Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis.

Abstract

The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD(+) (the INH-NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH-NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS-II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein-protein interactions involving InhA and how these ligand-induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild-type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross-linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross-linking across one of the dimer-dimer interfaces in the InhA tetramer. Interestingly, a similar ligand-induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present.

• Authors: Nicole A Kruh, Richa Rawat, Béla P Ruzsicska, Peter J Tonge
• Journal: Protein science : a publication of the Protein Society (Protein Sci) Vol. 16 Issue 8 Pg. 1617-27 (Aug 2007) ISSN: 0961-8368 [Print] United States
• PMID: 17600151 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Antitubercular Agents
• Bacterial Proteins
• Oxidoreductases
• InhA protein, Mycobacterium
• Lysine
• Isoniazid

Topics

• Antitubercular Agents (chemistry, pharmacology)
• Bacterial Proteins (antagonists & inhibitors, chemistry, genetics, metabolism)
• Drug Resistance, Bacterial
• Isoniazid (chemistry, pharmacology)
• Lysine (genetics, metabolism)
• Models, Molecular
• Mutation
• Mycobacterium tuberculosis (chemistry, enzymology)
• Oxidoreductases (antagonists & inhibitors, chemistry, genetics)
• Protein Conformation
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization