Crystal Structure of the Pleckstrin Homology Domain from the Ceramide Transfer Protein: Implications for Conformational Change upon Ligand Binding

Abstract

Ceramide transfer protein (CERT) is responsible for the nonvesicular trafficking of ceramide from the endoplasmic reticulum (ER) to the trans Golgi network where it is converted to sphingomyelin (SM). The N-terminal pleckstrin homology (PH) domain is required for Golgi targeting of CERT by recognizing the phosphatidylinositol 4-phosphate (PtdIns(4)P) enriched in the Golgi membrane. We report a crystal structure of the CERT PH domain. This structure contains a sulfate that is hydrogen bonded with residues in the canonical ligand-binding pocket of PH domains. Our nuclear magnetic resonance (NMR) chemical shift perturbation (CSP) analyses show sulfate association with CERT PH protein resembles that of PtdIns(4)P, suggesting that the sulfate bound structure likely mimics the holo form of CERT PH protein. Comparison of the sulfate bound structure with the apo form solution structure shows structural rearrangements likely occur upon ligand binding, suggesting conformational flexibility in the ligand-binding pocket. This structural flexibility likely explains CERT PH domain’s low affinity for PtdIns(4)P, a property that is distinct from many other PH domains that bind to their phosphoinositide ligands tightly. This unique structural feature of CERT PH domain is probably tailored towards the transfer activity of CERT protein where it needs to shuttle between ER and Golgi and therefore requires short resident time on ER and Golgi membranes.

Phosphoinositide binding by the PH domain in ceramide transfer protein (CERT) is inhibited by hyperphosphorylation of an adjacent serine-repeat motif

“…The three-dimensional structure of the PH domain of CERT has been resolved by NMR and X-ray diffraction analyses, and these structural biological studies have identified the subregions and amino acid residues that are crucial for PI4P-binding activity in the CERT PH domain (14,15). We measured the 2D 1 H-15 N heteronuclear single quantum correlation (HSQC) spectra of uniformly 15 N-labeled CERT derivatives (Fig.…”

“…The signals, which are visible only in PH-SRM(10E), are indicated by obelisks, whereas those invisible in both WT and 10E are denoted with double daggers. The asterisks indicate that 1 H- 15 N correlation signals were severely degenerated. P, proline residues (Pro-78 and Pro-102).…”

“…The CERT PH domain has two distinct but overlapping regions that are crucial for high-affinity binding to PI4P-embedded phospholipid membranes; one is a pocket that accommodates the negatively charged headgroup of PI4P, and the other is a basic groove that weakly associates with acidic phospholipid membranes (14). Seven amino acid residues (Lys-32, Trp-33, Thr-34, Asn-35, Trp-40, Gln-41, and Tyr-96) of the CERT PH domain are shared in both regions (15). Some of the shared residues (Trp-33, Thr-34, Trp-40, and Ser-93) were mapped as residues perturbed by the 10E mutation in SRM (Fig.…”

Cellular and molecular interactions of phosphoinositides and peripheral proteins

“…Of the seven PIs in the mammalian cell, the PH domain binds specifically to PI(3,4,5)P 3 , PI(4,5)P 2 , PI(3,4)P 2 (Lemmon 2007; Lemmon 2008; Kutateladze 2010; Moravcevic et al 2012). Binding of PH domains to monophosphorylated PIPs seems to be less selective and have lower affinity than to that of bisphosphorylated PIPs (Yu et al 2004; Lemmon et al 2007; Stahelin et al 2007; Prashek et al 2013) and others may be coincident detectors for PIPs and sphingolipids or bind sphingolipids alone (Gallego et al 2010). The membrane binding of PH domains is initially driven by non-specific electrostatic interactions, which is followed by specific PIP binding to achieve selective targeting and increase the membrane residence time.…”

Computational Analysis of the Binding Specificities of PH Domains

“…PH (pleckstrin homology) domains, consisting of about 100–120 amino acid residues, are found in a wide range of proteins involved in intracellular signalling or as constituents of the cytoskeleton [ 1 – 4 ]. Although their sequences bear very low similarity, all the determined three-dimensional structures have seven β -strands forming two perpendicular anti-parallel-sheets and one C-terminal α -helix [ 5 ]. PH domains bind either plasma-membrane phosphoinositides or cytosolic inositol phosphates with few exceptions.…”