Activation of moesin, a protein that links actin cytoskeleton to the plasma membrane, occurs by phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequentially to two sites and releasing an autoinhibitory linker

Abstract

Many cellular processes depend on ERM (ezrin, moesin, and radixin) proteins mediating regulated linkage between plasma membrane and actin cytoskeleton. Although conformational activation of the ERM protein is mediated by the membrane PIP2, the known properties of the two described PIP2-binding sites do not explain activation. To elucidate the structural basis of possible mechanisms, we generated informative moesin mutations and tested three attributes: membrane localization of the expressed moesin, moesin binding to PIP2, and PIP2-induced release of moesin autoinhibition. The results demonstrate for the first time that the POCKET containing inositol 1,4,5-trisphosphate on crystal structure (the "POCKET" Lys-63, Lys-278 residues) mediates all three functions. Furthermore the second described PIP2-binding site (the "PATCH," Lys-253/Lys-254, Lys-262/Lys-263) is also essential for all three functions. In native autoinhibited ERM proteins, the POCKET is a cavity masked by an acidic linker, which we designate the "FLAP." Analysis of three mutant moesin constructs predicted to influence FLAP function demonstrated that the FLAP is a functional autoinhibitory region. Moreover, analysis of the cooperativity and stoichiometry demonstrate that the PATCH and POCKET do not bind PIP2 simultaneously. Based on our data and supporting published data, we propose a model of progressive activation of autoinhibited moesin by a single PIP2 molecule in the membrane. Initial transient binding of PIP2 to the PATCH initiates release of the FLAP, which enables transition of the same PIP2 molecule into the newly exposed POCKET where it binds stably and completes the conformational activation.

FIGURE 1.

Moesin domain organization, conformational states, and binding sites. Correspondence between regions of sequence and the domain architecture of full-length moesin. Colors correspond to distinct functional parts: lobe A of FERM domain (green), lobe B (orange), lobe C (yellow), α-helical region (blue), FLAP (red), and C-terminal tail (gray). A, domains of moesin within the primary sequence. Residue numbers in human moesin are shown for boundaries and for the C-terminal phosphorylation site. B, schematic of full-length inactive and active conformations. Also shown in light blue and labeled in the figure are four known binding sites of the ERM protein that include two for PIP2 (1 and 2) and two for protein ligands (3 and 4). The two PIP-2 binding sites are shaded dark green. The two protein-binding sites are enclosed with dashed regions. a, schematic of inactive conformation (based on structure 2I1K) in which three binding sites are masked (by α-helical region, FLAP, and tail) but the PIP2-binding lysine PATCH (1) remains exposed. (The connecting peptide between the FLAP and C-terminal tail is poorly visible because it runs behind lobe C in this perspective.) b, schematic of active conformation in which three of the binding sites are unmasked. The helical region is shown with dashes in the middle to indicate it is longer than shown.

FIGURE 2.

The PIP2 binding POCKET defined by crystallography is necessary for ERM activation. A, the complex of radixin FERM domain with IP3 (PDB 1GC6) represented as ribbon drawing. The dashed box highlights the basic cleft between lobes A and C. The IP3 molecule is shown in a ball and stick model. The positively charged residues proposed to interact with the phosphate groups of IP3 are shown in space-filled and colored blue. The side chains of IP3 are shown in ball-and-stick models. Inset shows expanded view of this cleft. B, membrane enrichment of GFP-tagged constructs of WT or mutant moesin (and GFP-only as control) was assessed in representative midplane confocal images of transfected Jurkat cells. The large nucleus of Jurkat is indicated with “N.” Scale bar is 5 μm. C, quantitation of membrane enrichment of moesin constructs in Jurkat cells having the indicated mutations of lysines in the PIP2-binding POCKET. The enrichment characteristic of the WT protein is indicated by the upper dashed line. Proteins not enriched at the plasma membrane typically have ratios of membrane/cytoplasmic localization of ∼1.0 (lower dashed line). Statistical significance compared with Moesin WT membrane enrichment is shown as: *, p < 0.1; **, p < 0.01; ***, p < 0.001.

FIGURE 3.

Mutation of the PIP2 POCKET abolishes PIP2 binding. A, binding of WT and K63N/K278N moesin to PIP2 in LUV was measured by a cosedimentation assay over the indicated range of PIP2 concentrations. Dissociation constants (mean of two independent experiments ± S.D.) are presented in the lower panel. B and C, binding of moesin to the cytoplasmic tail of CD44 (B) and the tail of NHERF1 (C) were assessed by pulldown assays. Recombinant proteins WT and K63/278N moesin were tested in the presence of lipid control PS (phosphatidylserine) (upper panels) or PIP2 (lower panels). Regions shown are from the pellet using anti-His to detect moesin pulled down and Coomassie stain to validate equal loading of GST-CD44 or GST-NHERF1.

FIGURE 4.

The PATCH of four lysines is critical for membrane localization of ERM protein. A, electrostatic surface potential of full-length insect ERM protein (PDB 2I1K) with color charge scale on the left. The molecule is oriented to show the single area of highest positive charge on the entire molecule (dashed circle). Labels indicate locations of the A, B, and C lobes. Location and identity of positively charged resides in the area are indicated. B, membrane enrichment of GFP-tagged constructs of WT or mutant moesin was assessed in representative midplane confocal images of transfected Jurkat cells. The large nucleus of Jurkat is indicated with N. Scale bar is 5 μm. C, quantitation of membrane enrichment of moesin constructs in Jurkat cells. The enrichment characteristic of the WT protein is indicated by the upper dashed line. Proteins not enriched at the plasma membrane typically have ratios of membrane/cytoplasmic localization of ∼1.0 (lower dashed line). Statistical significance compared with Moesin WT membrane enrichment is shown as: *, p < 0.1; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

Binding of PATCH mutant constructs to PIP2. A, binding of WT and mutant moesin to PIP2 in LUV was measured by a cosedimentation assay over the indicated range of PIP2 concentrations. Moesin constructs tested included WT, each of the single lysine mutations (K253N, K254N, K262N, K263N), and double mutations of the two pairs (K253N/K254N and K262N/K263N) that strongly impaired membrane localization in cells and the combined mutation of the 4 lysines K253N/K254N/K262N/K263N (4N). B, tabulation of dissociation constant (mean of two independent experiments ± S.D.).

FIGURE 6.

Single lysine mutations in the PATCH impair PIP2-induced activation of moesin binding to CD44 and NHERF1. A, binding of moesin to CD44 was assessed using pulldown of moesin-His by CD44 tail-GST immobilized on beads. Moesin constructs tested included WT, each of the single lysine mutations that strongly impaired membrane binding in cells, and combined mutations of lysines in a pair. Gel regions shown are from the pellet using anti-His to detect moesin pulled down and Coomassie stain to validate equal loading of GST-CD44. B and C, similar analysis as A but using NHERF1 tail-GST (B) or NHERF1 full-length-GST (C) instead of CD44-GST.

FIGURE 7.

FLAP, sequence and structure analysis. A, electrostatic surface potential of full-length insect ERM protein (PDB 2I1K) and B, of unmasked moesin FERM domain with color charge scale on the left. The molecules are oriented to show the PIP2 binding POCKET, which is covered by the FLAP in the closed conformation of moesin (A) and uncovered in the FERM domain (B). Labels indicate locations of the A, B, and C lobes. Location of PIP2 binding POCKET is indicated by arrows. C, ribbon representation of the closed ERM structure. FLAP is red (N-terminal and C-terminal parts) or magenta (reconstructed tip, amino acids 473–485). β1 to β4 strands of the PH-like domain are green; β5-β7 strands are yellow; α-helix is blue. C-terminal tail is light blue. Light Blue circles indicate locations of the two critical residues, Lys-63 and Lys-278, in the POCKET, which are masked by the FLAP when moesin is closed. D, multiple sequence analysis of the FLAP of human ezrin, radixin, and moesin and flanking regions. Red dashed rectangle highlights the FLAP. Above the sequence is a row of symbols that scores the extent of sequence conservation scored by ClustalX (* = identity; : = all residues belong to a strong conservation group; · = all residues belong to a weak conservation group). Arrowheads indicate residues in moesin that are acidic (red) or short side chains (black). Statistical significance compared with Moesin WT membrane enrichment is shown as: *, p < 0.1 and **, p < 0.01.

FIGURE 8.

Role of the FLAP in membrane localization of ERM protein. A, Jurkat cells were transfected with GFP-tagged Moesin WT and mutants (FLAP deleted (ΔFLAP), L281A and G487V) followed by fluorescence quantitation as described in the legends to Figs. 2 and 4 (B). The enrichment characteristic of the WT protein is indicated by the upper dashed line. Proteins not enriched at the plasma membrane typically have ratios of membrane/cytoplasmic localization of ∼1.0 (lower dashed line).

FIGURE 9.

FLAP removal increases PIP2-induced protein ligands binding to moesin. A, SDS-PAGE analysis of the pellet from pulldown assays using soluble moesin-His and the cytoplasmic tail of CD44-GST (or NHERF1-GST) immobilized on beads. Moesin in the pellet is detected by anti-His antibody. CD44-GST (or NHERF1-GST) is detected by Coomassie Blue to verify equal loading. B, quantitation of the relative amounts of bound moesin determined by densitometry.

FIGURE 10.

Representation of a proposed additional mechanism for conformational change when PIP2 binds to the PATCH. Panel A shows a model of the PATCH of intact ERM interacting with a lipid bilayer including a PIP2 molecule. Lobe C, the PH-like domain, has been colored to facilitate visualization: β1–β4 strands in green, β5–β7 strands in yellow, and the α-helix in blue. The FLAP is colored red. The headgroup of PIP2 is accommodated between the two pairs of lysines (Lys-253/Lys-254 and Lys-262/Lys-263) whose side chains are shown in ball and stick representation. Panel B is a magnified view of the binding site.

FIGURE 11.

Analysis of stoichiometry and cooperativity of moesin binding to PIP2. A, cooperativity. Binding of moesin-Alexa 488 to LUVs composed of varying percentages (0–15%) of PIP₂ assessed by co-sedimentation assays followed by spectrofluorimetric analysis. LUVs having fixed total lipid concentration (0.28 mm accessible lipid) but vary in their mole fractions of PIP2 were added to a fixed concentration of moesin-Alexa 488 (0.4 μm). The percent of moesin bound is plotted as a function of the percentage of PIP₂ in the LUVs where each point is the average of two experiments. The solid line is the nonlinear least squares best fit of all the data. B, stoichiometry. A standard protein to membrane FRET assay was employed to quantitate membrane-bound moesin. The FRET measured occurs between intrinsic tryptophan donors in moesin and the dansyl-PE acceptors in the LUVs. LUVs containing 10 mol % PIP2 in a lipid mixture mimicking the plasma membrane inner leaflet (PE/PC/PS/l-α-phosphatidylinositol/sphingomyelin/cholesterol/dansyl-PE/PIP2 (23.8:9.1:18.1:4.5:4.5:25:5:10)) were titrated into a fixed concentration of nonlabeled moesin (3.6 μm). These conditions were chosen to drive high affinity PIP2 binding such that the titration yields a linear increase in membrane-associated moesin until all proteins had been bound and a plateau achieved. High variability (a time dependent signal loss we ascribe to vesicle aggregation) was observed when LUV concentrations exceeded 6.0 μm PIP2 (data point shown in parentheses). The intersection of the best-fit straight lines for the linear increase and plateau regions represent the saturation point, yielding 3.1 ± 0.25 μm PIP2 molecules per 3.6 μm moesin molecules equivalent to a PIP2/moesin ratio of about 0.86 ± 0.07 (average of 5 experiments ± S.E.).