Impact of C-terminal domains of paralogous single-stranded DNA binding proteins from Streptomyces coelicolor on their biophysical properties and biological functions

Pipalović, Goran; Filić, Želimira; Čehić, Mirsada; Paradžik, Tina; Zahradka, Ksenija; Crnolatac, Ivo; Vujaklija, Dušica (2024) Impact of C-terminal domains of paralogous single-stranded DNA binding proteins from Streptomyces coelicolor on their biophysical properties and biological functions. International Journal of Biological Macromolecules, 268 (1). p. 131544. ISSN 0141-8130

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Abstract

Single-stranded DNA-binding proteins (SSB) are crucial in DNA metabolism. While Escherichia coli SSB is extensively studied, the significance of its C-terminal domain has only recently emerged. This study explored the significance of C-domains of two paralogous Ssb proteins in S. coelicolor. Mutational analyses of C-domains uncovered a novel role of SsbA during sporulation-specific cell division and demonstrated that the C-tip is non-essential for survival. In vitro methods revealed altered biophysical and biochemical properties of Ssb proteins with modified C-domains. Determined hydrodynamic properties suggested that the C-domains of SsbA and SsbB occupy a globular position proposed to mediate cooperative binding. Only SsbA was found to form biomolecular condensates independent of the C-tip. Interestingly, the truncated C-domain of SsbA increased the molar enthalpy of unfolding. Additionally, calorimetric titrations revealed that C-domain mutations affected ssDNA binding. Moreover, this analysis showed that the SsbA C-tip aids binding most likely by regulating the position of the flexible C-domain. It also highlighted ssDNA-induced conformational mobility restrictions of all Ssb variants. Finally, the gel mobility shift assay confirmed that the intrinsically disordered linker is essential for cooperative binding of SsbA. These findings highlight the important role of the C-domain in the functioning of SsbA and SsbB proteins.

Item Type:	Article
Uncontrolled Keywords:	intrinsically disordered linker (IDL); S. coelicolor; SsbA/SsbB proteins
Subjects:	NATURAL SCIENCES NATURAL SCIENCES > Chemistry > Physical Chemistry NATURAL SCIENCES > Chemistry > Applied Chemistry NATURAL SCIENCES > Biology > Biochemistry and Molecular Biology NATURAL SCIENCES > Biology > Microbiology TECHNICAL SCIENCES INTERDISCIPLINARY AREAS OF KNOWLEDGE
Divisions:	Division of Molecular Biology Division of Organic Chemistry and Biochemistry Division of Physical Chemistry
Projects:	Project title Project leader Project code Project type Identifikacija interaktoma paralognih proteina SSB u višestaničnom prokariotu, Streptomyces coelicolor-id_ROBUST Dušica Vujaklija IP-2018-01-1754 HRZZ
Depositing User:	Ivo Crnolatac
Date Deposited:	08 Jan 2026 08:37
URI:	http://fulir.irb.hr/id/eprint/10738
DOI:	10.1016/j.ijbiomac.2024.131544

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