title: Modulation of cooperativity in Mycobacterium tuberculosis NADPH-ferredoxin reductase: cation-and pH-induced alterations in native conformation and destabilization of the NADP+-binding domain.
abstract: FprA, a Mycobacterium tuberculosis NADPH-ferredoxin reductase, consists of two structural domains, a FAD-binding and a NADP-binding domain, respectively. For the first time, we demonstrated that native FprA, on thermal treatment underwent partial denaturation with unfolding of only the FAD-binding domain and release of the protein-bound flavin. The NADP-binding domain of this protein is highly resistant to denaturation under these conditions. However, the presence of either 150 mM NaCl or KCl or 10 muM MgCl(2) or CaCl(2) or slightly acidic pH of 6.0 resulted in a highly cooperative and complete thermal unfolding of the protein. Physicochemical investigations showed that the monovalent cations or low concentrations of divalent cations induced compaction of the protein conformation. However, divalent cations at higher concentrations resulted in FAD release leading to stabilization of an enzymatically inactive apoenzyme. Detailed thermal denaturation studies on the native protein and the isolated NADP-binding domain showed that cations and pH 6.0 destabilized only the heat-stable NADP-binding domain. The experimental studies demonstrate that modulation of intramolecular ionic interactions induce significant conformational changes in the NADP-binding domain of FprA, resulting in a substantial increase in the structural cooperativity of the whole molecule. The results presented in this paper are of importance as they demonstrate alterations in the native three-dimensional structure of FprA and cooperativity in protein molecule on slight alteration of pH or modification of ionic interactions in protein.
authors.affiliation: Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India.
authors.forename: Anant Narayan
authors.initials: AN
authors.lastname: Bhatt
authors.forename: Nidhi
authors.initials: N
authors.lastname: Shukla
authors.forename: Alessandro
authors.initials: A
authors.lastname: Aliverti
authors.forename: Giuliana
authors.initials: G
authors.lastname: Zanetti
authors.forename: Vinod
authors.initials: V
authors.lastname: Bhakuni
mesh_terms: D002122:Calcium Chloride / Q000494:pharmacology; D002412:Cations; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
chemical_list: D002412:Cations; D012965:Sodium Chloride; D009249:NADP; C464438:FprA protein, M tuberculosis; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D002122:Calcium Chloride
references.citation: Infect Immun. 2000 Aug;68(8):4518-22
references.pmid: 10899850
references.citation: Yeast. 1998 Jun 30;14(9):839-46
references.pmid: 9818721
references.citation: Microbes Infect. 2001 Mar;3(3):249-55
references.pmid: 11358719
references.citation: Protein Sci. 2001 Sep;10(9):1712-28
references.pmid: 11514662
references.citation: Protein Sci. 2002 May;11(5):1260-73
references.pmid: 11967382
references.citation: J Biol Chem. 2002 May 10;277(19):17101-7
references.pmid: 11872744
references.citation: Eur J Biochem. 2002 Jun;269(12):3005-13
references.pmid: 12071965
references.citation: Biochemistry. 2002 Jul 16;41(28):8807-18
references.pmid: 12102623
references.citation: Biochem J. 2003 Jun 1;372(Pt 2):317-27
references.pmid: 12614197
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Biochemistry. 1974 Jan 29;13(3):589-97
references.pmid: 4149231
references.citation: Science. 1994 Feb 4;263(5147):678-81
references.pmid: 8303277
references.citation: J Immunol. 1994 Sep 15;153(6):2568-78
references.pmid: 8077667
references.citation: Biochim Biophys Acta. 1998 Feb 17;1382(2):191-206
references.pmid: 9540791
references.citation: Nature. 1998 Jun 11;393(6685):537-44
references.pmid: 9634230
references.citation: Biochemistry. 2001 Feb 20;40(7):1945-55
references.pmid: 11329261