Stoichiometry and topology of the complex of the endogenous ATP synthase inhibitor protein IF(1) with calmodulin.
IF(1), the natural inhibitor protein of F(O)F(1)ATP synthase able to regulate the ATP hydrolytic activity of both mitochondrial and cell surface enzyme, exists in two oligomeric states depending on pH: an inactive, highly helical, tetrameric form above pH 6.7 and an active, inhibitory, dimeric form below pH 6.7 [ Cabezon , E. , Butler , P. J. , Runswick , M. J. , and Walker , J. E. ( 2000 ) J. Biol. Chem. 275 , 25460 -25464 ]. IF(1) is known to interact in vitro with the archetypal EF-hand calcium sensor calmodulin (CaM), as well to colocalize with CaM on the plasma membrane of cultured cells. Low resolution structural data were herein obtained in order to get insights into the molecular interaction between IF(1) and CaM. A combined structural proteomic strategy was used which integrates limited proteolysis and chemical cross-linking with mass spectrometric analysis. Specifically, chemical cross-linking data clearly indicate that the C-terminal lobe of CaM molecule contacts IF(1) within the inhibitory, flexible N-terminal region that is not involved in the dimeric interface in IF(1). Nevertheless, native mass spectrometry analysis demonstrated that in the micromolar range the stoichiometry of the IF(1)-CaM complex is 1:1, thereby indicating that binding to CaM promotes IF(1) dimer dissociation without directly interfering with the intersubunit contacts of the IF(1) dimer. The relevance of the finding that only the C-terminal lobe of CaM is involved in the interaction is two fold: (i) the IF(1)-CaM complex can be included in the category of noncanonical structures of CaM complexes; (ii) it can be inferred that the N-terminal region of CaM might have the opportunity to bind to a second target.
Pagnozzi
Daniela
D
Department of Organic Chemistry and Biochemistry, University of Napoli Federico II, viaCynthia 6, 80126 Napoli, Italy.
Birolo
Leila
L
Leo
Gabriella
G
Contessi
Stefania
S
Lippe
Giovanna
G
Pucci
Pietro
P
Mavelli
Irene
I
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D013058:Mass Spectrometry; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D000097795:ATPase Inhibitory Protein
D000255:Adenosine Triphosphate; D002118:Calcium; D002147:Calmodulin; D011506:Proteins; D000097795:ATPase Inhibitory Protein