Structure of an enzyme-derived phosphoprotein recognition domain

Abstract

Membrane Associated Guanylate Kinases (MAGUKs) contain a protein interaction domain (GK(dom)) derived from the enzyme Guanylate Kinase (GK(enz)). Here we show that GK(dom) from the MAGUK Discs large (Dlg) is a phosphoprotein recognition domain, specifically recognizing the phosphorylated form of the mitotic spindle orientation protein Partner of Inscuteable (Pins). We determined the structure of the Dlg-Pins complex to understand the dramatic transition from nucleotide kinase to phosphoprotein recognition domain. The structure reveals that the region of the GK(dom) that once served as the GMP binding domain (GBD) has been co-opted for protein interaction. Pins makes significantly more contact with the GBD than does GMP, but primarily with residues that are conserved between enzyme and domain revealing the versatility of the GBD as a platform for nucleotide and protein interactions. Mutational analysis reveals that the GBD is also used to bind the GK ligand MAP1a, suggesting that this is a common mode of MAGUK complex assembly. The GK(enz) undergoes a dramatic closing reaction upon GMP binding but the protein-bound GK(dom) remains in the 'open' conformation indicating that the dramatic conformational change has been lost in the conversion from nucleotide kinase to phosphoprotein recognition domain.

Figure 1. The Guanylate Kinase domain is an enzyme-derived phosphoprotein recognition domain.

(A) The Dlg GK domain is a specific phosphoprotein recognition domain. A GST-pull down experiment shows that the Dlg SH3-GK region only interacts with the Pins Linker domain when it has been phosphorylated by Aurora A. (B) Change in fluorescence anisotropy of phosphorylated and unphosphorylated Pins Linker peptides as a function of Dlg GK domain concentration. The curves represent binding affinities of 0.8 µM (phosphorylated) and 206 µM (unphosphorylated). (C) Domain structure of Pins and Dlg. Pins consists of Tetratricopeptide repeats (TPR), a linker domain (L), and three GoLoco motifs (1–3). Dlg contains three PDZ domains, and SH3 domain, and the GK domain. The mitotic kinase Aurora A phosphorylates the Pins Linker domain initiating an interaction with the Dlg GK domain.

Figure 2. Phospho-Pins binds the vestigial GK GMP binding domain.

(A) Structure of Dlg-Pins. LID, CORE, and GBD subdomains, as described for the GK enzyme, are highlighted. The phosphomimetic D436 is shown in the Pins Linker. (B) The GMP nucleotide-binding pocket. The left panel shows the interaction of GMP with the GBD subdomain taken from the yeast Guanylate Kinase structure 1EX7 . The right panel shows only those residues of the Pins Linker domain that occupy the nucleotide binding pocket to show how the phosphorylated residue (D436 in the structure) and M437 mimic GMP interactions. An overlay of GMP and Pins residues 436 and 437 on the GK domain is also shown. (C) GK enzyme phosphorecognition. Three residues form the primary contacts with the GMP phosphate, as shown from the yeast Guanylate Kinase structure 1EX7 . (D) GK domain phosphorecognition. The residues that contact the phosphate in the GK enzyme are conserved in the domain and assume an identical configuration.

Figure 3. Extensive interactions between the Pins Linker and the GK domain GBD.

(A) Pins Linker makes extensive GBD contacts outside of the nucleotide binding site. The two residues that occupy the nucleotide binding site, D436 and M437, are shown along with the hydrophobic residues that form interactions along the GBD. (B) Alignment of GK enzyme and domain GMP-binding domains. (Ec = Escherichia coli; Sc = Saccharomyces cerevisiae; At = Arabidopsis thaliana; Dm = Drosophila melanogaster; Mm = Mus musculus; Hs = Homo sapiens). Residues that contact Pins are indicated by an arrow (a dot above the arrow indicates backbone contacts). The conserved alanine residue (number 852 in Discs large) is highlighted. Residues that contact the phosphate are shown by a red circle. Asterisk indicates residue that induces functional switch from enzyme to domain . (C) MAP1a residues 1862–1883 compete with Pins for binding to the GK domain. A GST-fusion of the Pins linker region that was incubated with Aurora A efficiently pulls down the Dlg SH3-GK module, but this interaction is displaced by a MAP1a peptide. (D) Aspartic acid 1874 within MAP1a residues 1862–1883 is required for interaction with the GK domain as assessed by GST-pull down.

Figure 4. Dlg GK domain contacts with Pins Linker are required for mitotic spindle orientation.

(A) GK domain mutations in residues at the binding interface lower the affinity for a phosphorylated Pins Linker peptide. The change in anisotropy of a rhodamine attached to the peptide is shown as a function of wild-type or the indicated mutant Dlg GK domains. (B) Pins Linker domain mutations in residues at the binding interface lower the affinity for the GK domain. GST-fusions of Pins linker regions containing the indicated mutations were incubated with Aurora A and their ability to pull-down the Dlg SH3-GK were assessed. (C) Schematic of Drosophila S2 cell induced polarity spindle orientation assay. Clustered cells polarize Echinoid-Pins (Ed-Pins) to sites of cell-cell contact. Ed-Pins with mutant Linker domains were assessed for their ability to orient the spindle by measuring the angle between the center of the crescent and the mitotic spindle. (D) Cumulative percentage plots of spindle orientation by Ed-Pins mutants. These plots show the cumulative percentage of cells that have a spindle angle below a particular value (x-axis). Cells expressing wild-type Ed-Pins have predominantly small angles between the Ed-Pins crescent and the spindle whereas cells expressing defective Ed-Pins have random distributions (diagonal distribution in the cumulative percentage plot). Mutation of Pins Linker residues that contact the GK domain (top panel) leads to loss of spindle orienting activity. In contrast, mutation of a residue that faces away from the domain (D441A) has little effect. (E) GK^dom-Pins^LINKER interactions are required for GK^dom recruitment to induced Ed-Pins crescents in Drosophila S2 cells. A GFP-fusion of the Dlg SH3-GK domain localizes to Ed-Pins crescents (white arrowhead). Mutation of Y831 or Y860 in the GK domain GBD to alanine prevents recruitment. (F) Quantification of GK^dom recruitment to induced Ed-Pins crescents in Drosophila S2 cells for the data in panel E. The ratio of the cortex and the cytoplasm for the GFP-Dlg SH3-GK signal is shown. Error bar represents one standard deviation. Asterisks represent p<0.001 using ANOVA with Dunn's post-hoc test.

Figure 5. Loss of a ligand-induced conformational change in the GK domain.

(A) Nucleotide binding to the GK enzyme induces a large conformational change. GMP binding causes a transition from an “open" conformation to a “closed" one allowing for long range communication between the ATP and GMP binding sites. The “hinge" region that undergoes dihedral angle changes during closing is shown. Recently it was found that mutation of a serine residue (S35) in this region to proline is sufficient to convert enzyme to domain . Structures 1EX6 and 1EX7 are shown . (B) The GK domain conformation does not change upon ligand binding. Unlike the GK enzyme, the GK domain does not assume a “closed" conformation upon Pins binding to the GBD. (C) GK closing is incompatible with Pins binding to the GBD. Ribbon and surface representations are shown of Pins aligned to the closed GK enzyme structure (1EX7) showing that closing would cause dramatic steric overlaps with the protein ligand. (D) A critical serine to proline mutation is in the GK “hinge" region. Mutation of a conserved GK enzyme serine is sufficient to convert it into a GK domain . The position of this proline, which lies in the region that undergoes dramatic dihedral angle changes in GK enzyme, is shown in a ribbon representation.