The schlafen core domain: from structure to function

Huber, Elisabeth (2018): The schlafen core domain: from structure to function. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

The immune system is a complex network of processes and structures to protect the organism against infectious diseases. All processes, which include the maturation of individual immune cells, must be strictly regulated to prevent malfunction. The schlafen protein family was recently reported to play key regulatory roles in T cell maturation and different subsets of individual schlafen family members are gradually upregulated during T cell maturation. The schlafen family belongs to the interferon-stimulated genes and various members play important roles in a plethora of cellular processes: regulation of the cell cycle, T cell quiescence, differentiation and proliferation of various cell types, tumorigenesis and inhibition of viral replication. All schlafen protein family members share the schlafen core domain, which is a highly conserved N- terminal region of approximately 340 amino acids. Based on sequence similarities, this region has been predicted to contain a divergent ATPase domain. Additionally, the longer schlafen protein members harbor a C-terminal domain with sequence homologies to superfamily 1 DNA/RNA helicases. Although the individual schlafen members play key roles in diverse cellular processes, they are poorly characterized in vitro and the underlying molecular mechanisms remain unknown. Since neither biochemical nor structural information were available, the overall goal of this study was to biochemically and structurally describe the schlafen proteins, and specifically their highly conserved schlafen core domain. Crystallographic studies performed during this thesis, revealed the structures of full length murine Schlafen 2 (Slfn2) and N-terminal human Schlafen 5 (SLFN51-336), which is the first high resolution structure of a human schlafen protein ever reported. Both crystal structures show a unique horseshoe shape with a novel fold harboring a highly conserved zinc finger. Interestingly, the schlafen core domain resembles no ATPase like fold and indeed, neither ATP-binding nor hydrolysis could be verified experimentally. Thus, structural information combined with experimental data prove that the schlafen core is no ATPase domain. Biochemical characterization discovered nucleic acid binding properties of the schlafen core domain. Binding affinities of SLFN51-336 increased in a length dependent manner for both, single-stranded DNA and RNA, although no preference for DNA or RNA could be identified. The highest affinity towards RNA was detected for a stem-loop structure and no binding for a RNA duplex, indicating that single-stranded regions are necessary for the interaction. The highest affinity of SLFN51-336 was measured with double-stranded DNA and the residues identified to be responsible for DNA binding were mapped to the zinc finger region by generating DNA-binding deficient mutants. Slfn2 was capable to bind both, DNA and RNA, with a 10 times higher affinity for both substrates than SLFN51-336. The 5S rRNA was identified by co-purification with Slfn2 as a potential nucleic acid substrate. Further biochemical investigations discovered a new function for SLFN51-336 and Slfn2, which is a 3’- 5’ exonuclease activity on single-stranded DNA in a metal ion-dependent, but ATP-independent manner. The importance of three conserved sites for DNA hydrolysis was confirmed by generating exonuclease-inactive mutants. However, the cleavage mechanism is not entirely solved and therefore a co-crystal structure of a substrate bound schlafen core domain would be of great benefit. Besides investigations of the schlafen core domain, preliminary biochemical studies of full length SLFN5 revealed increased nucleic acid binding properties towards DNA compared to SLFN51-336, as well as ATP-binding. These results imply that the predicted C-terminal helicase domain significantly contributes to DNA binding and is responsible for ATP turnover. Furthermore, 5.8 S rRNA was determined as potential substrate for full length SLFN5, since it was co-purified with SLFN5 from insect cells. Collectively, these results not only provide structural insights into the highly conserved slfn core domain, but also propose a new function on molecular basis for the schlafen protein family.

• 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: 6. November 2018
• 1. Bericht­erstatter:in: Hopfner, Karl-Peter