Characterization of the mammalian cell entry 1 (Mce1) complex of Mycobacterium tuberculosis

Mammalian cell entry (Mce) proteins are among the virulence-associated proteins of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). In Mtb, there are four homologous mce operons encoding four Mce complexes (Mce1, Mce2, Mce3, and Mce4). Each of these mce operons encodes two permease subunits (YrbEA-B), six substrate-binding proteins (SBPs; MceA-F), and up to four Mce-associated membrane (Mam) proteins. The permease subunits associate with the ATPase MceG (also known as Mkl) encoded elsewhere in the Mtb genome to form a functional ATP binding cassette (ABC)-transporter. Additionally, lipid uptake coordinator (LucA) and orphaned-Mam (Omam) proteins were identified to be involved in the stabilization of the Mce complexes. The current body of the literature suggests that the Mce1 complex is involved in the import of fatty acids including mycolic acid which can then be used as a source of carbon and energy and, in the homeostasis of the Mtb cell envelope. In this study, we performed structural characterization of the recombinant as well as the endogenous Mtb Mce1 complex.
Despite the challenges of expressing a large multi-membrane protein complex as well as the complexity of the genetic organization of Mce1 transporters, we successfully established a recombinant expression system that allowed the co-expression and co-purification of the 14 known components of the Mce1 complex. First, we demonstrated that the six Mce1 SBPs interact with each other forming the Mce1ABCDEF subcomplex and the helical domains play an essential role in their interaction. Furthermore, we recombinantly co-expressed 14 known components of the Mce1 complex and demonstrated the formation of the Mce1 complex in vitro. Single particle electron microscopy (EM) studies on recombinant Mce1ABCDEF subcomplex and the Mce1 complex were challenging due to the observed heterogeneity, low yields as well as their stability. However, the EM analysis of the endogenous Mce1 complex isolated from Mtb H37Ra revealed an elongated curved needle-like structure to which an AlphaFold generated Mce1 complex fitted well and agreed with the recently published structure of Mycobacterium smegmatis Mce1 complex by another group. The needle-like structure formed by the helical domains of Mce1A-1F shows a hydrophobic interior through which the lipids could be imported across the Mtb cell envelop.