Structural and Biochemical Analysis of OrfG: The VirB8-like Component of the Conjugative Type IV Secretion System of ICESt3 From Streptococcus thermophilus

Abstract

Conjugative transfer is a major threat to global health since it contributes to the spread of antibiotic resistance genes and virulence factors among commensal and pathogenic bacteria. To allow their transfer, mobile genetic elements including Integrative and Conjugative Elements (ICEs) use a specialized conjugative apparatus related to Type IV secretion systems (Conj-T4SS). Therefore, Conj-T4SSs are excellent targets for strategies that aim to limit the spread of antibiotic resistance. In this study, we combined structural, biochemical and biophysical approaches to study OrfG, a protein that belongs to Conj-T4SS of ICESt3 from Streptococcus thermophilus. Structural analysis of OrfG by X-ray crystallography revealed that OrfG central domain is similar to VirB8-like proteins but displays a different quaternary structure in the crystal. To understand, at a structural level, the common and the diverse features between VirB8-like proteins from both Gram-negative and -positive bacteria, we used an in silico structural alignment method that allowed us to identify different structural classes of VirB8-like proteins. Biochemical and biophysical characterizations of purified OrfG soluble domain and its central and C-terminal subdomains indicated that they are mainly monomeric in solution but able to form an unprecedented 6-mer oligomers. Our study provides new insights into the structural analysis of VirB8-like proteins and discusses the interplay between tertiary and quaternary structures of these proteins as an essential component of the conjugative transfer.

Structure of the enterococcal T4SS protein PrgL reveals unique dimerization interface in the VirB8 protein family

“…The only exception is the recently described OrfG, a VirB8 homolog from the Gram-positive Streptococcus thermophilus (Cappele et al, 2021). OrfG is shown to be able to form hexamers in vitro, albeit the equilibrium is heavily geared toward monomers (Cappele et al, 2021). PrgL on the other hand easily multimerizes in vitro by its NTF2-like domain alone, as shown by our SEC-MALS data.…”

“…While it is well known that VirB8-like proteins like to crystallize as dimers or trimers, higher order oligomers were only described in vivo and for full-length protein (Bailey et al, 2006;Fercher et al, 2016;Terradot et al, 2005). The only exception is the recently described OrfG, a VirB8 homolog from the Gram-positive Streptococcus thermophilus (Cappele et al, 2021). OrfG is shown to be able to form hexamers in vitro, albeit the equilibrium is heavily geared toward monomers (Cappele et al, 2021).…”

Application of the switchSENSE® technology for real-time study of pesticides interaction with biological molecules

“…Size exclusion chromatography-multiangle light scattering (SEC-MALS) analysis was performed on MiniDAWN TREOS II (Wyatt Technology, Toulouse, France) coupled to Superdex 200 10/300 Increase column mounted on ÄKTA-Purifier™ system (Cytiva, Uppsala, Sweden) for protein molecular mass and oligomerization determination according to previous work [14]. A covalent conjugate between BSA and a 48 mer ssDNA (complementary nanolever cNLB48) was prepared using the amine coupling kit provided by Dynamic Biosensors (Munich, Germany).…”