Identification of the domains within the N2A region of titin that regulate binding to actin.

Titin, the largest muscle protein, plays an important role in passive tension, sarcomeric integrity and cell signaling within the muscle. Recent work has also highlighted a role for titin in active muscle and the N2A region found in skeletal muscle titin and in some isoforms of cardiac titin has been linked to this function. The N2A region is a multi-domain region composed of four immunoglobulin domains (I80-I83) and a disordered region called the insertion sequence. Previously, our lab has shown that the N2A region binds F-actin in a calcium dependent manner, but it is not known which domains within this region are critical for this binding to occur. In this work, we have used co-sedimentation to demonstrate that only constructs containing the I80 domain are capable of binding F-actin. In addition, binding was only observed in constructs containing at least 3 immunoglobulin domains suggesting a length-dependence to binding. Finally, the calcium-dependence of N2A binding is lost when I83 is not present, consistent with the calcium stabilization that has been reported for this domain. Based on these results, we propose that I80 is critical for initiating binding to F-actin and that I83 is responsible for the calcium dependence.