The post-mortem brains of individuals with Parkinson’s disease (PD) and other synucleinopathy disorders are characterized by the presence of aggregated forms of the presynaptic protein α-synuclein (aSyn). Understanding the molecular mechanism of aSyn aggregation is essential for the development of neuroprotective strategies to treat these diseases. In this study, we examined how interactions between aSyn and phospholipid vesicles influence the protein’s aggregation and toxicity to dopaminergic neurons. Two-dimensional NMR data revealed that two familial aSyn mutants, A30P and G51D, populated an exposed, membrane-bound conformer in which the central hydrophobic region was dissociated from the bilayer to a greater extent than in the case of wild-type aSyn. A30P and G51D had a greater propensity to undergo membrane-induced aggregation and elicited greater toxicity to primary dopaminergic neurons compared to the wild-type protein. In contrast, the non-familial aSyn mutant A29E exhibited a weak propensity to aggregate in the presence of phospholipid vesicles or to elicit neurotoxicity, despite adopting a relatively exposed membrane-bound conformation. Our findings suggest that the aggregation of exposed, membrane-bound aSyn conformers plays a key role in the protein’s neurotoxicity in PD and other synucleinopathy disorders.

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...

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate-specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein

Androcam is a D. melanogaster calmodulin-like protein expressed exclusively in the testis that interacts with myosin VI and is critical to spermatogenesis. At micromolar free Ca(2+), androcam binds two calcium ions using its C-terminal lobe but its N-terminal lobe is Ca(2+)-free. We are pursuing structural studies on androcam at physiological (10 μM) and high (10 mM) calcium. Here we report the (1)H, (15)N, and (13)C chemical shifts of androcam at 10 μM free Ca(2+) determined using multi-dimensional NMR experiments.

Androcam is a calmodulin-like protein that acts as a testis-specific light chain to myosin VI during spermatogenesis in D. melanogaster. Modest, localized chemical shift changes that accompany Ca(2+) binding to the androcam N-terminal lobe indicate that unlike calmodulin, androcam does not undergo a dramatic conformational change upon binding calcium. Here we report the (1)H, (15)N and (13)C resonances of androcam in the high calcium (10 mM) state and show the extent of chemical shift changes for backbone resonances relative to the low calcium state.

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