This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Google Scholar
Right arrow Articles by Kumar, C. M. S.
Right arrow Articles by Mande, S. C.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kumar, C. M. S.
Right arrow Articles by Mande, S. C.

 Previous Article  |  Next Article 

Journal of Bacteriology, November 2009, p. 6525-6538, Vol. 191, No. 21
0021-9193/09/$08.00+0     doi:10.1128/JB.00652-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Facilitated Oligomerization of Mycobacterial GroEL: Evidence for Phosphorylation-Mediated Oligomerization{triangledown} ,{dagger}

C. M. Santosh Kumar,1 Garima Khare,3 C. V. Srikanth,1,§ Anil K. Tyagi,3 Abhijit A. Sardesai,2* and Shekhar C. Mande1*

Laboratory of Structural Biology,1 Laboratory of Bacterial Genetics, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500001, India,2 Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, India3

Received 18 May 2009/ Accepted 21 August 2009

The distinctive feature of the GroES-GroEL chaperonin system in mediating protein folding lies in its ability to exist in a tetradecameric state, form a central cavity, and encapsulate the substrate via the GroES lid. However, recombinant GroELs of Mycobacterium tuberculosis are unable to act as effective molecular chaperones when expressed in Escherichia coli. We demonstrate here that the inability of M. tuberculosis GroEL1 to act as a functional chaperone in E. coli can be alleviated by facilitated oligomerization. The results of directed evolution involving random DNA shuffling of the genes encoding M. tuberculosis GroEL homologues followed by selection for functional entities suggested that the loss of chaperoning ability of the recombinant mycobacterial GroEL1 and GroEL2 in E. coli might be due to their inability to form canonical tetradecamers. This was confirmed by the results of domain-swapping experiments that generated M. tuberculosis-E. coli chimeras bearing mutually exchanged equatorial domains, which revealed that E. coli GroEL loses its chaperonin activity due to alteration of its oligomerization capabilities and vice versa for M. tuberculosis GroEL1. Furthermore, studying the oligomerization status of native GroEL1 from cell lysates of M. tuberculosis revealed that it exists in multiple oligomeric forms, including single-ring and double-ring variants. Immunochemical and mass spectrometric studies of the native M. tuberculosis GroEL1 revealed that the tetradecameric form is phosphorylated on serine-393, while the heptameric form is not, indicating that the switch between the single- and double-ring variants is mediated by phosphorylation.

* Corresponding author. Mailing address: Centre for DNA Fingerprinting and Diagnostics, Bldg. 7, Gruhakalpa, 5-4-399/B Nampally, Hyderabad 500001, India. Phone: 91-40-24749401. Fax: 91-40-24785447. E-mail for Shekhar C. Mande: shekhar{at}cdfd.org.in. E-mail for Abhijit A. Sardesai: abhijit{at}cdfd.org.in. Correspondent for reprint requests: Shekhar C. Mande

{triangledown} Published ahead of print on 28 August 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

§ Present address: Mucosal Immunology Laboratory, Massachusetts General Hospital, Charlestown, MA, 02129-4404.

Journal of Bacteriology, November 2009, p. 6525-6538, Vol. 191, No. 21
0021-9193/09/$08.00+0     doi:10.1128/JB.00652-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»