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

2011 ◽  
Vol 392 (1-2) ◽  
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.

scholarly journals Holo- and Apo-bound Structures of Bacterial Periplasmic Heme-binding Proteins

2007 ◽  
Vol 282 (49) ◽  
pp. 35796-35802 ◽  
Author(s):  
Winny W. Ho ◽  
Huiying Li ◽  
Suntara Eakanunkul ◽  
Yong Tong ◽  
Angela Wilks ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Binding Protein ◽  
Heme Iron ◽  
Shigella Dysenteriae ◽  
Class Iii ◽  
Heme Pocket ◽  
Heme Transport ◽  
Binding Domains ◽  
Periplasmic Binding Proteins ◽  
Common Architecture

An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an “in” position where it can coordinate the heme iron to an “out” orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg228 in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg228, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B12-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B12, compared with ligands for FhuD, a peptide siderophore.

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

2020 ◽  
Author(s):  
Pooja Asthana ◽  
Dhirendra Singh ◽  
Jan Skov Pedersen ◽  
Mikko J. Hynönen ◽  
Ramita Sulu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mammalian Cell ◽  
Binding Proteins ◽  
Hydrophobic Interactions ◽  
Substrate Binding ◽  
Mycolic Acid ◽  
Cell Entry ◽  
Transport Systems ◽  
Tail Domain ◽  
Helical Domain

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.

Atomic structures of periplasmic binding proteins and the high-affinity active transport systems in bacteria

1990 ◽  
Vol 326 (1236) ◽  
pp. 341-352 ◽  
Keyword(s):  
Ligand Binding ◽  
Active Transport ◽  
Binding Proteins ◽  
Osmotic Shock ◽  
Transport Systems ◽  
Binding Studies ◽  
Tertiary Structures ◽  
Atomic Structures ◽  
High Affinity ◽  
Periplasmic Binding Proteins

We have determined and refined the X-ray crystal structures of six periplasmic binding proteins that serve as initial receptors for the osmotic-shock sensitive, active transport of L-arabinose, D-galactose/D-glucose, maltose, sulphate, leucine/isoleucine/ valine and leucine. The tertiary structures and atomic interactions between proteins and ligands show common features that are important for understanding the function of the binding proteins. All six structures are ellipsoidal, consisting of two similar, globular domains. The ligand-binding site is located deep in the cleft between the two domains. Irrespective of the nature of the ligand (e.g. saccharide, sulphate dianion or leucine zwitterion), the specificities and affinities of the binding sites are achieved mainly through hydrogen-bonding interactions. Binding of ligands induces a large protein conformational change. Three different structures have been observed among the binding proteins: unliganded ‘open cleft´, liganded ‘open cleft’, and liganded ‘closed cleft5. Here we discuss the functions of binding proteins in the light of numerous crystallographic and ligand-binding studies and propose a mechanism for the binding protein-dependent, high-affinity active transport.

scholarly journals Polyamine transport in bacteria and yeast

1999 ◽  
Vol 344 (3) ◽  
pp. 633-642 ◽  
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.

scholarly journals Phosphorylation of the Periplasmic Binding Protein in Two Transport Systems for Arginine Incorporation in Escherichia coli K-12 Is Unrelated to the Function of the Transport System

1998 ◽  
Vol 180 (18) ◽  
pp. 4828-4833 ◽  
Author(s):  
Roberto T. F. Celis ◽  
Peter F. Leadlay ◽  
Ipsita Roy ◽  
Anne Hansen
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Transport System ◽  
Binding Proteins ◽  
Binding Protein ◽  
Transport Systems ◽  
Periplasmic Binding Protein ◽  
Transport Atpase ◽  
Periplasmic Binding Proteins ◽  
K 12

ABSTRACT In Escherichia coli K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system). Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system. The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins. Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R. T. F. Celis, J. Biol. Chem. 265:1787–1793, 1990). The gene encoding the transport ATPase enzyme (argK) has been cloned and sequenced. The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da). The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters. We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant. We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored. In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level. This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in E. coli K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.

scholarly journals The carboxy-terminal domain of Gs alpha is necessary for anchorage of the activated form in the plasma membrane.

1990 ◽  
Vol 111 (4) ◽  
pp. 1427-1435 ◽  
Author(s):  
Y Audigier ◽  
L Journot ◽  
C Pantaloni ◽  
J Bockaert
Keyword(s):  
Adenylate Cyclase ◽  
Binding Proteins ◽  
Alpha Subunit ◽  
Amino Terminus ◽  
Gtp Binding Proteins ◽  
Amino Terminal ◽  
Alpha Subunits ◽  
Gtp Binding ◽  
Carboxy Terminal ◽  
Gs Alpha

GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the alpha beta gamma-type. In the activated state, the alpha subunit dissociates from the beta gamma complex but remains anchored in the membrane. The alpha subunits of several GTP-binding proteins, such as Go and Gi, are myristoylated at the amino terminus (Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Proc. Natl. Acad. Sci. USA. 84:7493-7497). This hydrophobic modification is crucial for their membrane attachment. The absence of fatty acid on the alpha subunit of Gs (Gs alpha), the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gs alpha, we used a reconstitution model in which posttranslational addition of in vitro-translated Gs alpha to cyc- membranes (obtained from a mutant of S49 cell line which does not express Gs alpha) restores the coupling between the beta-adrenergic receptor and adenylate cyclase. The consequence of deletions generated by proteolytic removal of amino acid sequences or introduced by genetic removal of coding sequences was determined by analyzing membrane association of the proteolyzed or mutated alpha chains. Proteolytic removal of a 9-kD amino-terminal domain or genetic deletion of 28 amino-terminal amino acids did not modify the anchorage of Gs alpha whereas proteolytic removal of a 1-kD carboxyterminal domain abolished membrane interaction. Thus, in contrast to the myristoylated alpha subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gs alpha are required for association of this protein with the membrane.

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