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The interaction between alpha-actinin and titin, two modular muscle proteins, is essential for sarcomere assembly. We have solved the solution structure of a complex between the calcium-insensitive C-terminal EF-hand domain of alpha-actinin-2 and the seventh Z-repeat of titin. The structure of the complex is in a semi-open conformation and closely resembles that of myosin light chains in their complexes with heavy chain IQ motifs. However, no IQ motif is present in the Z-repeat, suggesting that the semi-open conformation is a general structural solution for calcium-independent recognition of EF-hand domains.

Titin and alpha-actinin, two modular muscle proteins, are with actin the major components of the Z-band in vertebrate striated muscles where they serve to organize the antiparallel actin filament arrays in adjacent sarcomeres and to transmit tension between sarcomeres during activation. Interactions between titin and alpha-actinin have been mainly localized in a 45-amino acid multiple motif (Z-repeat) in the N-terminal region of titin and the C-terminal region of alpha-actinin. In this study, we provide the first quantitative characterization of alpha-actinin-Z-repeat recognition and dissect the interaction to its minimal units. Different complementary techniques, such as circular dichroism, calorimetry, and nuclear magnetic spectroscopy, were used. Two overlapping alpha-actinin constructs (Act-EF34 and Act-EF1234) containing two and four EF-hand motifs, respectively, were produced, and their folding properties were examined. Complex formation of Act-EF34 and Act-EF1234 with single- and double-Z-repeat constructs was studied. Act-EF34 was shown quantitatively to be necessary and sufficient for binding to Z-repeats, excluding the presence of additional high-affinity binding sites in the remaining part of the domain. The binding affinities of the different Z-repeats for Act-EF34 range from micromolar to millimolar values. The strongest of these interactions are comparable to those observed in troponin C-troponin I complexes. The binding affinities for Act-EF34 are maximal for Zr1 and Zr7, the two highly homologous sequences present in all muscle isoforms. No cooperative or additional contributions to the interaction were observed for Z-repeat double constructs. These findings have direct relevance for evaluating current models of Z-disk assembly.

Titin is an exceptionally large protein (M.Wt. approximately 3 MDa) that spans half the sarcomere in muscle, from the Z-disk to the M-line. In the Z-disk, it interacts with alpha-actinin homodimers that are a principal component of the Z-filaments linking actin filaments. The interaction between titin and alpha-actinin involves repeating approximately 45 amino acid sequences (Z-repeats) near the N-terminus of titin and the C-lobe of the C-terminal calmodulin-like domain of alpha-actinin. The conformation of Z-repeat 7 (ZR7) of titin when complexed with the 73-amino acid C-terminal portion of alpha-actinin (EF34) was studied by heteronuclear NMR spectroscopy using (15)N-labeling of ZR7 and found to be helical over a stretch of 18 residues. Complex formation resulted in the protection of one site of preferential cleavage of EF34 at Phe14-Leu17, as determined by limited proteolysis experiments coupled to mass spectrometry measurements. Intermolecular NOEs show Val16 of ZR7 to be positioned close in space to the backbone of EF34 around Phe14. These observations suggest that the mode of binding of ZR7 to EF34 is similar to that of troponin I to troponin C and of peptide C20W to calmodulin. These complexes would appear to represent a general alternative binding mode of calmodulin-like domains to target peptides.

Titin, a giant muscle protein, forms filaments that span half of the sarcomere and cover, along their length, quite diversified functions. The region of titin located in the sarcomere I-band is believed to play a major rôle in extensibility and passive elasticity of muscle. In the I-band, the titin sequence contains tandem immunoglobulin-like (Ig) modules intercalated by a potentially non-globular region. By a combined approach making use of small angle X-ray scattering and nuclear magnetic resonance techniques, we have addressed the questions of what are the average mutual orientation of poly-Igs and the degree of flexibility around the domain interfaces. Various recombinant fragments containing one, two and four titin I-band tandem domains were analysed. The small-angle scattering data provide a picture of the domains in a mostly extended configuration with their long axes aligned head-to-tail. There is a small degree of bending and twisting of the modules with respect to each other that results in an overall shortening in their maximum linear dimension compared with that expected for the fully extended, linear configurations. This shortening is greatest for the four module construct ( approximately 15%). 15N NMR relaxation studies of one and two-domain constructs show that the motions around the interdomain connecting regions are restricted, suggesting that titin behaves as a row of beads connected by rigid hinges. The length of the residues in the interface seems to be the major determinant of the degree of flexibility. Possible implications of our results for the structure and function of titin in muscles are discussed., (Copyright 1998 Academic Press)

Cyclic GMP dependent protein kinase exists as a dimer in its native form. A peptide corresponding to the dimerization domain in the N-terminal segment has been characterized by circular dichroism, ultracentrifugation, and 1H NMR spectroscopy. The peptide (G-kinase1-39 amide) is shown to be dimeric in solution. Determination of the molecular weight of the species in solution from the sedimentation coefficient and diffusion coefficient yields a value more than twice that of the monomeric species. Circular dichroism studies show G-kinase1-39 amide to be largely helical in aqueous solution and stable over a wide range of pH and temperature. The conformational stability is found to be concentration dependent, the peptide having a melting temperature of 75 degrees C (at 20 microM and pH 4.0). The assignment of the 1H NMR spectrum and analysis of the patterns of nuclear Overhauser enhancements confirm the helical nature of the conformation. Distance geometry calculations result in a well-defined helical structure containing a kink near Ser 26. The dimerization of G-kinase is most likely to occur through the hydrophobic interaction of leucine and isoleucine side chains located on one face of a helical structure with supporting electrostatic interactions between flanking side chains. The dimerization domain of G-kinase is clearly analogous to the "leucine zipper" motifs found in a number of DNA transcriptional activators.