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ABSTRACT
Cholinesterase-like adhesion molecules (CLAMs) are a family of neuronal cell adhesion molecules with important roles in synaptogenesis, and in maintaining structural and functional integrity of the nervous system. Our earlier study on the cytoplasmic domain of one of these CLAMs, the Drosophila protein, gliotactin, showed that it is intrinsically unstructured in vitro. Bioinformatic analysis suggested that the cytoplasmic domains of other CLAMs are also intrinsically unstructured, even though they bear no sequence homology to each other or to any known protein. In this study, we overexpress and purify the cytoplasmic domain of human neuroligin 3, notwithstanding its high sensitivity to the Escherichia coli endogenous proteases that cause its rapid degradation. Using bioinformatic analysis, sensitivity to proteases, size exclusion chromatography, fluorescence correlation spectroscopy, analytical ultracentrifugation, small angle x-ray scattering, circular dichroism, electron spin resonance, and nuclear magnetic resonance, we show that the cytoplasmic domain of human neuroligin 3 is intrinsically unstructured. However, several of these techniques indicate that it is not fully extended, but becomes significantly more extended under denaturing conditions.
INTRODUCTION
The neuroligins (NLs) are a set of neuronal adhesion proteins belonging to the family of cholinesterase-like adhesion molecules (CLAMs). All CLAMs contain an extracellular domain that bears high sequence homology to acetylcholinesterase (AChE), and share the same conserved intrachain disulfides. However, they all lack one or more of the members of the catalytic triad of AChE, and are thus believed to be catalytically inactive ( 1 ). To date, three transmembrane CLAMs have been identified: the mammalian and invertebrate NLs, and the insect proteins, gliotactin (GIi) and neurotactin (Nrt) (2) (Fig. 1 ). All three are single-pass transmembrane proteins whose cytoplasmic domains contain -120-320 amino-acid residues, and bear no sequence similarity to any known protein, or even to each other.
NLs are expressed in both neurons and glia in the nervous system, as well as in a variety of other tissues (3-5). At synapses, they are associated with the postsynaptic membrane, and have been shown to bind through their AChE-like domains to the extracellular domains of both a- and /3-neurexin, which are also synaptic adhesion proteins (3,6,7). The cytoplasmic domains of the NLs, as well as that of GIi, that is, NL-cyt and Gli-cyt, contain PDZ recognition motifs at their C-termini (2)....