processing remain unclear. Here, we use a combination of biochemistry and electron cryomicroscopy (cryo-EM) to unveil the structural and functional basis for full-length human mitochondrial Lons degradation of mitochondrial transcription factor A (TFAM). We show how opposing protomers in the Lon hexamer barrel interact through their N-terminal domains to give what resembles three feet above the barrel and help to form a triangular pore located just above the entry pore to the barrel. The interactions between opposing protomers constitute a primary allosteric regulation of Lon activity. A secondary allosteric regulation consists of an inter-subunit signaling element in the ATPase domains. By considering the ATP or ADP load in each protomer, we show how this dual allosteric mechanism in Lon achieves coordinated ATP hydrolysis and substrate processing. This mechanism enforces sequential anti-clockwise ATP hydrolysis resulting in a coordinated hand-over-hand translocation of the substrate towards the protease active sites.

biochemistry↗

bioRxiv2021

Structural basis for late maturation steps of the human mitoribosomal large subunit

Mitochondrial ribosomes (mitoribosomes) synthezise a critical set of proteins essential for oxidative phosphorylation. Therefore, their function is vital to cellular energy supply and mitoribosomal defects give rise to a large and diverse group of human diseases 1. The architecture of mitoribosomes is strikingly different from that of their bacterial and eukaryotic cytosolic counterparts and display high divergence between species 2-6. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determined the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5. This allowed us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis that involve normally short-lived assembly intermediates. Our structures provide important insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangments leading to PTC formation. Moreover, our data reveal an unexpected role for the elongation factor mtEF-Tu in mt-LSU assembly, in which mt-EF-Tu interacts with GTPBP5 in a manner similar to its interaction with tRNA during translational elongation. Together, our approaches provide detailed understanding of the last stages of mt-LSU biogenesis that are unique to mammalian mitochondria.

biochemistry↗