Molecular characterization of secretory proteins Rv3619c and Rv3620c from Mycobacterium tuberculosis H37Rv.
Rv3619c and Rv3620c are the secretory, antigenic proteins of the ESAT-6/CFP-10 family of Mycobacterium tuberculosis H37Rv. In this article, we show that Rv3619c interacts with Rv3620c to form a 1 : 1 heterodimeric complex with a dissociation constant (K(d)) of 4.8 × 10(-7) M. The thermal unfolding of the heterodimer was completely reversible, with a T(m) of 48 °C. The comparative thermodynamics and thermal unfolding analysis of the Rv3619c-Rv3620c dimer, the ESAT-6-CFP-10 dimer and another ESAT family heterodimer, Rv0287-Rv0288, revealed that the binding strength and stability of Rv3619c-Rv3620c are relatively lower than those of the other two pairs. Molecular modeling and docking studies predict the structure of Rv3619c-Rv3620c to be similar to that of ESAT-6-CFP-10. Spectroscopic studies revealed that, in an acidic environment, Rv3619c and Rv3620c lose their secondary structure and interact weakly to form a complex with a lower helical content, indicating that Rv3619c-Rv3620c is destabilized at low pH. These results, combined with those of previous studies, suggest that unfolding of the proteins is required for dissociation of the complex and membrane binding. In the presence of membrane mimetics, the α-helical contents of Rv3619c and Rv3620 increased by 42% and 35%, respectively. In mice, the immune response against Rv3619c protein is characterized by increased levels of interferon-γ, interleukin-12 and IgG(2a) , indicating a dominant Th1 response, which is mandatory for protection against mycobacterial infection. This study therefore emphasizes the potential of Rv3619c as a subunit vaccine candidate.
Molecular and Structural Biology Division, Central Drug Research Institute, Lucknow, India.
Anjum
A
Mahmood
Shubhra
S
Srivastava
Sarita
S
Tripathi
Mairaj Ahmed
MA
Ansari
Mohammad
M
Owais
Ashish
A
Arora
D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D002151:Calorimetry; D002942:Circular Dichroism; D005260:Female; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D007371:Interferon-gamma / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D058849:Protein Refolding; D017433:Protein Structure, Secondary / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018417:Th1 Cells / Q000276:immunology / Q000378:metabolism; D013816:Thermodynamics; D014270:Trifluoroethanol / Q000494:pharmacology; D014611:Vaccination
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D007074:Immunoglobulin G; D011994:Recombinant Proteins; C560899:Rv3619c protein, Mycobacterium tuberculosis; C560900:Rv3620c protein, Mycobacterium tuberculosis; D010767:Phosphorylcholine; D018664:Interleukin-12; D015847:Interleukin-4; C028810:dodecylphosphocholine; D014270:Trifluoroethanol; D007371:Interferon-gamma