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Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16.

Barathy DV, Suguna K - FEBS Open Bio (2013)

Bottom Line: The three-dimensional structure of one of them, Rv0977 (PE_PGRS16) of M. tuberculosis revealed the characteristic pepsin-fold and catalytic site architecture.However, the active site was completely blocked by the N-terminal His-tag.A comparison of the structure with pepsins showed significant differences in the critical substrate binding residues and in the flap tyrosine conformation that could contribute to the lack of proteolytic activity of Rv0977.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.

ABSTRACT
We report the crystal structure of the first prokaryotic aspartic proteinase-like domain identified in the genome of Mycobacterium tuberculosis. A search in the genomes of Mycobacterium species showed that the C-terminal domains of some of the PE family proteins contain two classic DT/SG motifs of aspartic proteinases with a low overall sequence similarity to HIV proteinase. The three-dimensional structure of one of them, Rv0977 (PE_PGRS16) of M. tuberculosis revealed the characteristic pepsin-fold and catalytic site architecture. However, the active site was completely blocked by the N-terminal His-tag. Surprisingly, the enzyme was found to be inactive even after the removal of the N-terminal His-tag. A comparison of the structure with pepsins showed significant differences in the critical substrate binding residues and in the flap tyrosine conformation that could contribute to the lack of proteolytic activity of Rv0977.

No MeSH data available.


Related in: MedlinePlus

Surface representation of the active site of (A) Mtb-AP and (B) rhizopuspepsin (PDB code: 3APR). A modified peptide inhibitor of rhizopuspepsin is shown at a structural equivalent site in Mtb-AP. Ile193 of Mtb-AP which forms the mouth of S3 pocket is shown with arrow.
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fig0009: Surface representation of the active site of (A) Mtb-AP and (B) rhizopuspepsin (PDB code: 3APR). A modified peptide inhibitor of rhizopuspepsin is shown at a structural equivalent site in Mtb-AP. Ile193 of Mtb-AP which forms the mouth of S3 pocket is shown with arrow.

Mentions: A comparison of the structure of Mtb-AP with its structural equivalents human pepsin (PDB code: 1PSO) and rhizopuspepsin (PDB code: 3APR) in complex with inhibitors showed that the subsites of Mtb-AP appear to be similar to those of reported pepsins [28,29] with some small but significant differences (Fig. 8 and Table S2). S1: The flap residue tyrosine (Tyr64) along with glycine (Gly66) is well conserved in the family of eukaryotic aspartic proteinases. Mtb-AP has an additional alanine (Ala65) between Tyr64 and Gly66 (Fig. S4). A phenylalanine insertion corresponding to Ala65 of Mtb-AP was observed in the case of cockroach allergen Bla g 2 which was found to be inactive [30]. When pepstatin was docked in the binding pocket of Mtb-AP, the Ala65 side chain and the flap Tyr64 were found to make short contacts with it. The side chain of the conserved flap tyrosine has a different conformation (χ1 = 173°) when compared to that (χ1 = −54° in porcine pepsin) of eukaryotic aspartic proteinases (Fig. S5A). This conformation is stabilized by the hydrogen bond between tyrosine hydroxyl group and the side chain of the catalytic Asp36. Similar conformation of tyrosine was also observed in the crystal structures of chymosin [31] and saccharopepsin [32] (Fig. S5B) and was postulated to be responsible for keeping the enzymes in a self-inhibited state. The alanine insertion along with the altered conformer of tyrosine makes the S1 pocket narrower and hence, it is difficult for the substrate to enter the active site. S2: The Oγ atom of Thr77 of porcine pepsin makes a hydrogen bond with the amide nitrogen of P2 residue. This threonine is either an aspartate or a serine in other pepsins, but is replaced by a glycine (Gly67) in Mtb-AP (Fig. 8). S3: The position occupied by Ile193 of Mtb-AP is generally a Thr/Ser/Asn, the side chain of which makes a conserved hydrogen bond with substrate backbone nitrogen atom of P3 residue. This substrate stabilizing interaction is lost in Mtb-AP due to the presence of Ile193. Moreover, Ile193, which forms the mouth of S3 subsite projects out, occluding the S3 subsite (Fig. 9). S1′: This subsite contains the first hydrophobic residue present in the conserved C-terminal HHG motif of eukaryotic aspartic proteinases. In Mtb-AP, the hydrophilic residue Asn247 which replaces the first residue of the C-terminal HHG motif forms the S1′ pocket (Fig. 8). Thus we can speculate that Mtb-AP might require a hydrophilic residue at P1′ position. S2′: Some of the residues of S2′ pocket which can interact with and stabilize the substrate are either not conserved or absent because of the deletion of the loops and thus making this binding pocket wide open (Fig. 9). These differences could contribute to the lack of activity of Mtb-AP.


Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16.

Barathy DV, Suguna K - FEBS Open Bio (2013)

Surface representation of the active site of (A) Mtb-AP and (B) rhizopuspepsin (PDB code: 3APR). A modified peptide inhibitor of rhizopuspepsin is shown at a structural equivalent site in Mtb-AP. Ile193 of Mtb-AP which forms the mouth of S3 pocket is shown with arrow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3722594&req=5

fig0009: Surface representation of the active site of (A) Mtb-AP and (B) rhizopuspepsin (PDB code: 3APR). A modified peptide inhibitor of rhizopuspepsin is shown at a structural equivalent site in Mtb-AP. Ile193 of Mtb-AP which forms the mouth of S3 pocket is shown with arrow.
Mentions: A comparison of the structure of Mtb-AP with its structural equivalents human pepsin (PDB code: 1PSO) and rhizopuspepsin (PDB code: 3APR) in complex with inhibitors showed that the subsites of Mtb-AP appear to be similar to those of reported pepsins [28,29] with some small but significant differences (Fig. 8 and Table S2). S1: The flap residue tyrosine (Tyr64) along with glycine (Gly66) is well conserved in the family of eukaryotic aspartic proteinases. Mtb-AP has an additional alanine (Ala65) between Tyr64 and Gly66 (Fig. S4). A phenylalanine insertion corresponding to Ala65 of Mtb-AP was observed in the case of cockroach allergen Bla g 2 which was found to be inactive [30]. When pepstatin was docked in the binding pocket of Mtb-AP, the Ala65 side chain and the flap Tyr64 were found to make short contacts with it. The side chain of the conserved flap tyrosine has a different conformation (χ1 = 173°) when compared to that (χ1 = −54° in porcine pepsin) of eukaryotic aspartic proteinases (Fig. S5A). This conformation is stabilized by the hydrogen bond between tyrosine hydroxyl group and the side chain of the catalytic Asp36. Similar conformation of tyrosine was also observed in the crystal structures of chymosin [31] and saccharopepsin [32] (Fig. S5B) and was postulated to be responsible for keeping the enzymes in a self-inhibited state. The alanine insertion along with the altered conformer of tyrosine makes the S1 pocket narrower and hence, it is difficult for the substrate to enter the active site. S2: The Oγ atom of Thr77 of porcine pepsin makes a hydrogen bond with the amide nitrogen of P2 residue. This threonine is either an aspartate or a serine in other pepsins, but is replaced by a glycine (Gly67) in Mtb-AP (Fig. 8). S3: The position occupied by Ile193 of Mtb-AP is generally a Thr/Ser/Asn, the side chain of which makes a conserved hydrogen bond with substrate backbone nitrogen atom of P3 residue. This substrate stabilizing interaction is lost in Mtb-AP due to the presence of Ile193. Moreover, Ile193, which forms the mouth of S3 subsite projects out, occluding the S3 subsite (Fig. 9). S1′: This subsite contains the first hydrophobic residue present in the conserved C-terminal HHG motif of eukaryotic aspartic proteinases. In Mtb-AP, the hydrophilic residue Asn247 which replaces the first residue of the C-terminal HHG motif forms the S1′ pocket (Fig. 8). Thus we can speculate that Mtb-AP might require a hydrophilic residue at P1′ position. S2′: Some of the residues of S2′ pocket which can interact with and stabilize the substrate are either not conserved or absent because of the deletion of the loops and thus making this binding pocket wide open (Fig. 9). These differences could contribute to the lack of activity of Mtb-AP.

Bottom Line: The three-dimensional structure of one of them, Rv0977 (PE_PGRS16) of M. tuberculosis revealed the characteristic pepsin-fold and catalytic site architecture.However, the active site was completely blocked by the N-terminal His-tag.A comparison of the structure with pepsins showed significant differences in the critical substrate binding residues and in the flap tyrosine conformation that could contribute to the lack of proteolytic activity of Rv0977.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.

ABSTRACT
We report the crystal structure of the first prokaryotic aspartic proteinase-like domain identified in the genome of Mycobacterium tuberculosis. A search in the genomes of Mycobacterium species showed that the C-terminal domains of some of the PE family proteins contain two classic DT/SG motifs of aspartic proteinases with a low overall sequence similarity to HIV proteinase. The three-dimensional structure of one of them, Rv0977 (PE_PGRS16) of M. tuberculosis revealed the characteristic pepsin-fold and catalytic site architecture. However, the active site was completely blocked by the N-terminal His-tag. Surprisingly, the enzyme was found to be inactive even after the removal of the N-terminal His-tag. A comparison of the structure with pepsins showed significant differences in the critical substrate binding residues and in the flap tyrosine conformation that could contribute to the lack of proteolytic activity of Rv0977.

No MeSH data available.


Related in: MedlinePlus