Interaction of caldesmon with actin subdomain-2.
The polymerization-resistant maleimidobenzoyl-G-actin (MBS-G-actin), which behaves as a functional analogue of native G-actin [Bettache, N., Bertrand, R. & Kassab, R. (1989) Proc. Natl Acad. Sci. USA 86, 6028-6032; Bettache, N., Bertrand, R. & Kassab, R. (1990) Biochemistry 29, 9085-9091) has been employed to probe the solution interaction between monomeric actin and smooth muscle caldesmon, using fluorescence measurements, limited proteolysis and covalent cross-linking reactions. MBS-G-actin associates, without polymerization, to turkey gizzard caldesmon, at about 50 mM ionic strength and 25 degrees C, with a high affinity (Kd approximately 0.04 microM) and with a 1:1 stoichiometry. However, the binding strength of the complex including caldesmon and MBS-G-actin cleaved at the subdomain-2 loop with subtilisin decreased fivefold (Kd approximately 0.20 microM). Conversely, caldesmon strongly protected subdomain-2 of MBS-G-actin from tryptic digestion at the susceptible peptide bond at positions 68-69. Furthermore, caldesmon induced the dissociation of native G-actin from its complex with DNase I, as assessed by cosedimentation assays, and increasing concentrations of the latter protein inhibited the MBS-G-actin-caldesmon interaction, suggesting mutual exclusion binding of caldesmon and DNase I to monomeric actin. MBS-G-actin was specifically coupled, via a maleimidobenzoyl group incorporated into its subdomain-2, to caldesmon, producing in high yield a 205-kDa covalent complex consisting of one actin monomer joined to Cys 580 of caldesmon. A similar conjugation process was observed with the complex of caldesmon and polymerized MBS-F-actin. MBS-G-actin could be also cross-linked to caldesmon by 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide, producing a three-band pattern identical to that of F-actin and caldesmon and previously shown to reflect the covalent union between the NH2-terminal segment of actin and the COOH-terminal actin-binding domain of caldesmon. The overall data point to a direct interaction of the latter region with actin subdomain-2 and suggest that during its binding to monomeric or filamentous actin, the caldesmon functional domain spans the entire length of a single actin and closely contacts the bottom of its subdomain-1 as well as the top portion of its subdomain-2.
Centre de Recherches de Biochimie Macromoléculaire du CNRS, UPR 1086, Université de Montpellier 1, France.
A
A
Bartegi
C
C
Roustan
R
R
Bertrand
R
R
Kassab
A
A
Fattoum
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000378:metabolism; D011817:Rabbits; D013388:Succinimides / Q000737:chemistry
D000199:Actins; D002148:Calmodulin-Binding Proteins; D003432:Cross-Linking Reagents; D013388:Succinimides; C072252:m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester; D003850:Deoxyribonuclease I