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Steric regulation of tandem calponin homology domain actin-binding affinity

Journal Article · · Molecular Biology of the Cell
 [1];  [1];  [1];  [1];  [2];  [3];  [4]
  1. Univ. of California, Berkeley, CA (United States)
  2. Department of Mechanical Engineering, University of California, Berkeley, Berkeley, CA 94720
  3. Lehrstuhl für Biophysik (E27), Technische Universität München, Garching 85748, Germany
  4. Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Chan Zuckerberg Biohub, San Francisco, CA (United States)

Tandem calponin homology (CH1–CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1–CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for F-actin. To investigate features that affect CH1–CH2 affinity for F-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1–CH2 interface, replacing the linker domain, and adding a polyethylene glycol (PEG) polymer to CH2. Consistent with a previous model describing CH2 as a steric negative regulator of actin binding, we find that utrophin CH1–CH2 affinity is both increased and decreased by modifications that change the effective “openness” of CH1 and CH2 in solution. We also identified interface mutations that caused a large increase in affinity without changing solution “openness,” suggesting additional influences on affinity. Interestingly, we also observe nonuniform subcellular localization of utrophin CH1–CH2 that depends on the N-terminal flanking region but not on bulk affinity. Finally, these observations provide new insights into how small sequence changes, such as those found in diseases, can affect CH1–CH2 binding properties.

Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division (SUF)
Grant/Contract Number:
AC02-05CH11231
OSTI ID:
1615283
Journal Information:
Molecular Biology of the Cell, Journal Name: Molecular Biology of the Cell Journal Issue: 26 Vol. 30; ISSN 1059-1524
Publisher:
American Society for Cell BiologyCopyright Statement
Country of Publication:
United States
Language:
English

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