Qingqing Yang1, Haiyang Liu2, Zhiwei Li1, Yue Wang1, Wei Liu3. 1. Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, 518036, Guangdong province, China. 2. Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, 518036, Guangdong province, China; Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. 3. Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen, 518036, Guangdong province, China. Electronic address: liuwei@sphmc.org.
Abstract
TANC1 and its close relative TANC2 are two important synaptic scaffold proteins which play critical roles in regulating densities of synaptic spines and excitatory synapse strength. Recent studies indicated TANC1 and TANC2 are candidate genes of several neurodevelopmental disorders (NDD). So far, the biochemical properties of TANC1/2 proteins remain largely unknown. In this study, Ankyrin-repeats (AR) domain of TANC1 was expressed and purified using Escherichia coli. (E. coli.) cells, which showed low solubility and stability after removing the maltose binding protein (MBP) tag. Sequence analysis revealed that the TANC1 AR domain is lack of canonical N, C-capping units. By introducing two point mutations in the C-capping unit and replacing the N-capping unit, monomeric and well-folded TANC1 AR domain was purified and characterized by size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) and circular dichroism spectroscopy (CD). In addition, mutations from intellectual disability (ID) patients and cancer patients were imported into the TANC1 AR domain. The ID mutant exhibited marginal effects in terms of conformation and protein folding stability changes. By contrast, the cancer mutants dramatically decreased protein solubility. Combined with structural prediction, we speculated that mis-sense variants tested in this study may either affect protein folding or disrupt the interaction between TANC1/2 AR domains and their binding partners.
TANC1 and its close relative n class="Gene">TANC2 are two important synaptic scaffold proteins which play critical roles in regulating densities of synaptic spines and excitatory synapse strength. Recent studies indicated TANC1 and TANC2 are candidate genes of several neurodevelopmental disorders (NDD). So far, the biochemical properties of TANC1/2 proteins remain largely unknown. In this study, Ankyrin-repeats (AR) domain of TANC1 was expressed and purified using Escherichia coli. (E. coli.) cells, which showed low solubility and stability after removing the maltose binding protein (MBP) tag. Sequence analysis revealed that the TANC1 AR domain is lack of canonical N, C-capping units. By introducing two point mutations in the C-capping unit and replacing the N-capping unit, monomeric and well-folded TANC1 AR domain was purified and characterized by size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) and circular dichroism spectroscopy (CD). In addition, mutations from intellectual disability (ID) patients and cancerpatients were imported into the TANC1 AR domain. The ID mutant exhibited marginal effects in terms of conformation and protein folding stability changes. By contrast, the cancer mutants dramatically decreased protein solubility. Combined with structural prediction, we speculated that mis-sense variants tested in this study may either affect protein folding or disrupt the interaction between TANC1/2 AR domains and their binding partners.
Authors: Ezequiel A Galpern; Jacopo Marchi; Thierry Mora; Aleksandra M Walczak; Diego U Ferreiro Journal: Proc Natl Acad Sci U S A Date: 2022-07-29 Impact factor: 12.779