From Proteopedia

Revision as of 23:10, 21 April 2020

Background

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 ions are present 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 in that it exists mainly in erythrocytes in the heme compound hemoglobin, though it also exists in storage compounds such as ferritin, lactoferrin, transferrin, and hemosiderin4. As a result, pathogens have evolved several means by which heme and hemoglobin can be uptaken by cells and degraded for abstraction of iron.

Mycobacterium 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 mortality7. 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, Mycobacterium tuberculosis have approximately 35 known genes alone associated only with the production of salicylate-derivative iron siderophores termed mycobactins1.

Heme Transport Into M. Tuberculosis

Mycobacterium Tuberculosis 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, relative to the recent developments pertaining to the DPP complex, 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. The protein involved in the movement of heme through the periplasmic space, though, was unknown until September 2019, when the structure of DppA was elucidated. DppA is a type of periplasmic binding protein specific to Mycobacterium Tuberculosum.

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 cytoplasm2. These sorts of proteins are ubiquitous in both gram-negative and gram-positive bacteria, appearing in gram-positive bacteria as membrane-anchored lipoproteins2. The glucose/galactose binding protein (GGBP) of E. Coli is amongst the best studied of these proteins3. These proteins typically exhibit a “Venus fly-trap” appearance, consisting of two gobular domains connected by a small hinge region2. 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. PhuT of Pseudomonas aeruginosa and ShuT of Shigella dysenteriae were among the earliest of these proteins to be elucidated in 20075. 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 DppA5.

DPP System in Mycobacterium Tuberculosis

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 transports 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. 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.

General Information about DppA

Bacterial DppA proteins have signature Sec signal peptides specific to heme binding6. Research indicates the Sec signal peptide present on DppAmtb must be present for heme binding to occur6. DppA exhibits a much lower dissociation constant than other PBPs, around ~1.5 uM. This is significantly less than functionally similar proteins such as Haemophilus influenzae’s HbpA or E. Coli’s DppA (HbpAHaemophilus = ~655 uM and DppAEC = ~10 uM).

Crystal Structure of DppA

Crystal structure was obtained at 1.27Å resolution, with Rwork/free = 12.8/16.5%. 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 flexible α-helical hinge connects the N-terminal half (residues 1-249) to the C-terminal half (residues 267-541)6. This is speculated to function similar to a “clothespin spring,” maintaining a closed conformation.

Tetrapeptide Core

Between the two halves of the protein, buried inside the core, is a tetrapeptide composed of Ser-Ser-Val-Thr. The function of this is not as of yet fully understood.

= 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 268Å6. Though this contains a few heme-binding residues, including His131 and Arg 179, 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 R179, H131, S134, E481, and L477.

Further Elucidation of the Binding Pocket through Specific Residues

Amino acids H131 and R179 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.

Role of Tetrapeptide Binding in Core

The highly conserved residues W442 and D445 in the peptide-binding pocket of DppA were mutated to alanine6. Coli did not yield any D445 mutant protein, suggesting it did not fold and subsequently degraded. E. Coli did yield W442 mutant which, under spectroscopic analysis, appeared to bind and rapidly dissociate from heme. This suggests that this residue perhaps plays a role in maintaining a specific flexibility of the DppA halves.

Genetic Homology with Other PBPs

Other periplasmic binding proteins have been isolated and studied. DppA shares no homology with HemT of S. marcescens6. The Mycobacterium tuberculosis 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 space6.

Structural Homology with Other PBPs

DppA is similar structurally to a few homologous proteins, especially to the Salmonella typhimurium ortholog that superimposes the structure with an RMSD ~1.45Å6.

References