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X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
Published on: May 13, 2020
Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen
Deivanayaga V Barathy1, Kaza Suguna
1Molecular 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.
Insights
Researchers determined the crystal structure of a novel aspartic proteinase-like domain in Mycobacterium tuberculosis. This domain, Rv0977, exhibits a pepsin-fold but lacks proteolytic activity due to structural differences.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- The PE family proteins in Mycobacterium tuberculosis are crucial virulence factors.
- Aspartic proteinases are essential enzymes involved in various biological processes.
- Identifying novel proteinase domains can reveal new therapeutic targets.
Purpose of the Study:
- To determine the crystal structure of the first identified prokaryotic aspartic proteinase-like domain in Mycobacterium tuberculosis.
- To investigate the structural basis for the proteolytic activity of the Rv0977 protein.
Main Methods:
- X-ray crystallography was used to determine the three-dimensional structure of Rv0977.
- Bioinformatic searches were conducted across Mycobacterium species genomes.
- Enzyme activity assays were performed before and after His-tag removal.
Main Results:
- The crystal structure of Rv0977 revealed a characteristic pepsin-fold and catalytic site architecture.
- The active site of Rv0977 was initially blocked by an N-terminal His-tag.
- Rv0977 demonstrated no proteolytic activity, even after His-tag removal, due to structural variations.
Conclusions:
- Rv0977 represents a novel aspartic proteinase-like domain in prokaryotes with a unique structural fold.
- Significant differences in substrate-binding residues and flap tyrosine conformation likely account for Rv0977's inactivity.
- Further studies are needed to elucidate the precise function of Rv0977 in Mycobacterium tuberculosis.
