Structure of the split PH domain and distinct lipid-binding properties of the PH-PDZ supramodule of alpha-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 alpha-syntrophin. The split PH domain of alpha-syntrophin adopts a canonical PH domain fold. The isolated partial PH domains of alpha-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.

Figure 1

Comparison of the split PH and PDZ domains in the PH_N–PDZ–PH_C tandem and in their respective isolated state. (A) Schematic diagram showing the domain organization of syntrophins. (B) Superposition plots of ¹H, ¹⁵N HSQC spectra of the PH_N–PDZ–PH_C tandem (black), the isolated PDZ domain (green), and the joint PH_N–PH_C domain (red). (C) Plot of chemical shift changes as a function of residue number of the split PH and PDZ domains in the PH_N–PDZ–PH_C tandem and in their respective isolated forms. The combined ¹H and ¹⁵N chemical shift changes are defined as: Δδ_HN and Δδ_N represent chemical shift differences of amide proton and nitrogen chemical shifts between the PH_N–PDZ–PH_C tandem and the isolated PH and PDZ domains, respectively. The scaling factor (α_N) used to normalize the ¹H and ¹⁵N chemical shifts is 0.17. The domain organization of the PH_N–PDZ–PH_C tandem is indicated at the top of the plot.

Figure 2

Structure of the PH_N–PDZ–PH_C tandem. (A) Stereo view showing the backbones of 20 superimposed NMR-derived structures of the split PH domain from the PH_N–PDZ–PH_C tandem. Since the orientation between the split PH domain and the PDZ domain in the PH_N–PDZ–PH_C tandem is not fixed, we chose to present the structures of each domain separately. (B) Ribbon diagram of a representative NMR structure of the split PH domain. The insertion of the PDZ domain in the β3/β4-loop of the split PH domain is indicated. (C) Stereo view plot of 20 superimposed NMR structures of the PDZ domain from the PH_N–PDZ–PH_C tandem. (D) Ribbon diagram drawing of the PDZ domain structure. The split PH domain connected to the βA and βF of the domain is indicated. (E) Ribbon diagram drawing of a representative NMR structure of the PH_N–PDZ–PH_C tandem. In this drawing, the split PH domain is in green, and the PDZ domain is in gold.

Figure 3

Folding and interaction of the two isolated, partial PH domain fragments. (A) SDS–PAGE showing the purification of the joined PH_N–‘C'–PH_C domain and cleavage of the domain into PH_N and PH_C fragments. (B) Overlay plot of the ¹H, ¹⁵N HSQC spectra of the joined PH_N–‘C'–PH_C (black) and its protease cleaved form (red). ¹H, ¹⁵N HSQC spectra of the isolated PH_N (C) and PH_C (D) fragments. (E) Overlay plot of the ¹H, ¹⁵N HSQC spectra of the joined PH_N–‘C'–PH_C (black) and the 1:1 mixture of the two halves of the split PH domain (red).

Figure 4

The PH_N–PDZ–PH_C supramodule binds to brain liposomes with enhanced avidity. (A) Schematic diagrams showing the various forms of proteins purified for liposome-binding assays. (B) Brain liposome-binding assays of the PH_N–PDZ–PH_C tandem and its fragments. ‘S' and ‘P' denote proteins recovered in the supernatants and pellets, respectively, in the centrifugation-based liposome-binding assays. (C) Dose-dependent binding between the PH_N–PDZ–PH_C tandem and the brain liposome. In this assay, the amount of the PH_N–PDZ–PH_C tandem is fixed at 10 μM, and the concentration of liposome varies. The liposome-bound PH_N–PDZ–PH_C tandem is recovered in the pellet in the binding assay. (D) Lipid binding of the two mutants of the PH_N–PDZ–PH_C tandem. In these two mutants, the PDZ domain was placed either at the front or after the split PH domain. In these mutants, the PH and the PDZ domains were connected by a flexible ‘Gly–Ser–Gly–Gly–Ser–Gly–Gly–Ser–Gly–Ser' linker. (E) Lipid binding of a PH_N–PDZ–PH_C tandem mutant with the two connecting linkers shortened to six residues. Amino-terminal His₆-tagged proteins were used in all of the brain liposome-binding assays.

Figure 5

Lipid-binding specificity of the PH_N–PDZ–PH_C supramodule using phospholipid strip overlay assay. (A) Binding of the PH_N–PDZ–PH_C tandem to the phospholipids spotted on a cellulose membrane. The amount of lipid per spot was 100 pmol. Abbreviations for the lipids: S1P: sphingosine-1-phosphate; LPA: lysophosphatidic acid; LPC: lysophosphocholine; PE: phosphatidylethanolamine; PS: phosphatidylserine; PA: phosphatidic acid; PC: phosphatidylcholine; PtdIns: phosphatidylinositol. The amount of protein used in the assay is 0.5 μg/ml. (B) Binding isotherms of PH_N–PDZ–PH_C with PC/PS liposomes containing either 10 or 20% PI(3,5)P₂. (C) Identification of the residues in the split PH domain of the PH_N–PDZ–PH_C tandem that are involved in the lipid binding.

Figure 6

The PDZ ligands do not affect lipid binding of the PH_N–PDZ–PH_C tandem. (A) Addition of excess amounts (up to ∼10 equivalents) of a PDZ-binding peptide to the liposome-binding assay buffer did not alter the lipid-binding capacity of the PH_N–PDZ–PH_C tandem. The amino-acid sequence of the PDZ ligand peptide is: KLSSIESDV. (B) Mutation of the two positively charged residues (Arg85 and Arg86) in the carboxyl group-binding loop of the target recognition groove of the PDZ domain did not change lipid-binding capacity of the PH_N–PDZ–PH_C tandem. (C) Binding of lipids to the PH_N–PDZ–PH_C tandem does not change the binding of PDZ domain to its ligand, the nNOS PDZ domain. Three different liposome concentrations (0.4, 0.6, and 0.8 mg/ml) were tested in this assay.