Conjugative Coupling Proteins and the Role of Their Domains in Conjugation, Secondary Structure and in vivo Subcellular Location.

Itxaso Álvarez-Rodríguez,José Luis R Arrondo,Carlos Garbisu,Begoña Ugarte-Uribe,Itziar Alkorta,Igor De La Arada

doi:10.3389/fmolb.2020.00185

Itxaso Álvarez-Rodríguez, José Luis R Arrondo + Show 4 more

Open Access

https://doi.org/10.3389/fmolb.2020.00185

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Abstract

Type IV Coupling Proteins (T4CPs) are essential elements in many type IV secretion systems (T4SSs). The members of this family display sequence, length, and domain architecture heterogeneity, being the conserved Nucleotide-Binding Domain the motif that defines them. In addition, most T4CPs contain a Transmembrane Domain (TMD) in the amino end and an All-Alpha Domain facing the cytoplasm. Additionally, a few T4CPs present a variable domain at the carboxyl end. The structural paradigm of this family is TrwBR388, the T4CP of conjugative plasmid R388. This protein has been widely studied, in particular the role of the TMD on the different characteristics of TrwBR388. To gain knowledge about T4CPs and their TMD, in this work a chimeric protein containing the TMD of TraJpKM101 and the cytosolic domain of TrwBR388 has been constructed. Additionally, one of the few T4CPs of mobilizable plasmids, MobBCloDF13 of mobilizable plasmid CloDF13, together with its TMD-less mutant MobBΔTMD have been studied. Mating studies showed that the chimeric protein is functional in vivo and that it exerted negative dominance against the native proteins TrwBR388 and TraJpKM101. Also, it was observed that the TMD of MobBCloDF13 is essential for the mobilization of CloDF13 plasmid. Analysis of the secondary structure components showed that the presence of a heterologous TMD alters the structure of the cytosolic domain in the chimeric protein. On the contrary, the absence of the TMD in MobBCloDF13 does not affect the secondary structure of its cytosolic domain. Subcellular localization studies showed that T4CPs have a unipolar or bipolar location, which is enhanced by the presence of the remaining proteins of the conjugative system. Unlike what has been described for TrwBR388, the TMD is not an essential element for the polar location of MobBCloDF13. The main conclusion is that the characteristics described for the paradigmatic TrwBR388 T4CP should not be ascribed to the whole T4CP family. Specifically, it has been proven that the mobilizable plasmid-related MobBCloDF13 presents different characteristics regarding the role of its TMD. This work will contribute to better understand the T4CP family, a key element in bacterial conjugation, the main mechanism responsible for antibiotic resistance spread.

Highlights

Type IV coupling proteins (T4CPs) are essential elements in the conjugative type IV secretion systems (T4SSs) and are key elements in many pathogenic T4SSs
Published papers have highlighted the role of the Transmembrane Domain (TMD) on different characteristics of TrwBR388, such as plasmid conjugation (Moncalián et al, 1999), subcellular localization (Segura et al, 2014), nucleotide-binding (Hormaeche et al, 2006), hexamerization (Hormaeche et al, 2002; Matilla et al, 2010), protein stability (Hormaeche et al, 2004), interaction with other proteins of the T4SS of R388 (Segura et al, 2013), and ATP hydrolase activity (Tato et al, 2005, 2007)
Several studies performed with TrwBR388, the full-length T4CP of the conjugative plasmid R388, have proven that the TMD is more than a mere anchor to the membrane and that it has a role in protein activity, stability, oligomerization and subcellular localization (Moncalián et al, 1999; Hormaeche et al, 2002, 2004, 2006; Vecino et al, 2010, 2011; Segura et al, 2013, 2014)

Summary

Introduction

Type IV coupling proteins (T4CPs) are essential elements in the conjugative type IV secretion systems (T4SSs) and are key elements in many pathogenic T4SSs. TrwBR388 is the only full-length T4CP that has been successfully purified to date (Hormaeche et al, 2002; Redzej et al, 2017), while trials for purifying other membrane T4CPs have not rendered the sufficient amounts of high quality protein for performing in vitro assays (Chen et al, 2008). For this reason most of the in vitro studies of T4CPs have been achieved using deletion mutant proteins that lack the TMD (Schroder and Lanka, 2003; Tato et al, 2007; Larrea et al, 2017). The TMD deletion mutant protein of TrwBR388, TrwB N70, was resolved by X-ray crystallography, showing that the CD of TrwBR388 contains an NBD with the Walker A and Walker B motifs and a small membrane-distal All-Alpha Domain (AAD) (Gomis-Rüth et al, 2001)

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