Characterization of the SAM-key – a conserved regulatory domain of the Fun30 nucleosome remodeler

Karl, Leonhard (2023): Characterization of the SAM-key – a conserved regulatory domain of the Fun30 nucleosome remodeler. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Cells need to constantly access their genetic material. However, in eukaryotic cells, DNA is compactly wrapped around nucleosomes and their presence poses a barrier for DNA transactions. To facilitate access, eukaryotes use ATP-driven molecular machines that dynamically shape chromatin structure, called nucleosome remodelers. Budding yeast Fun30 is the prototype member of the Fun30-SMARCAD1-ETL sub-family of nucleosome remodelers important for DNA repair and gene silencing. While the catalytic mechanism has been elucidated for several remodelers, for this family of single-subunit remodelers we lack mechanistic understanding. Here we report the discovery of the SAM-key, an evolutionary conserved domain with a sterile alpha motif (SAM)-like fold with one characteristic, long, protruding helix, using structure prediction, multiple sequence alignment and biochemical characterization. The SAM-key is crucial for Fun30 function, as deletion of the SAM-key from FUN30 in budding yeast leads to DNA repair and gene silencing defects similar to a deletion of FUN30. Biochemical and biophysical characterization of the SAM-key mutant in vitro showed similar folding and stability as wildtype Fun30 as well as wildtype-level binding to DNA and nucleosomes. However, the mutant is deficient in DNA-stimulated ATP hydrolysis as well as nucleosome sliding and eviction. Structure prediction using AlphaFold2 models interaction of the long helix of the SAM-key with protrusion I, a structural element of the conserved 2-lobed ATPase domain that controls catalytic activity in other remodelers. We verified the model and the interaction by crosslinking-mass spectrometry and mutation of the interface with a double point mutant Fun30-ICRR, which phenocopies the SAM-key deletion with defective ATPase activity and nucleosome remodeling. This confirms a regulatory role for the interaction of the SAM-key helix with protrusion I. Our data thereby demonstrate a central role of the SAM-key domain in mediating the activation of Fun30 catalytic activity, a new insight into the biology of this protein and highlighting the importance of allosteric activation for nucleosome remodelers.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 1. März 2023
• 1. Bericht­erstatter:in: Conti, Elena