Structure And Function Of Cpn60.2 from Mycobacterium Tuberculosis
Written by Anat Shahar
Anat Shahar, Yechezkel Kashi* and Noam Adir
Shulich Faculty of Chemistry and Faculty of Biotechnology and Food Engineering, Technion, Technion City, Haifa 32000 Israel
Cpn60.2 from Mycobacterium tuberculosis (Mt) belongs to the Heat Shock Proteins 60 family which is also called Chaperonins. These proteins are involved in the process of folding of a large number of proteins in an ATP dependent manner. In addition, Cpn60.2 has a medical importance due to its high immunogenity. It elicits a significant immune response when whole Mt cells are used in vaccination. We have isolated Cpn60.2 by over expression of the cloned gene encoding for Cpn60.2 into pQE60 vector to enable metal chelate affinity purification. Crystals of His-Cpn60.2 grow in 2-14 days in 10% 2-propanol, 20% PEG 4K, 0.1M Hepes pH 7.5. Crystallographic analysis showed the crystal to be monoclinic (P21) with unit cell dimensions of a=58.4, b=112.2, c=77.5, β=95.5º and containing a dimer in the asymmetric unit. We have collected a complete 2.75Ǻ data set using synchrotron radiation. The structure has been solved by molecular replacement, using a lower resolution model. The Cpn60.2 shares 60% homology with the heat shock protein GroEL from E.coli. GroEL appears in solution as a cylinder composed of two seven subunits rings stacked back to back. Size analysis by various methods indicates that Cpn60.2 is primarily a monomer in solution. Measurement of ATP hydrolysis rates by the Cpn60.2 show that this protein possesses ATPase activity in resemblance to that of GroEL in contradiction to results in the literature that have claimed that the Mt chaperonin functions in an ATP independent manner. Since Mt posses another copy of the Cpn60 gene, the Cpn60.1, it raises the question regarding the necessity of these two copies and the mechanism of action of these proteins.