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

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.

Multivalent Angiomotin‐like 1 and Yes‐associated protein form a dynamic complex

“…We conclude that the apo AMOTL1 PPxY domain, A 123 , has a limited folded structure, localized in two short helical segments, residues 193–197 and 245–257 as shown by CD and NMR. This first experimental demonstration that the full AMOTL1 PPxY segment is partially disordered is consistent with the structures of other multivalent PPxY proteins 24–26 . Second, the AMOTL1‐YAP PPxY‐WW complex is formed by one molecule of AMOTL1 bound to one molecule of YAP, as shown by the hydrodynamic method of SEC‐MALS and ITC.…”

“…Proteins were overexpressed at 20°C for 5 (A 123 variants) or 16 (YWW TD proteins) hours after induction with 0.1 mM IPTG. His 6 ‐tagged recombinant proteins were purified as previously reported 25 . The A 123 polypeptides were prone to proteolytic degradation, therefore, to minimize degradation the purification tag which is part of the expression vector was not removed from the N‐terminal end of the polypeptide.…”

“…Heteronuclear NOE (HetNOE), longitudinal ( R 1 ), and transverse ( R 2 ) spin relaxation rates were measured using TROSY‐based interleaved pulse sequences 44 . HetNOE experiments were collected with and without proton saturation using a relaxation delay of 8 s. NOE errors were calculated as previously reported 25 . R 1 spin relaxation rates were measured with relaxation delays of 0.02, 0.06 (×3), 0.1, 0.2, 0.4, 0.6, 0.8, and 1.2 s. R 2 spin relaxation rates were obtained with delays of 17, 34 (×3), 51, 68, 85, 140, 170, and 240 ms with a recycle delay of 1.5 s. R 1 and R 2 data were fit to a single exponential decay function, I ( t ) = I 0 e − Rt , where t is the variable relaxation delay and R is the relaxation rate.…”

The Effect of Interleukin-4 and Dexamethasone on RNA-Seq-Based Transcriptomic Profiling of Human Podocytes: A Potential Role in Minimal Change Nephrotic Syndrome

“…The knockdown of WWC1 in an in vitro model promoted podocyte apoptosis by inducing nuclear relocation of dendrin protein [ 48 ] and impaired directed cell migration [ 49 ]. Conversely, KIBRA has also been shown to promote podocyte injury by inhibiting YAP signaling, by disrupting actin cytoskeletal dynamics [ 50 ], and by interacting with synaptopodin [ 51 ]. While the published results on the role of WWC1 in podocytes appear contradictory, WWC1 expression was downregulated in the present study.…”

PSINDB: A comprehensive database of postsynaptic protein-protein interactions

“…Intrinsically disordered regions (IDRs) were shown to be critical components for network assembly in the PSD [8], often by containing short linear motifs mediating transient interactions [9], post-translational modification sites regulating interactions as switches [10] and more. These regions play crucial roles in forming higher-order assemblies of the PSD, such as fuzzy complexes [11] and membraneless organelles via liquid-liquid phase separation [12].…”