Non-canonical Interaction of Phosphoinositides with Pleckstrin Homology Domains of Tiam1 and ArhGAP9

Derek F.J Ceccarelli,Ivan M Blasutig,Marilyn Goudreault,Zhiqin Li,Julie Ruston,Tony Pawson,Frank Sicheri

doi:10.1074/jbc.m700505200

Derek F.J Ceccarelli, Ivan M Blasutig + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m700505200

Copy DOI

Abstract

Pleckstrin homology (PH) domains are phosphoinositide (PI)-binding modules that target proteins to membrane surfaces. Here we define a family of PH domain proteins, including Tiam1 and ArhGAP9, that demonstrates specificity for PI(4,5)P(2), as well as for PI(3,4,5)P(3) and PI(3,4)P(2), the products of PI 3-kinase. These PH domain family members utilize a non-canonical phosphoinositide binding pocket related to that employed by beta-spectrin. Crystal structures of the PH domain of ArhGAP9 in complex with the headgroups of Ins(1,3,4)P(3), Ins(1,4,5)P(3), and Ins(1,3,5)P(3) reveal how two adjacent phosphate positions in PI(3,4)P(2), PI(4,5)P(2), and PI(3,4,5)P(3) are accommodated through flipped conformations of the bound phospholipid. We validate the non-canonical site of phosphoinositide interaction by showing that binding pocket mutations, which disrupt phosphoinositide binding in vitro, also disrupt membrane localization of Tiam1 in cells. We posit that the diversity in PI interaction modes displayed by PH domains contributes to their versatility of use in biological systems.

Highlights

The spatial and temporal regulation of protein localization plays an important role in the transduction of signals between sub-cellular compartments
A basic consensus motif within the ␤1–␤2 loop region is characteristic of Pleckstrin homology (PH) domains that bind phosphoinositides with high affinity and specificity (13)
The unique PI-binding profile displayed by Tiam[1], together with the absence of sequence motifs characteristic of PH domains that bind phosphoinositides with high affinity and selectivity, suggests a novel mode of phospholipid recognition

Summary

EXPERIMENTAL PROCEDURES

Construct Generation—Murine TIAM1 constructs C1199 and C1199⌬PH in the vector pEGFP were obtained from Peter Downes The maltose-binding protein (MBP) fusion proteins MBPTIAM1 PHn, MBP-Tiam[1] PHn-cc-Ex, MBP-Akt, and MBPAG9, and point mutants thereof were expressed per GST-AG9 as above except the bacterial cell supernatant was applied to amylose resin (New England Biolabs). MBP proteins were eluted with 20 mM maltose, concentrated, and further purified by size exclusion chromatography over a Superdex S200 column (Amersham Biosciences) in 20 mM HEPES, pH 7.5, 100 mM NaCl, and 5 mM ␤-mercaptoethanol, concentrated, and stored at Ϫ70 °C. Crystallization, Data Collection, and Structure Determination—Crystals of the AG9 PH domain were grown by vapor diffusion in hanging drops obtained by mixing 1 ␮l of a solution containing 0.5– 0.8 mM protein and 1 mM Ins(1,4,5)P3, Ins(1,3,4)P3, or Ins(1,3,5)P3 with 0.5 ml of a reservoir solution containing 20 –26% polyethylene glycol 4000, 100 mM HEPES, pH 7.0, 10% glycerol, 5 mM dithiothreitol.

RESULTS

MBP fusion protein construct

Unit cell

Hypothetical Binding Mode of

To further validate the phosphoinositide interactions observed