Biophysical characterization of the unstructured cytoplasmic domain of the human neuronal adhesion protein neuroligin 3.
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.
Paz
Aviv
A
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
Zeev-Ben-Mordehai
Tzviya
T
Lundqvist
Martin
M
Sherman
Eilon
E
Mylonas
Efstratios
E
Weiner
Lev
L
Haran
Gilad
G
Svergun
Dmitri I
DI
Mulder
Frans A A
FA
Sussman
Joel L
JL
Silman
Israel
I
D001703:Biophysics / Q000379:methods; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D015816:Cell Adhesion Molecules, Neuronal; D003198:Computer Simulation; D003593:Cytoplasm / Q000737:chemistry*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D008956:Models, Chemical; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D015815:Cell Adhesion Molecules; D015816:Cell Adhesion Molecules, Neuronal; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C098004:neuroligin 3