The Relationship between Structural Symmetry and Function in Membrane Proteins

Abstract

Multivalent interactions of intrinsically disordered segments of proteins are essential in many cellular functions, but challenges in recombinant protein production has limited understanding of how the complexes assemble. One example is the interaction between Angiomotin-like 1 (AMOTL1), Yesassociated protein (YAP), and kidney and brain expressed protein (KIBRA), three multivalent proteins with roles in protein scaffolding, cell polarity, memory performance, and cell proliferation. YAP and KIBRA each contain two tandem WW domains, a 40-residue interaction module composed of three antiparallel b-strands which recognize the Leucine/Proline-Proline-any amino acid-Tyrosine (L/PPXY) motif, three of which are present in a primarily disordered segment of AMOTL1. While several studies support co-localization of AMOTL1 with YAP and/or KIBRA in cultured cells, the structural basis of assembly in the context of all binding-competent domains is not well understood. Here, we show by multiple approaches including circular dichroism (CD), isothermal titration calorimetry (ITC), analytical size exclusion chromatography (SEC), and solution NMR that YAP and KIBRA bind AMOTL1 with micromolar affinity and have mapped the binding interfaces to the N and C-terminal L/PPXY motifs, respectively. The two KIBRA WW domains synergize to enhance binding to the AMOTL1 motifs but only one WW domain was required for assembly of the YAP-AMOTL1 complex. Our findings provide novel, molecular-level insights that extend understanding of intrinsically disordered regions and multivalent binding modules in protein complexes.