2؉ and myristoylation. The dimerization site is composed of residues in EF4 and the loop region between EF3 and EF4, confirmed by mutagenesis. We present the structure of the VILIP-1 dimer and a Ca 2؉ -myristoyl switch to provide structural insights into Ca 2؉ -induced trafficking of nicotinic acetylcholine receptors.
Visinin-like protein 1 (VILIP-1)2 is a neuronal Ca 2ϩ sensor (NCS) protein belonging to the calmodulin superfamily of calcium sensor proteins (1-4). VILIP-1 is expressed throughout the brain with high expression levels in the rat hippocampus (5, 6), where it may play a role in regulating synaptic plasticity relevant for learning and memory (7,8). VILIP-1 regulates several neuronal signaling pathways implicated in synaptic plasticity, such as cyclic nucleotide cascades (9 -12) and nicotinergic signaling (13). Moreover, VILIP-1 binds to the ␣-subunit of the ␣ 4  2 nicotinic acetylcholine receptor (nAChR) and promotes surface expression and trafficking of ␣ 4  2 nAChR in oocytes (13) and hippocampal neurons (14). VILIP-1 has been implicated in the modulation of neuronal excitability by influencing ␣ 4  2 nAChR signaling in hippocampal neurons (14 -16). VILIP-1 has also been implicated in the pathology of CNS diseases (17), including Alzheimer's disease (18, 19) and schizophrenia. Indeed, VILIP-1 expression shows cell-specific changes in the brains of schizophrenics and in animal models of the disease (16,20,21). Thus, VILIP-1 appears to be an important Ca 2ϩ sensor for controlling neuronal excitability by modulating nicotinergic neurotransmission important for synaptic plasticity and disease processes (17,22).VILIP-1 belongs to the NCS family of Ca 2ϩ -myristoyl switch proteins (Fig. 1). The three-dimensional structures are known for NCS-1 (23), recoverin (24, 25), and neurocalcin (26). The common structural features of NCS proteins are an ϳ200-residue chain containing four EF-hand motifs (EF1, EF2, EF3, and EF4), the sequence CPXG in the first EF-hand that eliminates its capacity to bind Ca 2ϩ and an N-terminal myristoylation consensus sequence. The binding of Ca 2ϩ to NCS proteins (e.g. NCS-1 (27), recoverin (28), and neurocalcin (29)) induces their binding to cellular membranes (29 -31). The N-terminal myristoyl group has been shown to be sequestered structurally inside Ca 2ϩ -free recoverin (32, 33), whereas the binding of two Ca 2ϩ to recoverin leads to the extrusion of the covalently attached myristoyl group (24). The Ca 2ϩ -induced exposure of the myristoyl group, termed Ca 2ϩ -myristoyl switch, enables recoverin and related NCS proteins to bind membranes only at high Ca 2ϩ . In this study, we report on the structural analysis of Ca 2ϩ and Mg 2ϩ binding to VILIP-1 to characterize the structural mechanism of the Ca 2ϩ -myristoyl switch and determine its dimeric structure. We show that VILIP-1 binds functionally to Mg 2ϩ at EF3 and binds cooperatively to Ca 2ϩ at EF2 and EF3. VILIP-1 sequesters its N-terminal myristoyl group inside the apo-protein core and exhibits Ca 2ϩ -induced extrusion o...
