The interface between caldesmon domain 4b and subdomain 1 of actin studiedby nuclear magnetic resonance spectroscopy

The ability of caldesmon to inhibit actomyosin ATPase activity involves the interaction of three nonsequential segments of caldesmon domain 4 (amino a cids 600-756) with actin. Two of these contacts are located in the C-termin al half of this region of caldesmon which has been designated domain 4b (65 8-756). To investigate the spatial relationship between the two sites and t o determine whether their corresponding contacts on actin are sequentially distinct, we have used NMR spectroscopy to compare the actin binding proper ties of the minimal inhibitory peptide LW30 comprising residues 693-722 wit h those of the recombinant domain 4b constructs 658C (658-756) and Cg1 (a m utant of 658C in which the sequence (691)Glu-Trp-Leu-Thr-Lys-Thr(696) is ch anged to Pro-Gly-His-Tyr-Asn-Asn). Cg1 retains dual-sited actin attachment but displays lowered actin affinity. In the presence of tropomyosin, domain 4b-actin contacts were stronger but not qualitatively different, indicatin g that tropomyosin affected the conformational equilibrium of caldesmon bin ding. Simultaneous dual-sited attachment of domain 4b to actin is enabled b y the conformational properties of the site-spanning sequence common to 658 C, Cg1, and LW30 as reflected in the corresponding NOE and other NMR spectr al parameters. A backbone turn region ((713)Gly-Asp-Val-Ser(716)) preceded by an extended segment (Ser702-Pro-Ala-Pro-Lys-Pro) acts to constrain the r elative disposition of the flanking actin contact sites of domain 4b. In te sts with a library of actin peptides, only the C-terminus, 350-375, bound t o 658C and LW30. The use of CU2+ as a paramagnetic spectral probe bound to the unique His-371 provided evidence of a well-defined geometry for the com plex between LW30 and actin residues 350-375 with the N-terminal, site B of domain 4b close to the C-terminal residues of actin. The data are discusse d in the context of the potentiation of inhibitory activity by tropomyosin.