Dataset Information

Molecular basis of PIP2-dependent conformational switching of phosphorylated CD44 in binding FERM.

ABSTRACT: Association of the cellular adhesive protein CD44 and the N-terminal (FERM) domain of cytoskeleton adaptors is critical for cell proliferation, migration, and signaling. Phosphorylation of the cytoplasmic domain (CTD) of CD44 acts as an important regulator of the protein association, but the structural transformation and dynamics mechanism remain enigmatic. In this study, extensive coarse-grained simulations were employed to explore the molecular details in the formation of CD44-FERM complex under S291 and S325 phosphorylation, a modification path known to exert reciprocal effects on the protein association. We find that phosphorylation of S291 inhibits complexation by causing the CTD of CD44 to adopt a more closed structure. In contrast, S325 phosphorylation liberates the CD44-CTD from the membrane surface and promotes the linkage with FERM. The phosphorylation-driven transformation is found to occur in a PIP2-dependent manner, with PIP2 effecting the relative stability of the closed and open conformation, and a replacement of PIP2 by POPS greatly abrogates this effect. The revealed interdependent regulation mechanism by phosphorylation and PIP2 in the association of CD44 and FERM further strengthens our understanding of the molecular basis of cellular signaling and migration.

SUBMITTER: Ren M

PROVIDER: S-EPMC10397572 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Molecular basis of PIP2-dependent conformational switching of phosphorylated CD44 in binding FERM.

Ren Meina M Zhao Lina L Ma Ziyi Z An Hailong H Marrink Siewert Jan SJ Sun Fude F

Biophysical journal 20230522 13

Association of the cellular adhesive protein CD44 and the N-terminal (FERM) domain of cytoskeleton adaptors is critical for cell proliferation, migration, and signaling. Phosphorylation of the cytoplasmic domain (CTD) of CD44 acts as an important regulator of the protein association, but the structural transformation and dynamics mechanism remain enigmatic. In this study, extensive coarse-grained simulations were employed to explore the molecular details in the formation of CD44-FERM complex und ...[more]

PMID: 37218130

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Project description:Interferon-gamma stimulation triggers tyrosine phosphorylation of the transcription factor STAT1 at position 701, which is associated with switching from carrier-independent nucleocytoplasmic shuttling to carrier-mediated nuclear import. Unlike most substrates that carry a classical nuclear localization signal (NLS) and bind to importin alpha1, STAT1 possesses a nonclassical NLS recognized by the isoform importin alpha5. In the present study, we have analyzed the mechanisms by which importin alpha5 binds phosphorylated STAT1 (pSTAT1). We found that a homodimer of pSTAT1 is recognized by one equivalent of importin alpha5 with K(d)=191+/-20 nM. Whereas tyrosine phosphorylation at position 701 is essential to assemble a pSTAT1-importin alpha5 complex, the phosphate moiety is not a direct binding determinant for importin alpha5. In contrast to classical NLS substrates, pSTAT1 binding to importin alpha5 is not displaced by the N-terminal importin beta binding domain and requires the importin alpha5 C-terminal acidic tail (505-EEDD-508). A local unfolding of importin alpha5 Armadillo (ARM) repeat 10 accompanies high-affinity binding to pSTAT1. This unfolding is mediated by a single conserved tyrosine at position 476 of importin alpha5, which is inserted between ARM repeat 10 helices H1-H2-H3, thereby preventing intramolecular helical stacking essential to stabilize the folding conformation of ARM 10. Introducing a glycine at this position, as in importin alpha1, disrupts high-affinity binding to pSTAT1, suggesting that pSTAT1 recognition is dependent on the intrinsic flexibility of ARM 10. Using the quantitative stoichiometry and binding data presented in this article, together with mutational information available in the literature, we propose that importin alpha5 binds between two STAT1 monomers, with two major binding determinants in the SH2 and DNA binding domains. In vitro, this model is supported by the observation that a 38-mer DNA oligonucleotide containing two tandem cfosM67 promoters can displace importin alpha5 from pSTAT1, suggesting a possible role for DNA in releasing activated STAT1 in the cell nucleus.

Dataset Information

Molecular basis of PIP2-dependent conformational switching of phosphorylated CD44 in binding FERM.

Publications

Molecular basis of PIP2-dependent conformational switching of phosphorylated CD44 in binding FERM.

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