Electrostatic interactions mediate binding of obscurin to small ankyrin 1: biochemical and molecular modeling studies.

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TitleElectrostatic interactions mediate binding of obscurin to small ankyrin 1: biochemical and molecular modeling studies.
Publication TypeJournal Article
Year of Publication2011
AuthorsBusby, B, Oashi, T, Willis, CD, Ackermann, MA, Kontrogianni-Konstantopoulos, A, Mackerell, AD, Bloch, RJ
JournalJ Mol Biol
Volume408
Issue2
Pagination321-34
Date Published2011 Apr 29
ISSN1089-8638
KeywordsAnkyrins, Guanine Nucleotide Exchange Factors, Humans, Maltose-Binding Proteins, Models, Molecular, Molecular Dynamics Simulation, Muscle Proteins, Muscle, Skeletal, Mutagenesis, Site-Directed, Mutation, Protein Conformation, Recombinant Fusion Proteins, Rho Guanine Nucleotide Exchange Factors, Sarcoplasmic Reticulum, Surface Plasmon Resonance
Abstract

Small ankyrin 1 (sAnk1; also known as Ank1.5) is an integral protein of the sarcoplasmic reticulum (SR) in skeletal and cardiac muscle cells, where it is thought to bind to the C-terminal region of obscurin, a large modular protein that surrounds the contractile apparatus. Using fusion proteins in vitro, in combination with site-directed mutagenesis and surface plasmon resonance measurements, we previously showed that the binding site on sAnk1 for obscurin consists, in part, of six lysine and arginine residues. Here we show that four charged residues in the high-affinity binding site on obscurin for sAnk1 (between residues 6316 and 6345), consisting of three glutamates and a lysine, are necessary, but not sufficient, for this site on obscurin to bind to sAnk1 with high affinity. We also identify specific complementary mutations in sAnk1 that can partially or completely compensate for the changes in binding caused by charge-switching mutations in obscurin. We used molecular modeling to develop structural models of residues 6322-6339 of obscurin bound to sAnk1. The models, based on a combination of Brownian and molecular dynamics simulations, predict that the binding site on sAnk1 for obscurin is organized as two ankyrin-like repeats, with the last α-helical segment oriented at an angle to nearby helices, allowing lysine 6338 of obscurin to form an ionic interaction with aspartate 111 of sAnk1. This prediction was validated by double-mutant cycle experiments. Our results are consistent with a model in which electrostatic interactions between specific pairs of side chains on obscurin and sAnk1 promote binding and complex formation.

DOI10.1016/j.jmb.2011.01.053
Alternate JournalJ. Mol. Biol.
PubMed ID21333652
PubMed Central IDPMC3367564
Grant ListT32 GM008181 / GM / NIGMS NIH HHS / United States
GM051501 / GM / NIGMS NIH HHS / United States
T32 AR07592 / AR / NIAMS NIH HHS / United States
T32 GM08181 / GM / NIGMS NIH HHS / United States
T32 AR007592 / AR / NIAMS NIH HHS / United States
F32 AR058079 / AR / NIAMS NIH HHS / United States
S10 RR015890 / RR / NCRR NIH HHS / United States
CA120215 / CA / NCI NIH HHS / United States
R01 CA120215 / CA / NCI NIH HHS / United States
R01 GM051501 / GM / NIGMS NIH HHS / United States
R29 GM051501 / GM / NIGMS NIH HHS / United States
R01 AR056330 / AR / NIAMS NIH HHS / United States
R01 AR052768 / AR / NIAMS NIH HHS / United States