Structural insights on the substrate-binding proteins of the Mycobacterium tuberculosis mammalian-cell-entry (Mce) 1 and 4 complexes

AbstractTuberculosis (Tb), caused by Mycobacterium tuberculosis (Mtb), is responsible for more than a million deaths annually. In the latent phase of infection, Mtb uses lipids as the source of carbon and energy for its survival. The lipid molecules are transported across the cell wall via multiple transport systems. One such set of widely present and less-studied transporters is the Mammalian-cell-entry (Mce) complexes. Here, we report the properties of the substrate-binding proteins (SBPs; MceA-F) of the Mce1 and Mce4 complexes from Mtb which are responsible for the import of mycolic acid/fatty acids, and cholesterol respectively. MceA-F are composed of four domains namely, transmembrane, MCE, helical and tail domains. Our studies show that MceA-F are predominantly monomeric when purified individually and do not form homohexamers unlike the reported homologs (MlaD, PqiB and LetB) from other prokaryotes. The crystal structure of MCE domain of Mtb Mce4A (MtMce4A39-140) determined at 2.9 Å shows the formation of an unexpected domain-swapped dimer in the crystals. Further, the purification and small-angle X-ray scattering (SAXS) analysis on MtMce1A, MtMce4A and their domains suggest that the helical domain requires hydrophobic interactions with the detergent molecules for its stability. Combining all the experimental data, we propose a heterohexameric arrangement of MtMceA-F SBPs, where the soluble MCE domain of the SBPs would remain in the periplasm with the helical domain extending to the lipid layer forming a hollow channel for the transport of lipids across the membranes. The tail domain would reach the cell surface assisting in lipid recognition and binding.

Download Full-text

Structural insights into the substrate-binding proteins Mce1A and Mce4A from Mycobacterium tuberculosis

IUCrJ ◽

10.1107/s2052252521006199 ◽

2021 ◽

Vol 8 (5) ◽

Author(s):

Pooja Asthana ◽

Dhirendra Singh ◽

Jan Skov Pedersen ◽

Mikko J. Hynönen ◽

Ramita Sulu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Structural Properties ◽

Binding Proteins ◽

Substrate Binding ◽

Cell Entry ◽

X Ray Scattering ◽

Solution Studies ◽

Hydrophobic Pore ◽

Saxs Analysis ◽

Structural Insights

Mycobacterium tuberculosis (Mtb), which is responsible for more than a million deaths annually, uses lipids as the source of carbon and energy for its survival in the latent phase of infection. Mtb cannot synthesize all of the lipid molecules required for its growth and pathogenicity. Therefore, it relies on transporters such as the mammalian cell entry (Mce) complexes to import lipids from the host across the cell wall. Despite their importance for the survival and pathogenicity of Mtb, information on the structural properties of these proteins is not yet available. Each of the four Mce complexes in Mtb (Mce1–4) comprises six substrate-binding proteins (SBPs; MceA–F), each of which contains four conserved domains (N-terminal transmembrane, MCE, helical and C-terminal unstructured tail domains). Here, the properties of the various domains of Mtb Mce1A and Mce4A, which are involved in the import of mycolic/fatty acids and cholesterol, respectively, are reported. In the crystal structure of the MCE domain of Mce4A (MtMce4A39–140) a domain-swapped conformation is observed, whereas solution studies, including small-angle X-ray scattering (SAXS), indicate that all Mce1A and Mce4A domains are predominantly monomeric. Further, structural comparisons show interesting differences from the bacterial homologs MlaD, PqiB and LetB, which form homohexamers when assembled as functional transporter complexes. These data, and the fact that there are six SBPs in each Mtb mce operon, suggest that the MceA–F SBPs from Mce1–4 may form heterohexamers. Also, interestingly, the purification and SAXS analysis showed that the helical domains interact with the detergent micelle, suggesting that when assembled the helical domains of MceA–F may form a hydrophobic pore for lipid transport, as observed in EcPqiB. Overall, these data highlight the unique structural properties of the Mtb Mce SBPs.

Download Full-text

Polyamine transport in bacteria and yeast

Biochemical Journal ◽

10.1042/bj3440633 ◽

1999 ◽

Vol 344 (3) ◽

pp. 633-642 ◽

Cited By ~ 110

Author(s):

Kazuei IGARASHI ◽

Keiko KASHIWAGI

Keyword(s):

Binding Proteins ◽

Hydrophobic Interactions ◽

Substrate Binding ◽

Site Directed Mutagenesis ◽

Transport Systems ◽

Uptake System ◽

Polyamine Transport ◽

Polyamine Content ◽

Methylene Groups ◽

Preferential Uptake

The polyamine content of cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, the genes for three different polyamine transport systems have been cloned and characterized. Two uptake systems (putrescine-specific and spermidine-preferential) were ABC transporters, each consisting of a periplasmic substrate-binding protein, two transmembrane proteins and a membrane-associated ATPase. The crystal structures of the substrate-binding proteins (PotD and PotF) have been solved. They consist of two domains with an alternating β-α-β topology, similar to other periplasmic binding proteins. The polyamine-binding site is in a cleft between the two domains, as determined by crystallography and site-directed mutagenesis. Polyamines are mainly recognized by aspartic acid and glutamic acid residues, which interact with the NH2- (or NH-) groups, and by tryptophan and tyrosine residues that have hydrophobic interactions with the methylene groups of polyamines. The precursor of one of the substrate binding proteins, PotD, negatively regulates transcription of the operon for the spermidine-preferential uptake system, thus providing another level of regulation of cellular polyamines. The third transport system, catalysed by PotE, mediates both uptake and excretion of putrescine. Uptake of putrescine is dependent on membrane potential, whereas excretion involves an exchange reaction between putrescine and ornithine. In Saccharomyces cerevisiae, the gene for a polyamine transport protein (TPO1) was identified. The properties of this protein are similar to those of PotE, and TPO1 is located on the vacuolar membrane.

Download Full-text

A structural and functional analysis of type III periplasmic and substrate binding proteins: their role in bacterial siderophore and heme transport

Biological Chemistry ◽

10.1515/bc.2011.012 ◽

2011 ◽

Vol 392 (1-2) ◽

Cited By ~ 32

Author(s):

Byron C.H. Chu ◽

Hans J. Vogel

Keyword(s):

Binding Proteins ◽

Substrate Binding ◽

Transport Systems ◽

Complex Structures ◽

Type Iii ◽

Heme Transport ◽

Periplasmic Binding Proteins ◽

Outer Membrane Transporter ◽

Α Helix ◽

Carboxy Terminal

AbstractInEscherichia colithe Fhu, Fep and Fec transport systems are involved in the uptake of chelated ferric iron-siderophore complexes, whereas in pathogenic strains heme can also be used as an iron source. An essential step in these pathways is the movement of the ferric-siderophore complex or heme from the outer membrane transporter across the periplasm to the cognate cytoplasmic membrane ATP-dependent transporter. This is accomplished in each case by a dedicated periplasmic binding protein (PBP). Ferric-siderophore binding PBPs belong to the PBP protein superfamily and adopt a bilobal type III structural fold in which the two independently folded amino and carboxy terminal domains are linked together by a single long α-helix of approximately 20 amino acids. Recent structural studies reveal how the PBPs of the Fhu, Fep, Fec and Chu systems are able to bind their corresponding ligands. These complex structures will be discussed and placed in the context of our current understanding of the entire type III family of Gram-negative periplasmic binding proteins and related Gram-positive substrate binding proteins.

Download Full-text