Structural and functional characterization of TraI from pKM101 reveals basis for DNA processing

Introduction

Antibiotic resistance is one of the most pressing health challenges in today’s world. One of the main drivers of this worldwide problem is the ability of pathogens to spread resistance genes using horizontal gene transfer. This is a process that is often facilitated by conjugative type 4 secretion systems (T4SSs) (Juhas, 2015; Gonzalez-Rivera et al, 2016). These are megadalton-sized, multicomponent systems that transport DNA and proteins from a bacterial donor cell into a recipient cell (Grohmann et al, 2018; Waksman, 2019). In Gram-negative bacteria, T4SS consist of a channel that is made up of an inner membrane complex that is connected with an outer-membrane core complex by a stalk (Low et al, 2014; Chandran Darbari & Waksman, 2015; Costa et al, 2021). Other important features are the pilus, which extends out from the cell and is involved in mediating attachment to recipient cells, and the type IV coupling protein, an ATPase that helps to recruit the single-stranded DNA substrate which is coupled to a relaxase (Arutyunov & Frost, 2013; Grohmann et al, 2018). In recent years, researchers have generated structural and functional insight into these systems, especially from Gram-negative (G−) bacteria, among others the R388 plasmid and the pKM101 system (Khara et al, 2021; Macé et al, 2022). For a recent review of the structure and function of T4SSs, please see Costa et al (2021). The focus on this work is the T4SS of pKM101, which belongs to the class of minimal T4SS that consist of 12 proteins homologous to the paradigmatic VirB/VirD4 T4SS from Agrobacterium tumefaciens (Christie, 2004, 2016).

For all known T4SSs that transfer conjugative plasmids, the plasmid DNA must be processed before it can be transported. This is done via the relaxosome complex, which consists of a relaxase, accessory factor protein(s), and the DNA (Grohmann et al, 2018). To form this complex, one or several accessory proteins bind to the origin of transfer (oriT) on the DNA and locally melt the double-stranded DNA to promote relaxase binding to a defined sequence, often forming a hairpin, close to the nicking site (Zechner et al, 2017). The relaxase binds this single-stranded oriT DNA via its N-terminal trans-esterase domain (Garcillán-Barcia et al, 2009). This domain reacts with the DNA via a transesterification reaction at the specific nic-site, which generates the transfer intermediate consisting of the relaxase covalently bound to the 5′ end of the cleaved transfer strand (T-strand) (Byrd & Matson, 1997). Many relaxases have a second functional domain in the more variable C-terminal part of the enzyme. This is often a helicase domain, which unwinds the DNA to allow for transport of the single-stranded transfer-strand DNA (Garcillán-Barcia et al, 2009). The relaxase-transfer-strand complex (T-complex) is recruited to the T4SS by the type IV coupling protein and is transported through the T4SS channel into the recipient cell (Alvarez-Martinez & Christie, 2009). Once present in the recipient cell, the trans-esterase domain religates the DNA to regenerate the circularized plasmid (Waksman, 2019).

Relaxases have been phylogenetically classified into eight MOB families: MOBF, MOBH, MOBQ, MOBC, MOBP, MOBV, MOBT, and MOBB. Of these, pKM101-encoded TraI (TraIpKM101) belongs to the MOBF11 subclade together with its closest relative TrwC from plasmid R388 (TrwCR388) and the identical TraI from the sister plasmid pCU1 (TraIpCU1) (Paterson et al, 1999; Garcillán-Barcia et al, 2009; Guglielmini et al, 2011). Although structural information is available for the trans-esterase domains of several MOBF relaxases (De La Cruz et al, 2010; Zechner et al, 2017), structural data for the substrate DNA-bound state is only available for TrwCR388 and TraI from the F-plasmid (TraIF), which belongs to subclade MOBF12 (Larkin et al, 2005; Boer et al, 2006). Furthermore, there is only very limited data on full-length relaxases, again mostly from TraIF. TraIF consists of one trans-esterase domain, a vestigial helicase domain, and an active helicase domain (Fig 1A) and binds oriT DNA as a heterogenous dimer. One TraIF monomer adapts an open conformation and binds oriT at a hairpin positioned 5′ of the nic-site with the trans-esterase domain, whereas a second TraIF binds ssDNA with the helicase domains in a closed conformation. These two states are incompatible with each other in a single monomer (Ilangovan et al, 2017). A cryo-EM structure of the helicase-bound state is available and shows how the single-stranded DNA is almost entirely surrounded by the helicase domains. This structure is of full-length TraIF and visualizes all of the protein except the very flexible C-terminal domain (Ilangovan et al, 2017). It is not clear to what extent this structure and mode of action apply to TraIpKM101 as it is much shorter and completely lacks the vestigial helicase domain (Fig 1A).

In this study, we present the crystal structure of the trans-esterase domain of TraIpKM101 bound to its substrate oriT DNA. This structure confirms the highly conserved DNA-binding mode of the trans-esterase domain observed in other MOBF family relaxases. We also present the apo structure with the flexible thumb-subdomain defined, which shows a large conformational change between the apo and substrate-bound structures. TraI is further characterized via electrophoretic mobility shift assays (EMSAs) and nicking and religation assays.