pH-Sensitive Monomer-Dimer Equilibrium and Self-Association of the Cytoplasmic, N-Terminal Domain of the NBCe1-A Cotransporter

Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking.

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Periphilin self-association underpins epigenetic silencing by the HUSH complex

10.1101/2019.12.18.881300 ◽

2019 ◽

Cited By ~ 2

Author(s):

Daniil M. Prigozhin ◽

Anna Albecka ◽

Christopher H. Douse ◽

Iva A. Tchasovnikarova ◽

Richard T. Timms ◽

...

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Low Complexity ◽

Core Complex ◽

Terminal Domain ◽

Genome Wide ◽

Repressive Mark ◽

Self Association ◽

Nascent Transcripts

AbstractTranscription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR core complex shows Periphilin forms α-helical homodimers, which each bind a single TASOR molecule. The NTD binds RNA non-specifically and forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation on nascent transcripts.

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pH-sensitive Self-associations of the N-terminal Domain of NBCe1-A Suggest a Compact Conformation under Acidic Intracellular Conditions

Protein and Peptide Letters ◽

10.2174/092986612802762642 ◽

2012 ◽

Vol 19 (10) ◽

pp. 1054-1063 ◽

Cited By ~ 5

Author(s):

Harindarpal S. Gill

Keyword(s):

Ph Sensitive ◽

Terminal Domain ◽

Compact Conformation

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Functional Domains of Yeast Plasmid-Encoded Rep Proteins

Journal of Bacteriology ◽

10.1128/jb.183.7.2306-2315.2001 ◽

2001 ◽

Vol 183 (7) ◽

pp. 2306-2315 ◽

Cited By ~ 21

Author(s):

A. Sengupta ◽

K. Blomqvist ◽

A. J. Pickett ◽

Y. Zhang ◽

J. S. K. Chew ◽

...

Keyword(s):

Amino Acids ◽

Protein Interaction ◽

Yeast Plasmid ◽

Amino Terminal ◽

Plasmid Segregation ◽

Terminal Domain ◽

Rep Proteins ◽

Self Association ◽

Carboxy Terminal

ABSTRACT Both of the Saccharomyces cerevisiae 2μm circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division. In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2. Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions. Amino- or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2μm-based stability vector withrep1 deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxy-terminal portion of Rep1. The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association. The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2μm circle plasmid stability locus, forming a structure that mediates plasmid segregation. In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex.

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