Androcam replaces calmodulin as a tissue-specific myosin VI light chain on the actin cones that mediate D. melanogaster spermatid individualization. We show that the androcam structure and its binding to the myosin VI structural (Insert 2) and regulatory (IQ) light chain sites are distinct from those of calmodulin and provide a basis for specialized myosin VI function. The androcam N lobe noncanonically binds a single Ca 2þ and is locked in a "closed" conformation, causing androcam to contact the Insert 2 site with its C lobe only. Androcam replacing calmodulin at Insert 2 will increase myosin VI lever arm flexibility, which may favor the compact monomeric form of myosin VI that functions on the actin cones by facilitating the collapse of the C-terminal region onto the motor domain. The tethered androcam N lobe could stabilize the monomer through contacts with C-terminal portions of the motor or recruit other components to the actin cones. Androcam binds the IQ site at all calcium levels, constitutively mimicking a conformation adopted by calmodulin only at intermediate calcium levels. Thus, androcam replacing calmodulin at IQ will abolish a Ca 2þ -regulated, calmodulin-mediated myosin VI structural change. We propose that the N lobe prevents androcam from interfering with other calmodulin-mediated Ca 2þ signaling events. We discuss how gene duplication and mutations that selectively stabilize one of the many conformations available to calmodulin support the molecular evolution of structurally and functionally distinct calmodulin-like proteins. 2þ signals, calmodulin, is ubiquitously expressed and completely conserved across vertebrates; only three of its 148 residues differ between man and D. melanogaster. The flexible calmodulin central linker connects two globular lobes, each with two EF-hand motifs and a short antiparallel β sheet (1, 2). Ca 2þ binding to EF-hand motif side chain and backbone oxygens changes each lobe from a "closed" antiparallel four helix bundle to an "open" conformation that exposes a hydrophobic binding cleft (3, 4). Calmodulin binds hundreds of proteins in many interaction modes (5), but despite this versatility, all metazoans express additional Ca 2þ binding EFhand proteins similar to calmodulin. What roles these proteins play in Ca 2þ signaling are the subject of ongoing research.We are studying the structure and binding properties of the testis-specific calmodulin-like D. melanogaster protein androcam (6), which is conserved across the genus (7), to understand its role as a tissue-specific light chain for myosin VI in fly spermatogenesis (8). In mammals, the light chain calmodulin binds to two myosin VI motifs within the lever arm: the myosin VI-specific Insert 2 sequence and an IQ motif (9, 10). In the Drosophila ovary, calmodulin binds to myosin VI, but in the testis, androcam (and not calmodulin) coimmunoprecipitates with myosin VI (8).Androcam colocalizes with myosin VI at the flat leading edge of specialized structures (actin cones) as they move along the axonemes during spermat...