Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c)

Area of Science:

• Biochemistry
• Structural Biology
• Immunology

Background:

• Mycobacterium tuberculosis (Mtb) serine protease Hip1 (Rv2224c) contributes to tuberculosis (TB) pathogenesis.
• Hip1 impairs host immune responses by degrading the Mtb GroEL2 substrate.
• Hip1's cell surface localization and immunomodulatory role suggest it as a potential drug target for adjunctive TB therapies.

Purpose of the Study:

• To determine the crystal structure of the Mtb serine protease Hip1.
• To investigate the enzymatic activity of Hip1 against model substrates and Mtb GroEL2.
• To elucidate the structural basis for Hip1's function and guide inhibitor design.

Main Methods:

• X-ray crystallography to determine the 2.6 Å resolution structure of Hip1.
• Enzymatic kinetic studies using model substrates and the Mtb GroEL2 protein.
• Site-directed mutagenesis (Thr466 to alanine) to assess functional importance.

Main Results:

• The crystal structure reveals Hip1 as a two-domain protein, with one domain housing the serine protease catalytic residues.
• A threonine residue (Thr466) was identified within the active site, capable of hydrogen bonding with catalytic residues Asp463 and His490.
• Mutation of Thr466 to alanine significantly impaired Hip1's enzymatic function, confirming its critical role.

Conclusions:

• This study provides the first structural insights into Hip1, a novel serine protease.
• The identified active site threonine is crucial for Hip1's proteolytic activity.
• The Hip1 structure provides a foundation for designing specific inhibitors to block its activity and aid in TB treatment.