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Binding Specificity of EspG5 to PE25-PPE41 Proteins in Mycobacterium tuberculosis

Figure 1: EspG3 protein (from 4w4i)

Biological Function

Through specific binding factors, an EspG binds to its PE-PPE ligand to be secreted through the ESAT pathway.

Clinical Relevance

Though the ESAT-6 secretion system is poorly understood, it is known that PE-PPE proteins and EspG proteins influence virulence and pathogenicity of the infection.

General Structure and Function

The EspG₃ protein shown in Figure 1 has a mass of 33.7kD ^[1]. The protein's beta sheets (yellow) make up the core of the protein. The 2-fold pseudo-symmetry implied by this cartoon view of the secondary structure is not supported by sequence similarity. As a monomeric protein, this binds to its ligand with high specificity. A key beta sheet region on the will vary between EspG subtypes to influence what the random loop on the PE-PPE protein will interact with, since this region includes key residues that interact with the random coil of the PE-PPE ligand. The β-sheet core is surrounded by 8 alpha helices (red). One key will vary among different EspG proteins to stericly limit binding to PE-PPE ligand. Variations in the long can also impact binding to the EspG's ligand.

Structural Differences between EspG subtypes

Figure 2: EspG5 (blue) and EspG3 (yellow)

Featured in Figure 2 are the key differences found on the tertiary structure of EspG3 (yellow) with EspG5 (blue). The highlighted alpha helix (lower right) shows a difference in length between EspG3 and EspG5. This difference in length contributes to the steric hinderance when binding to a PE-PPE ligand. The random loop highlighted toward the bottom of this protein also varies in length between EspG proteins, and this influences ligand binding. The small random turn highlighted shows inconceivable difference to the EspG5 protein. The sequence similarity between EspG5 and EspG3 at the β-2 and β-3 strands is very low.

Excretion

EspG PE-PPE excretion is done through the ESX secretion pathway. Mycobacterium tuberculosis uses this ESX-1 secretion system to deliver virulence factors into host macrophage and monocyte white blood cells during the 'Mycobacterium tuberculosis' infection. Though the exact excreted protein is currently unknown, several different PE-PPE complexes and EspG complexes have been identified. In the image "Specificity of EspG Binding", it can be noted how the specificity of specific PE-PPE complexes only bind to certain EspG proteins. To exemplify this, notice the factors that influence binding in EspG5 with PE25-PPE41 and see the contrast when EspG3 attempts PE25-PPE41. ^[2]

"Specificity of EspG Binding"

Binding

The EspG-PE-PPE binding is highly specific. The specific pair we were looking at was the EspG5-PE25-PPE41 complex. A variety of binding factors influence the EspG that will bind to a specific PE-PPE. The secretion pathway carried out needs the coupled protein.

Residue Interactions:

The random loop on the cigar shaped PE-PPE ligand binds to the β2-β3 sheets on this EspG protein. The residues on the random turn in the PE-PPE protein are key for the specificity to the EspG protein. Coils between PE-PPE proteins vary greatly and influence binding affinity. Combined with its β-2 & β-3 interactions on the EspG protein, the EspG protein is PE-PPE specific. These make up the bulk of residue interactions in the complex. An EspG protein will not bind to its non-corresponding PE-PPE complex. The residue hinderance image shows the hypothetical consequences of a non-corresponding EspG-PE-PPE bound complex. Here we see specific amino acid residue contact hinderance between the Asp36 and Gln172 on the EspG, with Asn122, His2, Phe3 and Met1 on the PE25-PPE41 complex.

Residue Hinderance

Concavity:

The concave region on the C-terminal half of the EspG protein facilitates binding of the tip of the cigar shaped PE-PPE. The tight bind between the protein and ligand works with the hydrophobic effect to increase binding affinity for the specific EspG-PE-PPE complex. These hydrophobic regions are buried after the binding of PE-PPE to EspG, which exemplifies to the hydrophobic effect due to the increase in entropy of water.

Binding Pocket Residues

EspG5 can bind to PE25-PPE41 due to a hand full of amino acid interactions. Most notably we have a Pro51 on α-2 helix of the EspG5 protein. Also we have various contact residues on the random turn that interact with the β2-β3 subunit. Particularly between the Glu127 of the random turn on the PE-PPE ligand interacts with the Glu256 on the EspG protein. There are a few hydrophobic residues on the PE-PPE protein involved with binding affinity, the specific residues are .

Electrostatics: There is an overall negative charge on the PE-PPE complex, and the binding tip is only partially negative. On the EspG5 protein, the binding pocket is partially positive, which aids coupling of the EspG & PE-PPE complex. The other EspG proteins found in 'Mycobacterium tuberculosis' have different electrostatic pocket charges which prevent binding of the PE25-PPE41 ligand.

Electrostatics

Relevance

The binding affinity between EspG and PE-PPE ligands is needed for excretion into the ESAT-6 pathway in Mycobacterium tuberculosis. This pathway is of popular study of Mtb because it is linked to the virulence of the virus. Hindering this pathway has been found to induce apoptosis in cells infested with Mtb.