Structure of the split PH domain and distinct lipid-binding properties of the PH-PDZ supramodule of α-syntrophin

Abstract

Pleckstrin homology (PH) domains play diverse roles in cytoskeletal dynamics and signal transduction. Split PH domains represent a unique subclass of PH domains that have been implicated in interactions with complementary partial PH domains 'hidden' in many proteins. Whether partial PH domains exist as independent structural units alone and whether two halves of a split PH domain can fold together to form an intact PH domain are not known. Here, we solved the structure of the PH N -PDZ-PH C tandem of a-syntrophin. The split PH domain of a-syntrophin adopts a canonical PH domain fold. The isolated partial PH domains of a-syntrophin, although completely unfolded, remain soluble in solution. Mixing of the two isolated domains induces de novo folding and yields a stable PH domain. Our results demonstrate that two complementary partial PH domains are capable of binding to each other to form an intact PH domain. We further showed that the PH N -PDZ-PH C tandem forms a functionally distinct supramodule, in which the split PH domain and the PDZ domain function synergistically in binding to inositol phospholipids.

Atypical Membrane-embedded Phosphatidylinositol 3,4-Bisphosphate (PI(3,4)P2)-binding Site on p47 Phox Homology (PX) Domain Revealed by NMR

“…phox PX domain for insoluble long lipid chains, C 16 -PI(3,4)P 2 embedded in liposomes, a 7-diethylamino-3-(4Ј-maleimidylphenyl)-4-methylcoumarin (CPM)-based fluorescence assay (16,42) was conducted. The increase of fluorescence of the CPM-labeled PX domain was observed upon the addition of C 16 -PI(3,4)P 2 -containing liposomes, whereas the CPM-labeled PX domain showed no interaction in an identical experiment, using control liposomes without PI(3,4)P 2 (composed of POPC/POPE ϭ 80:20) (Fig.…”

Membrane-Microdomain Localization of Amyloid β-Precursor Protein (APP) C-terminal Fragments Is Regulated by Phosphorylation of the Cytoplasmic Thr668 Residue

“…We extracted endogenous lipids from membrane fractions of mouse brain to prepare liposomes to serve as a model for neural membranes (34,(43)(44)(45). Synthetic cytoplasmic peptides of APP sequence 648 -695, one with phosphate at Thr 668 (pC47) and one without phosphate at Thr 668 (nC47) were incubated with the liposomes prepared from mouse brain lipids and the liposome-bound peptides were recovered and analyzed by immunoblotting.…”

Interaction of α1‐syntrophin with multiple isoforms of heterotrimeric G protein α subunits

“…In a1-syntrophin, the primary sequence of the PH1 domain is split into N-and C-terminal halves by the intervening PDZ domain: PH1a domain creates b1-3, whereas PH1b domain creates b4-7 strands and the helix. Each half of the PH domain is in an unfolded state in solution, but the two subdomains form a stable structure when they interact [54]. Because Gas interacted with the PH1a domain, the binding surface for Ga is likely to include the N-terminal b1-3 strands.…”