Periplasmic dipeptide-binding protein
From Proteopedia
Introduction
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Pathogenic bacteria require several metal cofactors for enzymatic activity and, therein, performance of biochemical processes. As a result, these parasites have evolved mechanisms by which they can uptake essential nutrients from their host. Though many of these nutrients are present as ions in the cytosol of host cells or in the extracellular matrix of host tissue at various concentrations, thereby making sequestering these materials relatively simple, iron presents an interesting obstacle in terms of accessibility for bacteria. Iron in eukaryotes exists mainly in erythrocytes in the heme compound hemoglobin, though it also exists in storage compounds such as ferritin, lactoferrin, transferrin, and hemosiderin[1]. As a result, pathogens have evolved several means by which facilitated transport of heme and hemoglobin occurs for subsequent heme and hemoglobin degradation.
M. tuberculosis and Iron Uptake
Mycobacterium tuberculosis (Mtb) is a droplet-spread bacteria which causes tuberculosis. The bacterium lives and reproduces within the phagosomes of alveolar macrophages. In 2018 alone, nearly 1.5 million people died from tuberculosis, making it among the top 10 diseases in terms of mortality[2]. Being that iron is relatively scarce within alveolar macrophage phagosomes, Mtb has evolved intricate means by which iron is uptaken. The sheer number of genes dedicated to these processes is an indication of the complex evolution of this uptake. For instance, approximately 35 known genes in the Mtb genome are associated only with the production of salicylate-derivative iron siderophores termed mycobactins[3].
Heme Transport Into Mtb
Mtb has a two-membrane exterior, composed of a peptidoglycan exterior membrane and an interior cell membrane. Heme transport into the periplasmic space has been understood for some time in that several integral proteins used in the transport of heme from the extracellular matrix into the periplasmic space have been elucidated, specifically PPE36, PPE22, and PPE62[4]. DppA is a type of periplasmic binding protein specific to Mtb.
Periplasmic Binding Proteins (PBPs)
Periplasmic binding proteins (PBPs) are non-enzymatic receptors that bacteria use to sense small molecules such as carbohydrates, amino acids, and ions, and transport them into the cytoplasm[5]. These sorts of proteins are ubiquitous in both gram-negative and gram-positive bacteria, appearing in gram-positive bacteria as membrane-anchored lipoproteins[5]. The glucose/galactose binding protein () of E. coli is amongst the best studied of these proteins[6]. These proteins typically exhibit a “Venus fly-trap” appearance, consisting of two globular domains connected by a small hinge region[5]. The hinge-like appearance is evident in GBBP. These proteins often also work in conjunction with an ABC-binding cassette transporter which catalyzes the movement of the substance at hand across the cytoplasmic membrane.
Other Heme Binding PBPs
Researchers have elucidated a few other heme binding PBPs which are functionally similar to DppA of Mtb. ShuT of S. dysenteriae and PhuT of P. aeruginosa were among the earliest of these proteins to be elucidated in 2007[7]. A general mechanism has been proposed for the activity of these proteins, but these proteins differ significantly structurally from DppA, so it is unlikely the specific mechanism of these proteins relates to DppA[7].
DPP System in Mtb
The DPP system in Mtb is used for influx of heme across the cellular membrane. DppA is a member of the DPP system in Mtb. DppA or periplasmic dipeptide-binding protein or ABC transporter-associated periplasmic dipeptide-binding proteintransports heme across the periplasmic space of Mtb to the DppBCD transporter, which likely transfers the heme across the membrane as has been seen with other substrate-binding proteins of ABC transporters[4]. Research has shown the DPP complex is not involved in heme detoxification, but rather is involved in the import of heme across the cell membrane[4].
General Information about DppA
Bacterial DppA proteins have signature Sec signal peptides specific to heme binding[4]. Research indicates the Sec signal peptide present on DppA of Mtb must be present for heme binding to occur[4]. DppA exhibits a much lower dissociation constant than other PBPs, around ~1.5 uM. This is significantly less than functionally similar proteins such as H. influenzae’s HbpA or E. coli’s DppA (HbpAH. influenzae = ~655 uM and DppAE. coli = ~10 uM)[4].
Crystal Structure of DppA
Crystal structure was obtained at 1.27Å resolution, with R work/free = 12.8/16.5%[4]. The structure shows a globular, heart-like appearance. The tertiary structure is formed from two globular and mildly offset halves which are quite complementary. The two halves fold onto each other, similar to two shells of a mollusk.
“Clothespin Spring” α-helical Hinge
A connects the to the [4]. This is speculated to function similar to a “clothespin spring,” maintaining a closed conformation.
Role of Tetrapeptide Binding in Core
Between the two halves of the protein, buried inside the core, is a [4]. The function of this is not as of yet fully understood. The highly conserved residues in the peptide-binding pocket of DppA were mutated to alanine by researchers[4]. E. coli did not yield any D445 mutant protein, suggesting it did not fold and subsequently degraded[4]. E. coli did yield W442 mutant which, under spectroscopic analysis, appeared to bind and rapidly dissociate from heme[4]. This suggests that this residue perhaps plays a role in maintaining a specific flexibility of the DppA halves.
Solvent-Exposed Binding Sight
The CASTp software computed a solvent-accessible pocket on the closed conformation of the protein with a Richards’ solvent-accessible volume of 268Å[4]. Though this contains a few heme-binding residues, including , it is too small to accommodate heme. Normal mode analysis, though, showed that the first three lowest frequency modes produced a wide opening in the cleft which was brought on by a clamp-like, 10.7Å motion of the two halves, during which the halves slightly twisted in opposite directions. The generated pocket has a Richards’ solvent-accessible volume ~2583Å, which would be compatible with heme binding. Similarly, this conformation would place heme in bonding distance with several DppA residues such as .
Further Elucidation of the Binding Pocket through Specific Residues
Amino acids have been mutated experimentally to assess the effects of these specific residues on heme binding. H131 mutations resulted in a nonfunctional protein which would not fold. R179 mutations resulted in a crystalized protein nearly structurally identical to wild type DppA, with RMSD ~.11Å. After introduction to heme, spectroscopic findings showed that heme-binding abilities of the protein were abolished in the mutant, suggesting this residue plays a significant function in binding with heme.
Genetic Homology with Other PBPs
Other periplasmic binding proteins have been isolated and studied. DppA shares no homology with HemT of S. marcescens[4]. The Mtb rv3666c-rv3663c operon, though, does encode four proteins that share ~25-45% sequence similarity with DPP dipeptide transporter of E. coli, which similarly transports hemoglobin through the periplasmic space[4].
Structural Homology with Other PBPs
DppA is similar structurally to a few homologous proteins, especially to the S. typhimurium ortholog that superimposes the structure with an RMSD ~1.45Å[4].
3D structures of periplasmic dipeptide-binding protein
See ABC transporter 3D structures
References
- ↑ Ems, Thomas. “Biochemistry, Iron Absorption.” StatPearls [Internet]., U.S. National Library of Medicine, 21 Apr. 2019, www.ncbi.nlm.nih.gov/books/NBK448204/.
- ↑ “Tuberculosis (TB).” World Health Organization, World Health Organization, www.who.int/news-room/fact-sheets/detail/tuberculosis.
- ↑ Boelaert JR, Vandecasteele SJ, Appelberg R, Gordeuk VR. The effect of the host's iron status on tuberculosis. J Infect Dis. 2007 Jun 15;195(12):1745-53. doi: 10.1086/518040. Epub 2007 May 4. PMID:17492589 doi:http://dx.doi.org/10.1086/518040
- ↑ 4.00 4.01 4.02 4.03 4.04 4.05 4.06 4.07 4.08 4.09 4.10 4.11 4.12 4.13 4.14 4.15 Mitra A, Ko YH, Cingolani G, Niederweis M. Heme and hemoglobin utilization by Mycobacterium tuberculosis. Nat Commun. 2019 Sep 18;10(1):4260. doi: 10.1038/s41467-019-12109-5. PMID:31534126 doi:http://dx.doi.org/10.1038/s41467-019-12109-5
- ↑ 5.0 5.1 5.2 Borrok MJ, Zhu Y, Forest KT, Kiessling LL. Structure-based design of a periplasmic binding protein antagonist that prevents domain closure. ACS Chem Biol. 2009 Jun 19;4(6):447-56. PMID:19348466 doi:10.1021/cb900021q
- ↑ Contreras H, Chim N, Credali A, Goulding CW. Heme uptake in bacterial pathogens. Curr Opin Chem Biol. 2014 Apr;19:34-41. doi: 10.1016/j.cbpa.2013.12.014. Epub, 2014 Jan 4. PMID:24780277 doi:http://dx.doi.org/10.1016/j.cbpa.2013.12.014
- ↑ 7.0 7.1 Ho WW, Li H, Eakanunkul S, Tong Y, Wilks A, Guo M, Poulos TL. Holo- and apo-bound structures of bacterial periplasmic heme-binding proteins. J Biol Chem. 2007 Dec 7;282(49):35796-802. Epub 2007 Oct 9. PMID:17925389 doi:10.1074/jbc.M706761200
