Structure of a translocation signal domain mediating conjugative transfer by type IV secretion systems.

Adam Redzej,Christian J Gruber,Maya Topf,Klaus Zangger,Aravindan Ilangovan,Silvia Lang,Ellen L Zechner,Gabriel Waksman

doi:10.1111/mmi.12275

Abstract

Relaxases are proteins responsible for the transfer of plasmid and chromosomal DNA from one bacterium to another during conjugation. They covalently react with a specific phosphodiester bond within DNA origin of transfer sequences, forming a nucleo-protein complex which is subsequently recruited for transport by a plasmid-encoded type IV secretion system. In previous work we identified the targeting translocation signals presented by the conjugative relaxase TraI of plasmid R1. Here we report the structure of TraI translocation signal TSA. In contrast to known translocation signals we show that TSA is an independent folding unit and thus forms a bona fide structural domain. This domain can be further divided into three subdomains with striking structural homology with helicase subdomains of the SF1B family. We also show that TSA is part of a larger vestigial helicase domain which has lost its helicase activity but not its single-stranded DNA binding capability. Finally, we further delineate the binding site responsible for translocation activity of TSA by targeting single residues for mutations. Overall, this study provides the first evidence that translocation signals can be part of larger structural scaffolds, overlapping with translocation-independent activities.

Highlights

Type IV secretion systems (T4SS) are protein complexes spanning bacterial cell membranes (Alvarez-Martinez and Christie, 2009; Wallden et al, 2010; Zechner et al., 2012)
They can be divided into three subclasses: (i) the effector protein translocation T4SS which are responsible for the delivery of effector proteins into the cytoplasm of eukaryotic cells (Terradot and Waksman, 2011), (ii) the conjugative systems which transfer DNA or nucleoprotein complexes from a donor to a recipient strain in a cell contact-dependent manner (Fronzes et al, 2009), and (iii) T4SS which release DNA into or mediate uptake from the extracellular milieu (Hamilton et al, 2005)
T4SS in Gram-negative bacteria are typically composed of 12 proteins termed VirB1-11 and VirD4 based on the prototypical Agrobacterium tumefaciens T-DNA delivery system

Summary

Introduction

Type IV secretion systems (T4SS) are protein complexes spanning bacterial cell membranes (Alvarez-Martinez and Christie, 2009; Wallden et al, 2010; Zechner et al., 2012). They are used to transport biomolecules such as proteins, nucleic acids and nucleoprotein complexes across the cell envelope. T4SS substrates are thought to be recruited to the VirB machinery by the VirD4 ATPase, a protein which couples substrate recruitment to secretion and commonly referred to as the type IV coupling protein (T4CP) (Cabezón et al, 1997; Schröder and Lanka, 2003; Alvarez-Martinez and Christie, 2009). ATPase activity is associated with the release and unfolding of complexes between substrates and specific chaperones and is required to energize the secretion process

Results

Discussion

Conclusion