Characterization of the Ca2+-binding Sites of Annexin II Tetramer

Annexin II heterotetramer (AIIt) is a multifunctional Ca2+-binding protein composed of two 11-kDa subunits and two annexin II subunits. The annexin II subunit contains three type II and two type III Ca2+-binding sites which are thought to regulate the interaction of AIIt with anionic phospholipid, F-actin, and heparin. In the present study we utilized site-directed mutagenesis to create AIIt mutants with inactive type III (TM AIIt), type II (CM AIIt), and both type II and III Ca2+-binding sites (TCM AIIt). Surprisingly, we found that in the presence of Ca2+, the TM, CM, and TCM AIIt bound phospholipid and F-actin with similar affinity to the wild type AIIt (WT AIIt). Furthermore, the TCM mutant, and to a lesser extent the TM and CM AIIt displayed dose-dependent Ca2+-independent phospholipid aggregation and binding. While the TM and CM AIIt demonstrated Ca2+-dependent binding to F-actin, the binding of the TCM AIIt was Ca2+-independent. These results suggest that the type II or type III Ca2+-binding sites do not directly participate in anionic phospholipid or F-actin binding. We therefore propose that in the absence of Ca2+, the type II and type III Ca2+-binding sites of AIIt stabilize a conformation of AIIt that is unfavorable for binding phospholipid and F-actin. Ca2+ binding to these sites, or the inactivation of these Ca2+-binding sites by site-directed mutagenesis, results in a conformational change that promotes binding to anionic phospholipid and F-actin. Since the TM, CM, and TCM AIIt require Ca2+ for binding to heparin, we also propose that novel Ca2+-binding sites regulate this binding event. Annexin II heterotetramer (AIIt) is a multifunctional Ca2+-binding protein composed of two 11-kDa subunits and two annexin II subunits. The annexin II subunit contains three type II and two type III Ca2+-binding sites which are thought to regulate the interaction of AIIt with anionic phospholipid, F-actin, and heparin. In the present study we utilized site-directed mutagenesis to create AIIt mutants with inactive type III (TM AIIt), type II (CM AIIt), and both type II and III Ca2+-binding sites (TCM AIIt). Surprisingly, we found that in the presence of Ca2+, the TM, CM, and TCM AIIt bound phospholipid and F-actin with similar affinity to the wild type AIIt (WT AIIt). Furthermore, the TCM mutant, and to a lesser extent the TM and CM AIIt displayed dose-dependent Ca2+-independent phospholipid aggregation and binding. While the TM and CM AIIt demonstrated Ca2+-dependent binding to F-actin, the binding of the TCM AIIt was Ca2+-independent. These results suggest that the type II or type III Ca2+-binding sites do not directly participate in anionic phospholipid or F-actin binding. We therefore propose that in the absence of Ca2+, the type II and type III Ca2+-binding sites of AIIt stabilize a conformation of AIIt that is unfavorable for binding phospholipid and F-actin. Ca2+ binding to these sites, or the inactivation of these Ca2+-binding sites by site-directed mutagenesis, results in a conformational change that promotes binding to anionic phospholipid and F-actin. Since the TM, CM, and TCM AIIt require Ca2+ for binding to heparin, we also propose that novel Ca2+-binding sites regulate this binding event. annexin II tetramer recombinant AIIt composed of wild type p11 subunit and mutant annexin II subunit (Glu-52 → Ala and Glu-95 → Ala, Asp-161 → Ala, Glu-246 → Ala, and Asp-321 → Ala) p11 light chain of annexin II tetramer recombinant AIIt composed of wild type annexin II and wild type p11 subunits recombinant AIIt composed of wild type p11 subunit and mutant annexin II subunit (Glu-52 → Ala and Glu-95 → Ala) recombinant AIIt composed of wild type p11 subunit and mutant annexin II subunit (Asp-161 → Ala, Glu-246 → Ala, and Asp-321 → Ala) polyacrylamide gel electrophoresis dithiothreitol 4-morpholinepropanesulfonic acid The annexins are a family of about 13 proteins that bind to cellular membranes and anionic phospholipids in a Ca2+-dependent manner (1Siever D.A. Erickson H.P. Int. J. Biochem. Cell Biol. 1997; 29: 1219-1223Crossref PubMed Scopus (103) Google Scholar, 2Waisman D.M. Mol. Cell. Biochem. 1995; 149–150: 301-322Crossref PubMed Scopus (260) Google Scholar, 3Swairjo M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Crossref PubMed Scopus (188) Google Scholar). All annexins have four repeating, highly homogenous acid in the of annexin the annexin PubMed Scopus Google Scholar). The annexin contains in the highly acid that of a The of the annexin the protein which contains the binding sites for annexin Ca2+ and anionic the protein is the which in annexins and is therefore thought to of are of annexins of of for and of and of in 2Waisman D.M. Mol. Cell. Biochem. 1995; 149–150: 301-322Crossref PubMed Scopus (260) Google Cell Mol. 1997; PubMed Scopus Google Scholar, Biophys. 1997; PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar). The in of annexins have not of annexin II in the presence of a type II Ca2+-binding in of the and and two type III Ca2+-binding sites in the of the these Ca2+-binding sites, with the sites, are the of the the F-actin and sites are the of the the Annexin II is the annexins in that a for a of the family of Ca2+-binding p11 D.M. Mol. Cell. Biochem. 1995; 149–150: 301-322Crossref PubMed Scopus (260) Google Scholar). The by the interaction of the annexin II and p11 subunits is annexin II heterotetramer Since the of AIIt is not the or conformation of the annexin II subunits AIIt is is also or not the Ca2+-binding sites of the annexin II subunits AIIt are was that a mutant of annexin composed of type II Ca2+-binding sites displayed a Ca2+ for phospholipid binding. Furthermore, the inactivation of both the type II and type III Ca2+-binding sites in the of phospholipid binding by the mutant annexin II Biochem. J. 1994; PubMed Scopus Google Scholar). was therefore that type II and type III Ca2+-binding sites for the binding of these utilized a annexin II in which the was therefore the results these to the Since the annexin II not bind the p11 was also not to the of the type II and type III Ca2+-binding sites of have the and of annexin II and p11 subunits D.M. 1997; PubMed Scopus Google Scholar). The recombinant composed of the wild type recombinant subunits of annexin II and to that of AIIt In the present we the of AIIt in which the type II and type III Ca2+-binding sites have (TCM AIIt). inactivation of the type II and type III Ca2+-binding sites of annexin II in the Ca2+-independent of AIIt with both phospholipid and F-actin. Furthermore, the binding of the TCM AIIt to Ca2+, we that Ca2+-binding sites the type II and type III Ca2+-binding sites the is a multifunctional protein that in a Ca2+-dependent manner with anionic phospholipid, F-actin, and heparin. The binding sites for these are the annexin II subunit of In the present study we utilized site-directed mutagenesis to the type III TM type II CM and both type II and III Ca2+-binding sites of annexin II TCM The for inactivation of the annexin II Ca2+-binding sites was by Biochem. J. 1994; PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar, J. Biol. PubMed Google that the of the of Ca2+-binding with in the inactivation of the Ca2+-binding in to that which utilized a annexin we a recombinant annexin for the we to the TM, CM, and TCM mutant of AIIt by the mutant recombinant annexin II with wild type recombinant p11 In the present we have these mutant of AIIt to the of the type II and III sites of AIIt in the Ca2+-dependent interaction with phospholipid, F-actin, heparin, and the and three mutant of annexin II and we these not the conformation of annexin the mutant annexin II to bind to p11 and a that annexin II was in Furthermore, the recombinant AIIt and the TM, CM, and TCM mutants of AIIt gel and we have that the TM, CM, and TCM mutants of AIIt not The of the three mutants to bind and to was that the conformation of these proteins was not of the Ca2+-binding of the TM, CM, and TCM AIIt we found that in the presence of Ca2+, annexin II and AIIt bound about or of of Since the of annexin II the presence of two type III Ca2+-binding sites and three type II Ca2+-binding sites, was that annexin II bind of and AIIt bind of of in the presence of that annexin II and AIIt bound and of Ca2+ of J. J. Biol. PubMed Google Scholar). for the the results and the results is that of the Ca2+-binding sites require of AIIt in the presence of Ca2+ to of the and is therefore not to of Ca2+ is also that AIIt Ca2+ the conformation or of the annexin II subunits is that not Ca2+-binding sites of the annexin II subunit are the of the of is not to this was that the TCM annexin II and TCM AIIt bound of and of the the presence of type II and type III Ca2+-binding sites annexin II M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Crossref PubMed Scopus (188) Google Scholar, M.A. M.A. Seaton B.A. Struct. Biol. 1995; PubMed Scopus Google Scholar). for this is that the annexin II do not inactivation of the type II and type III Ca2+-binding We this is the of with in the of two Ca2+-binding that in for the Ca2+-binding sites to bind Ca2+, the type III Ca2+-binding sites of the TM AIIt to Ca2+ binding with two and type of for a Ca2+-binding is The type II Ca2+-binding sites of CM AIIt require the of Ca2+ by three and four this to a Ca2+-binding The in the of the for the CM AIIt is that the Glu-246 → Ala the type II Ca2+-binding of III of annexin is therefore to that similar in the type II sites in for the of the TCM annexin II and TCM AIIt to bind Ca2+ is that novel Ca2+-binding sites, type II or type III Ca2+-binding sites are present these Since the TCM annexin II and AIIt mutants Ca2+-dependent binding to heparin, is that the type II or type III Ca2+-binding sites are not in is therefore to propose that the Ca2+-binding present TCM annexin II or the two to three Ca2+-binding sites present the TCM AIIt are novel Ca2+-binding sites that are for the interaction of AIIt with is that the binding of of of AIIt are to Ca2+-binding sites the type II and type III Ca2+-binding sites is to the of of Ca2+ bound to the type II or type III sites for mutant Since TM AIIt of of protein and two of these Ca2+ are not type II or III Ca2+-binding sites, we that three Ca2+ bound to the type II sites present TM the CM AIIt four Ca2+ and two Ca2+ are not type II or III Ca2+-binding sites the CM AIIt of of protein to the type III sites present the the Ca2+-binding is with the binding of Ca2+ to of the annexin II subunits the that of the two annexin II subunits of AIIt have type II or type III Ca2+-binding sites of binding Ca2+ in the presence of annexin II subunit have type II Ca2+-binding sites and the subunit have type III Ca2+-binding that annexin II contains two type III Ca2+-binding sites in the of the protein and three type II Ca2+-binding sites in the and of the The acid of the type II Ca2+-binding is Ca2+ is by three two of the and two The of the annexin and was these that Ca2+ a the of the type II Ca2+-binding sites and the of the phospholipid M.A. M.A. Seaton B.A. Struct. Biol. 1995; PubMed Scopus Google Scholar). Furthermore, the of the of the type II Ca2+-binding sites with the of was therefore that TM and CM AIIt bound to phospholipid and that the binding of the CM and TCM mutant was Ca2+-independent. was also that the CM and TCM AIIt affinity for phospholipid in the presence or absence of is the of the of the TCM AIIt with to the interaction of AIIt with is to by the interaction of the of the with the of and by the interaction of Ca2+ with the of the affinity of the TM, CM, and TCM AIIt for phospholipid is is that these are not to the interaction of annexin II with of the of the interaction of annexin II or AIIt with not the of the is results by Biochem. J. 1994; PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar, J. Biol. PubMed Google that the TCM annexin II not bind to Since the TCM annexin II mutant in these was a that the of the is that protein was not in and PubMed Scopus Google have that of the of annexin II the Ca2+ of interaction of AIIt with was also that the TM and CM AIIt demonstrated Ca2+-dependent binding to F-actin. that the type II or III Ca2+-binding sites are not in the Ca2+-dependent interaction of AIIt with F-actin. was therefore that the TCM AIIt Ca2+-independent binding to F-actin. is to propose that in the absence of Ca2+, the of the type II Ca2+-binding sites and the of the type III Ca2+-binding sites with annexin interaction results in the of a conformation of annexin II which not for interaction with phospholipid or F-actin. The binding of Ca2+ to the Ca2+-binding sites results in the of these that participate in The change in the conformation also the interaction of annexin II with phospholipid or F-actin. of these by site-directed mutagenesis also these with acid and the conformation of annexin II that with phospholipid or F-actin. of this of the of The annexins are a family of about 13 proteins that bind to cellular membranes and anionic phospholipids in a Ca2+-dependent manner (1Siever D.A. Erickson H.P. Int. J. Biochem. Cell Biol. 1997; 29: 1219-1223Crossref PubMed Scopus (103) Google Scholar, 2Waisman D.M. Mol. Cell. Biochem. 1995; 149–150: 301-322Crossref PubMed Scopus (260) Google Scholar, 3Swairjo M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Crossref PubMed Scopus (188) Google Scholar). All annexins have four repeating, highly homogenous acid in the of annexin the annexin PubMed Scopus Google Scholar). The annexin contains in the highly acid that of a The of the annexin the protein which contains the binding sites for annexin Ca2+ and anionic the protein is the which in annexins and is therefore thought to of are of annexins of of for and of and of in 2Waisman D.M. Mol. Cell. Biochem. 1995; 149–150: 301-322Crossref PubMed Scopus (260) Google Cell Mol. 1997; PubMed Scopus Google Scholar, Biophys. 1997; PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar). The in of annexins have not of annexin II in the presence of a type II Ca2+-binding in of the and and two type III Ca2+-binding sites in the of the these Ca2+-binding sites, with the sites, are the of the the F-actin and sites are the of the the Annexin II is the annexins in that a for a of the family of Ca2+-binding p11 D.M. Mol. Cell. Biochem. 1995; 149–150: 301-322Crossref PubMed Scopus (260) Google Scholar). The by the interaction of the annexin II and p11 subunits is annexin II heterotetramer Since the of AIIt is not the or conformation of the annexin II subunits AIIt is is also or not the Ca2+-binding sites of the annexin II subunits AIIt are was that a mutant of annexin composed of type II Ca2+-binding sites displayed a Ca2+ for phospholipid binding. Furthermore, the inactivation of both the type II and type III Ca2+-binding sites in the of phospholipid binding by the mutant annexin II Biochem. J. 1994; PubMed Scopus Google Scholar). was therefore that type II and type III Ca2+-binding sites for the binding of these utilized a annexin II in which the was therefore the results these to the Since the annexin II not bind the p11 was also not to the of the type II and type III Ca2+-binding sites of We have the and of annexin II and p11 subunits D.M. 1997; PubMed Scopus Google Scholar). The recombinant composed of the wild type recombinant subunits of annexin II and to that of AIIt In the present we the of AIIt in which the type II and type III Ca2+-binding sites have (TCM AIIt). inactivation of the type II and type III Ca2+-binding sites of annexin II in the Ca2+-independent of AIIt with both phospholipid and F-actin. Furthermore, the binding of the TCM AIIt to Ca2+, we that Ca2+-binding sites the type II and type III Ca2+-binding sites the is a multifunctional protein that in a Ca2+-dependent manner with anionic phospholipid, F-actin, and heparin. The binding sites for these are the annexin II subunit of In the present study we utilized site-directed mutagenesis to the type III TM type II CM and both type II and III Ca2+-binding sites of annexin II TCM The for inactivation of the annexin II Ca2+-binding sites was by Biochem. J. 1994; PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar, J. Biol. PubMed Google that the of the of Ca2+-binding with in the inactivation of the Ca2+-binding in to that which utilized a annexin we a recombinant annexin for the we to the TM, CM, and TCM mutant of AIIt by the mutant recombinant annexin II with wild type recombinant p11 In the present we have these mutant of AIIt to the of the type II and III sites of AIIt in the Ca2+-dependent interaction with phospholipid, F-actin, heparin, and the and three mutant of annexin II and we these not the conformation of annexin the mutant annexin II to bind to p11 and a that annexin II was in Furthermore, the recombinant AIIt and the TM, CM, and TCM mutants of AIIt gel and we have that the TM, CM, and TCM mutants of AIIt not The of the three mutants to bind and to was that the conformation of these proteins was not of the Ca2+-binding of the TM, CM, and TCM AIIt we found that in the presence of Ca2+, annexin II and AIIt bound about or of of Since the of annexin II the presence of two type III Ca2+-binding sites and three type II Ca2+-binding sites, was that annexin II bind of and AIIt bind of of in the presence of that annexin II and AIIt bound and of Ca2+ of J. J. Biol. PubMed Google Scholar). for the the results and the results is that of the Ca2+-binding sites require of AIIt in the presence of Ca2+ to of the and is therefore not to of Ca2+ is also that AIIt Ca2+ the conformation or of the annexin II subunits is that not Ca2+-binding sites of the annexin II subunit are the of the of is not to this was that the TCM annexin II and TCM AIIt bound of and of the the presence of type II and type III Ca2+-binding sites annexin II M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Crossref PubMed Scopus (188) Google Scholar, M.A. M.A. Seaton B.A. Struct. Biol. 1995; PubMed Scopus Google Scholar). for this is that the annexin II do not inactivation of the type II and type III Ca2+-binding We this is the of with in the of two Ca2+-binding that in for the Ca2+-binding sites to bind Ca2+, the type III Ca2+-binding sites of the TM AIIt to Ca2+ binding with two and type of for a Ca2+-binding is The type II Ca2+-binding sites of CM AIIt require the of Ca2+ by three and four this to a Ca2+-binding The in the of the for the CM AIIt is that the Glu-246 → Ala the type II Ca2+-binding of III of annexin is therefore to that similar in the type II sites in for the of the TCM annexin II and TCM AIIt to bind Ca2+ is that novel Ca2+-binding sites, type II or type III Ca2+-binding sites are present these Since the TCM annexin II and AIIt mutants Ca2+-dependent binding to heparin, is that the type II or type III Ca2+-binding sites are not in is therefore to propose that the Ca2+-binding present TCM annexin II or the two to three Ca2+-binding sites present the TCM AIIt are novel Ca2+-binding sites that are for the interaction of AIIt with is that the binding of of of AIIt are to Ca2+-binding sites the type II and type III Ca2+-binding sites is to the of of Ca2+ bound to the type II or type III sites for mutant Since TM AIIt of of protein and two of these Ca2+ are not type II or III Ca2+-binding sites, we that three Ca2+ bound to the type II sites present TM the CM AIIt four Ca2+ and two Ca2+ are not type II or III Ca2+-binding sites the CM AIIt of of protein to the type III sites present the the Ca2+-binding is with the binding of Ca2+ to of the annexin II subunits the that of the two annexin II subunits of AIIt have type II or type III Ca2+-binding sites of binding Ca2+ in the presence of annexin II subunit have type II Ca2+-binding sites and the subunit have type III Ca2+-binding that annexin II contains two type III Ca2+-binding sites in the of the protein and three type II Ca2+-binding sites in the and of the The acid of the type II Ca2+-binding is Ca2+ is by three two of the and two The of the annexin and was these that Ca2+ a the of the type II Ca2+-binding sites and the of the phospholipid M.A. M.A. Seaton B.A. Struct. Biol. 1995; PubMed Scopus Google Scholar). Furthermore, the of the of the type II Ca2+-binding sites with the of was therefore that TM and CM AIIt bound to phospholipid and that the binding of the CM and TCM mutant was Ca2+-independent. was also that the CM and TCM AIIt affinity for phospholipid in the presence or absence of is the of the of the TCM AIIt with to the interaction of AIIt with is to by the interaction of the of the with the of and by the interaction of Ca2+ with the of the affinity of the TM, CM, and TCM AIIt for phospholipid is is that these are not to the interaction of annexin II with of the of the interaction of annexin II or AIIt with not the of the is results by Biochem. J. 1994; PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar, J. Biol. PubMed Google that the TCM annexin II not bind to Since the TCM annexin II mutant in these was a that the of the is that protein was not in and PubMed Scopus Google have that of the of annexin II the Ca2+ of interaction of AIIt with was also that the TM and CM AIIt demonstrated Ca2+-dependent binding to F-actin. that the type II or III Ca2+-binding sites are not in the Ca2+-dependent interaction of AIIt with F-actin. was therefore that the TCM AIIt Ca2+-independent binding to F-actin. is to propose that in the absence of Ca2+, the of the type II Ca2+-binding sites and the of the type III Ca2+-binding sites with annexin interaction results in the of a conformation of annexin II which not for interaction with phospholipid or F-actin. The binding of Ca2+ to the Ca2+-binding sites results in the of these that participate in The change in the conformation also the interaction of annexin II with phospholipid or F-actin. of these by site-directed mutagenesis also these with acid and the conformation of annexin II that with phospholipid or F-actin. of this of the of AIIt is a multifunctional protein that in a Ca2+-dependent manner with anionic phospholipid, F-actin, and heparin. The binding sites for these are the annexin II subunit of In the present study we utilized site-directed mutagenesis to the type III TM type II CM and both type II and III Ca2+-binding sites of annexin II TCM The for inactivation of the annexin II Ca2+-binding sites was by Biochem. J. 1994; PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar, J. Biol. PubMed Google that the of the of Ca2+-binding with in the inactivation of the Ca2+-binding in to that which utilized a annexin we a recombinant annexin for the we to the TM, CM, and TCM mutant of AIIt by the mutant recombinant annexin II with wild type recombinant p11 In the present we have these mutant of AIIt to the of the type II and III sites of AIIt in the Ca2+-dependent interaction with phospholipid, F-actin, heparin, and Since the and three mutant of annexin II and we these not the conformation of annexin the mutant annexin II to bind to p11 and a that annexin II was in Furthermore, the recombinant AIIt and the TM, CM, and TCM mutants of AIIt gel and we have that the TM, CM, and TCM mutants of AIIt not The of the three mutants to bind and to was that the conformation of these proteins was not of the Ca2+-binding of the TM, CM, and TCM AIIt we found that in the presence of Ca2+, annexin II and AIIt bound about or of of Since the of annexin II the presence of two type III Ca2+-binding sites and three type II Ca2+-binding sites, was that annexin II bind of and AIIt bind of of in the presence of that annexin II and AIIt bound and of Ca2+ of J. J. Biol. PubMed Google Scholar). for the the results and the results is that of the Ca2+-binding sites require of AIIt in the presence of Ca2+ to of the and is therefore not to of Ca2+ is also that AIIt Ca2+ the conformation or of the annexin II subunits is that not Ca2+-binding sites of the annexin II subunit are the of the of is not to this was that the TCM annexin II and TCM AIIt bound of and of the the presence of type II and type III Ca2+-binding sites annexin II M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Crossref PubMed Scopus (188) Google Scholar, M.A. M.A. Seaton B.A. Struct. Biol. 1995; PubMed Scopus Google Scholar). for this is that the annexin II do not inactivation of the type II and type III Ca2+-binding We this is the of with in the of two Ca2+-binding that in for the Ca2+-binding sites to bind Ca2+, the type III Ca2+-binding sites of the TM AIIt to Ca2+ binding with two and type of for a Ca2+-binding is The type II Ca2+-binding sites of CM AIIt require the of Ca2+ by three and four this to a Ca2+-binding The in the of the for the CM AIIt is that the Glu-246 → Ala the type II Ca2+-binding of III of annexin is therefore to that similar in the type II sites in for the of the TCM annexin II and TCM AIIt to bind Ca2+ is that novel Ca2+-binding sites, type II or type III Ca2+-binding sites are present these Since the TCM annexin II and AIIt mutants Ca2+-dependent binding to heparin, is that the type II or type III Ca2+-binding sites are not in is therefore to propose that the Ca2+-binding present TCM annexin II or the two to three Ca2+-binding sites present the TCM AIIt are novel Ca2+-binding sites that are for the interaction of AIIt with heparin. is that the binding of of of AIIt are to Ca2+-binding sites the type II and type III Ca2+-binding sites is to the of of Ca2+ bound to the type II or type III sites for mutant Since TM AIIt of of protein and two of these Ca2+ are not type II or III Ca2+-binding sites, we that three Ca2+ bound to the type II sites present TM the CM AIIt four Ca2+ and two Ca2+ are not type II or III Ca2+-binding sites the CM AIIt of of protein to the type III sites present the the Ca2+-binding is with the binding of Ca2+ to of the annexin II subunits the that of the two annexin II subunits of AIIt have type II or type III Ca2+-binding sites of binding Ca2+ in the presence of annexin II subunit have type II Ca2+-binding sites and the subunit have type III Ca2+-binding that annexin II contains two type III Ca2+-binding sites in the of the protein and three type II Ca2+-binding sites in the and of the The acid of the type II Ca2+-binding is Ca2+ is by three two of the and two The of the annexin and was these that Ca2+ a the of the type II Ca2+-binding sites and the of the phospholipid M.A. M.A. Seaton B.A. Struct. Biol. 1995; PubMed Scopus Google Scholar). Furthermore, the of the of the type II Ca2+-binding sites with the of was therefore that TM and CM AIIt bound to phospholipid and that the binding of the CM and TCM mutant was Ca2+-independent. was also that the CM and TCM AIIt affinity for phospholipid in the presence or absence of is the of the of the TCM AIIt with to the interaction of AIIt with is to by the interaction of the of the with the of and by the interaction of Ca2+ with the of the affinity of the TM, CM, and TCM AIIt for phospholipid is is that these are not to the interaction of annexin II with of the of the interaction of annexin II or AIIt with not the of the is results by Biochem. J. 1994; PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar, J. Biol. PubMed Google that the TCM annexin II not bind to Since the TCM annexin II mutant in these was a that the of the is that protein was not in and PubMed Scopus Google have that of the of annexin II the Ca2+ of interaction of AIIt with was also that the TM and CM AIIt demonstrated Ca2+-dependent binding to F-actin. that the type II or III Ca2+-binding sites are not in the Ca2+-dependent interaction of AIIt with F-actin. was therefore that the TCM AIIt Ca2+-independent binding to F-actin. is to propose that in the absence of Ca2+, the of the type II Ca2+-binding sites and the of the type III Ca2+-binding sites with annexin interaction results in the of a conformation of annexin II which not for interaction with phospholipid or F-actin. The binding of Ca2+ to the Ca2+-binding sites results in the of these that participate in The change in the conformation also the interaction of annexin II with phospholipid or F-actin. of these by site-directed mutagenesis also these with acid and the conformation of annexin II that with phospholipid or F-actin. of this of the of We and for and of the of the Ca2+-binding sites of annexin II of was the The is present of of

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