ClbM is a versatile, cation-promiscuous MATE transporter found in the colibactin biosynthetic gene cluster

Highlights

• ClbM is a cation promiscuous MATE multidrug transporter.

• The role of key residues were identified in both the cation and proton binding.

• The biologically relevant substrate for ClbM is the natural product precolibactin.

Abstract

Multidrug transporters play key roles in cellular drug resistance to toxic molecules, yet these transporters are also involved in natural product transport as part of biosynthetic clusters in bacteria and fungi. The genotoxic molecule colibactin is produced by strains of virulent and pathobiont Escherichia coli and Klebsiella pneumoniae. In the biosynthetic cluster is a multidrug and toxic compound extrusion protein (MATE) proposed to transport the prodrug molecule precolibactin across the cytoplasmic membrane, for subsequent cleavage by the peptidase ClbP and cellular export. We recently determined the X-ray structure of ClbM, and showed preliminary data suggesting its specific role in precolibactin transport. Here, we define a functional role of ClbM by examining transport capabilities under various biochemical conditions. Our data indicate ClbM responds to sodium, potassium, and rubidium ion gradients, while also having substantial transport activity in the absence of alkali cations.

Introduction

Multidrug transporters generally serve to remove toxic molecules from cellular environments, a process important to cell health and survival. However, these transporters can also mediate bacterial and cancer cell multidrug resistance to antibiotics and chemotherapeutic agents [1], [2]. Six distinct protein families harbor the known multidrug transporters [2], including the ATP-binding cassette superfamily (ABC), major facilitator superfamily (MFS), small multidrug resistance family, resistance nodulation division (RND) superfamily, multidrug and toxic compound extrusion family (MATE), and the proteobacterial antimicrobial compound efflux (PACE) family [3].

The MATE transporter family has been characterized recently [4] and growing evidence links the group to multidrug resistance in multiple systems [5], [6], [7], [8]. For example, human MATE transporters have been shown to export platinum-containing chemotherapeutics and additional members are directly involved in multidrug transport in diverse species [8]. Despite being broadly implicated in resistance mechanisms, relatively few structures of MATE transporters have been reported [9], [10], [11], [12], [13], [14]. We recently described a novel MATE transporter encoded by the clbM gene from the colibactin biosynthetic gene cluster (pks gene island) from certain strains of Escherichia coli [14]. The genotoxin colibactin is implicated in double-stranded DNA breaks and neoplastic changes [15], [16], [17], [18]. Colibactin is biosynthesized as an N-myristoyl-d-asparagine capped prodrug [19], [20], and the molecular structure of the active colibactin has been partially defined [17], [21], [22], [23], [24], [25]. Furthermore, a basis for DNA cross-linking by the colibactin “warhead” has been proposed [17]. We recently showed that the MATE transporter ClbM facilitates export of the prodrug scaffold precolibactin across the cytoplasmic membrane [14]. ClbM is conserved across pks gene islands of multiple strains of E. coli such as NC101 and Nissle 1917, and strains of Klebsiella pneumonia (Fig. 1A).

Including ClbM, five unique MATE structures have been determined [9], [10], [11], [12], [13], [14], and all share a conserved 12-transmembrane helical topology (Fig. 1B). PfMATE crystallized in two distinct conformations, whereby the N-terminal transmembrane helix (TH1) adopts a “straight” conformation at neutral pH, and a “bent” conformation at acidic pH [12]. This structural shift is hypothesized to be due to protonation of an aspartate residue on TH1 that is conserved among MATE transporters. This conserved residue has also been shown to be important for transporter function in the H⁺-coupled MATE transporter DinF [13]. ClbM has closest homology to PfMATE, a proton-coupled antiporter from P. furiosis. Despite the similarity, we characterized ClbM as a Na⁺-coupled transporter [14]. The Na⁺-coupled transporters NorM-VC and NorM-NG were crystallized in complex with Rb⁺ and Cs⁺, respectively, to define the cation binding site. NorM-VC utilizes π-cation interactions via two Phe residues to bind Rb⁺, and a nearby Asp is hypothesized to also interact upon drug binding [9]. NorM-NG utilizes similar π-cation interactions in addition to electrostatic interactions with a Glu residue to bind the cation with specificity [10]. Our initial predictions regarding the Na⁺-dependency of ClbM were based on the co-complex structure with Rb⁺ [14], whereby a cation-coupled transport mechanism was hypothesized based on comparison with the cation-binding pockets of NorM-VC and NorM-NG.