Crystal structure of a PP2A B56-BubR1 complex and its implications for PP2A substrate recruitment and localization

Abstract

Protein phosphatase 2A (PP2A) accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a catalytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kinetochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interaction plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attachment. Here we present the crystal structure of a PP2A B56-BubR1 complex, which demonstrates that a conserved BubR1 LxxIxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxIxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.

Keywords: BubR1; PP2A; cellular targeting; kinetochore; substrate recruitment.

Figure 1

Overall structure of the B56-BubR1 complex. (A) Schematic representations of domain structures of human BubR1 and PP2A B56γ1 proteins. Numbers above indicate amino acid position based on the sequence of human BubR1 and B56γ1. The fragments used for crystallization are indicated by red boxes. (B) Overall structure of the B56γ1(30–380)/BubR1(647–720)-3D complex, in two orthogonal views. (C) Superposition of the B56γ1-BubR1 structure with that of the Aα-B56γ1-Cα PP2A holoenzyme (PDB code: 2IAE). The red spheres are the two Mn²⁺ ions in the PP2A active site.

Figure 2

Details of the B56-BubR1 interface. (A) Stereo view of the interface interactions. BubR1 and B56 residues are labelled in red and blue, respectively. (B) The BubR1 KARD domain structure (in sticks) is laid on top of electrostatic surface representation of B56γ1.

Figure 3

Sequence alignment of B56γ1 and mutagenesis of B56γ1. (A) B56 sequence alignment. The upper half shows the alignment of the five different human B56 isoforms, whereas the lower half demonstrates the sequence conservation of B56γ from different organisms. Numbers above indicate amino acid position based on the sequence of human B56γ1. B56γ has three isoforms with different C-terminal domains. Only B56γ1 is shown here. The strictly conserved residues are in white letters with red background and the conserved residues are in red letters with yellow background. Residues involved in BubR1 interaction are indicated with red asterisks. (B) Pulldown of mutant B56γ1 proteins by WT GST-BubR1(647–720).

Figure 4

Sequence alignment of BubR1 and mutagenesis analysis of the B56γ1-BubR1 interface. (A) Sequence alignment of the KARD domain of BubR1. BubR1 residues essential for B56 binding are labelled with red stars, whereas phosphorylation sites are labeled green pound signs. (B) GST-pulldown of B56γ1 by mutant GST-BubR1(647–720) proteins. BubR1-AAAA represents GST-tagged BubR1(647–720; E678A/T680A/H681A).

Figure 5

Superposition of the B56-BubR1 complex with the PP2A-shugoshin complex. The Aα-B56γ1-Cα PP2A/Sgo1 complex structure (PDB code: 3FGA) is superimposed to the current B56γ1-BubR1 complex structure, based on the common B56γ1 subunit. The Sgo1 coiled-coil domain homodimer, which is responsible for PP2A binding, is in blue color.