A structural characterization of the interactions between titin Z-repeats and the alpha-actinin C-terminal domain

Titin and alpha -actinin, two modular muscle proteins, are with actin the m ajor components of the Z-band in vertebrate striated muscles where they ser ve to organize the antiparallel actin filament arrays in adjacent sarcomere s and to transmit tension between sarcomeres during activation. Interaction s 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 qua ntitative characterization of alpha -actinin-Z-repeat recognition and disse ct the interaction to its minimal units. Different complementary techniques , such as circular dichroism, calorimetry, and nuclear magnetic spectroscop y, were used. Two overlapping alpha -actinin constructs (Act-EF34 and Act-E F1234) 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 t o Z-repeats, excluding the presence of additional high-affinity binding sit es in the remaining part of the domain. The binding affinities of the diffe rent Z-repeats for Act-EF34 range from micromolar to millimolar values. The strongest of these interactions are comparable to those observed in tropon in 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 wer e observed for Z-repeat double constructs. These findings have direct relev ance for evaluating current models of Z-disk assembly.