Calorimetric Study of the Interaction of the C2 Domains of Classical Protein Kinase C Isoenzymes with Ca2+ and Phospholipids
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- 作者：Alejandro Torrecillas ; José ; Laynez ; Margarita Mené ; ndez ; Senena Corbalá ; n-Garcí ; a ; and Juan C. Gó ; mez-Ferná ; ndez
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：September 21, 2004
- 年：2004
- 卷：43
- 期：37
- 页码：11727 - 11739
- 全文大小：287K
- 年卷期：v.43,no.37(September 21, 2004)
- ISSN：1520-4995
文摘
The affinities of Ca2+ and anionic lipid vesicles from the C2 domains of classical proteinkinase C subfamily (
, 
II, and 
) were studied using isothermal titration calorimetry (ITC). In addition,the thermal stability of these C2 domains in the presence of different ligand concentrations was analyzedusing differential scanning calorimetry (DSC). These three closely related C2 domains bind Ca2+ in asimilar way, demonstrating the presence of two sets of sites. The first set of sites binds one Ca2+ ionexothermically with similar high affinity for the three proteins (Kd around 1 
M), while the second set ofsites binds endothermically approximately two Ca2+ ions with lower affinity, which varies for each C2domain: 22.2 M for the PKC-C2 domain, 17.2 M for the PKCII-C2 domain, and 4.3 M for thePKC-C2 domain. In the absence of Ca2+, the three C2 domains showed a weak interaction with vesiclescontaining anionic phospholipids. However, in the presence of a saturating Ca2+ concentration, the C2domains increased their affinities for the anionic lipid vesicles. In all cases, the C2 domains bound thevesicles exothermically and with similar affinities. A DSC thermal stability study of the C2 domains inthe presence of Ca2+ and anionic lipids provided further information about this protein-ligand interaction.The presence of increasing Ca2+ concentrations was matched by an increase in the Tm in all cases, whichwas even greater in the presence of anionic lipid vesicles. The extent of the change in Tm differed foreach C2 domain, reflecting the differing effect of the ligands bound during the protein stabilization.Denaturation of the C2 domains was irreversible both in the absence and in the presence of ligands,although the thermograms were not kinetically controlled. The dependence of the Tm on the Ca2+concentration indicates that the protein stabilization observed by DSC primarily reflects the saturation bythe cation of the low-affinity set of sites.
, II, and ) were studied using isothermal titration calorimetry (ITC). In addition,the thermal stability of these C2 domains in the presence of different ligand concentrations was analyzedusing differential scanning calorimetry (DSC). These three closely related C2 domains bind Ca2+ in asimilar way, demonstrating the presence of two sets of sites. The first set of sites binds one Ca2+ ionexothermically with similar high affinity for the three proteins (Kd around 1 M), while the second set ofsites binds endothermically approximately two Ca2+ ions with lower affinity, which varies for each C2domain: 22.2 M for the PKC-C2 domain, 17.2 M for the PKCII-C2 domain, and 4.3 M for thePKC-C2 domain. In the absence of Ca2+, the three C2 domains showed a weak interaction with vesiclescontaining anionic phospholipids. However, in the presence of a saturating Ca2+ concentration, the C2domains increased their affinities for the anionic lipid vesicles. In all cases, the C2 domains bound thevesicles exothermically and with similar affinities. A DSC thermal stability study of the C2 domains inthe presence of Ca2+ and anionic lipids provided further information about this protein-ligand interaction.The presence of increasing Ca2+ concentrations was matched by an increase in the Tm in all cases, whichwas even greater in the presence of anionic lipid vesicles. The extent of the change in Tm differed foreach C2 domain, reflecting the differing effect of the ligands bound during the protein stabilization.Denaturation of the C2 domains was irreversible both in the absence and in the presence of ligands,although the thermograms were not kinetically controlled. The dependence of the Tm on the Ca2+concentration indicates that the protein stabilization observed by DSC primarily reflects the saturation bythe cation of the low-affinity set of sites.