Abstract
The members of the avidin protein family are well known for their high affinity towards d-biotin and their structural stability. These properties make avidins a valuable tool for various biotechnological applications. In the present study, two avidin-like biotin-binding proteins (named streptavidin C1 and C2) from Streptomyces cinnamonensis were newly identified while exploring antifungal proteins against Fusarium oxysporum f. sp. cucumerinum. Streptavidin C1 reveals a low correlation (a sequence identity of approximately 64%) with all known streptavidins, whereas streptavidin C2 shares a sequence identity of approximately 94% with other streptavidins. Here, the crystal structures of streptavidin C1 in the mature form and in complex with biotin at 2.1 and 2.5 Å resolution, respectively, were assessed. The overall structures present similar tetrameric features with D 2 symmetry to other (strept)avidin structures. Interestingly, the long C-terminal region comprises a short α-helix (C-Lid; residues 169-179) andan extension C-terminal peptide (ECP; residues 180-191) which stretches into the biotin-binding sites of the same monomer. This ECP sequence (-180VTSANPPAS188-) is a newly defined biotin-binding site, which reduces the ability to bind to (strept)avidin family proteins. The novel streptavidin C1 could help in the development of an engineered tetrameric streptavidin with reduced biotin-binding capacity as well as other biomaterial tools.
Highlights
Avidin from chicken (Gallus gallus) egg white is a naturally glycosylated protein, whereas streptavidin is a nonglycosylated protein secreted by Streptomyces avidinii
Via protein identity analysis, the antifungal proteins were identified to be avidin-family proteins. To characterize these new avidin-like proteins, the full-length open reading frame (ORF) of streptavidin C1 (Met1–Glu191) from gDNA of S. cinnamonensis was cloned into the expression vector pET21a
Considering the sequence and structural similarities, we suggest that this extension C-terminal peptide (ECP) sequence (–180VTSANPPAS188–) is a newly defined biotinbinding site which reduces the binding ability of theavidin family proteins, with the –NP– sequence possibly acting as the key sequence in this binding site
Summary
Avidin from chicken (Gallus gallus) egg white is a naturally glycosylated protein, whereas streptavidin is a nonglycosylated protein secreted by Streptomyces avidinii. The discovery of highaffinity dimeric avidins from bacterial sources, including bacterial avidin-like proteins from Rhizobium etli (rhizavidin), Shewanella denitrificans (shwanavidin) and Hoeflea phototrophica DFL-43 (hoefavidin), has been reported, the high affinity for biotin was maintained in these proteins (Avraham et al, 2015; Helppolainen et al, 2007; Meir et al, 2009, 2012) Structural analyses of these proteins revealed that they maintain the unique basic features of the avidin family, in which each monomer displays a near-identical topology and quaternary structure, with similar interactions to the 1–4 interaction of the tetrameric avidins, forming a sandwichlike dimer. This novel member of the (strept)avidin family could be used in diverse biotin-based nanotechnologies or may act as a building block in the production of new bioinspired materials
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