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J Biol Chem. 2019 Nov 15;294(46):17383-17394. doi: 10.1074/jbc.RA119.009589. Epub 2019 Oct 9.

Intrinsic disorder and amino acid specificity modulate binding of the WW2 domain in kidney and brain protein (KIBRA) to synaptopodin.

Author information

1
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331.
2
Department of Molecular Nephrology, University Hospital, D-48149 Münster, Germany.
3
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331 nyarkoa@oregonstate.edu.

Abstract

The second WW domain (WW2) of the kidney and brain scaffolding protein, KIBRA, has an isoleucine (Ile-81) rather than a second conserved tryptophan and is primarily unstructured. However, it adopts the canonical triple-stranded antiparallel β-sheet structure of WW domains when bound to a two-PPXY motif peptide of the synaptic protein Dendrin. Here, using a series of biophysical experiments, we demonstrate that the WW2 domain remains largely disordered when bound to a 69-residue two-PPXY motif polypeptide of the synaptic and podocyte protein synaptopodin (SYNPO). Isothermal titration calorimetry and CD experiments revealed that the interactions of the disordered WW2 domain with SYNPO are significantly weaker than SYNPO's interactions with the well-folded WW1 domain and that an I81W substitution in the WW2 domain neither enhances binding affinity nor induces substantial WW2 domain folding. In the tandem polypeptide, the two WW domains synergized, enhancing the overall binding affinity with the I81W variant tandem polypeptide 2-fold compared with the WT polypeptide. Solution NMR results showed that SYNPO binding induces small but definite chemical shift perturbations in the WW2 domain, confirming the disordered state of the WW2 domain in this complex. These analyses also disclosed that SYNPO binds the tandem WW domain polypeptide in an antiparallel manner, that is, the WW1 domain binds the second PPXY motif of SYNPO. We propose a binding model consisting of a bipartite interaction mode in which the largely disordered WW2 forms a "fuzzy" complex with SYNPO. This binding mode may be important for specific cellular functions.

KEYWORDS:

PPXY motif; WW domain; circular dichroism (CD); intrinsically disordered protein; isothermal titration calorimetry (ITC); nuclear magnetic resonance (NMR); proline-rich motif; protein-protein interaction; scaffolding protein; synaptopodin (SYNPO)

PMID:
31597702
PMCID:
PMC6873182
[Available on 2020-11-15]
DOI:
10.1074/jbc.RA119.009589

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