Inositol 5-phosphatases: insights from the Lowe syndrome protein OCRL

Abstract

The precise regulation of phosphoinositide lipids in cellular membranes is crucial for cellular survival and function. Inositol 5-phosphatases have been implicated in a variety of disorders, including various cancers, obesity, type 2 diabetes, neurodegenerative diseases and rare genetic conditions. Despite the obvious impact on human health, relatively little structural and biochemical information is available for this family. Here, we review recent structural and mechanistic work on the 5-phosphatases with a focus on OCRL, whose loss of function results in oculocerebrorenal syndrome of Lowe and Dent 2 disease. Studies of OCRL emphasize how the actions of 5-phosphatases rely on both intrinsic and extrinsic membrane recognition properties for full catalytic function. Additionally, structural analysis of missense mutations in the catalytic domain of OCRL provides insight into the phenotypic heterogeneity observed in Lowe syndrome and Dent disease.

Figure 1. OCRL domain organization and interactions

a). Domain organization of the inositol 5-phosphatases. The gene names are indicated in italics, and alternate names are also given. The proline rich domains contain multiple protein interaction sites, such as for SH3 domains, EH domains, and clathrin adaptors, which are not shown for simplicity. b). Structural representation of OCRL, colored according to domains (orange= PH domain, 2KIE.pdb; green= 5-phosphatase domain, 3MTC.pdb; red= ASH domain, 3QIS.pdb; and blue= RhoGAP domain, 2QV2.pdb). Interaction partners are indicated adjacent to their respective binding surface (black text). Motifs within OCRL that bind clathrin and AP2 are indicated in gray. All molecular graphics were generated with Pymol, www.pymol.org [81]. c). Metabolic interconversions of phosphoinositide species. Substrates of the 5-phosphatases are indicated in green.

Figure 2. Structural analysis of the 5-phosphatase catalytic module

a). Structure-based[82]sequence alignment of 5-phosphatases (PDB codes: 1I9ZA, 3MTCA, 3NR8B, 2XSWA). Active site residues are indicated by red symbols, red squares represent metal coordinating residues (either direct or via a water molecule), and red circles represent phosphoinositide interacting residues. The acyl chain binding site in INPP5B and corresponding residues in other 5-phosphatases are indicated by a dashed black box. Patient mutations are indicated underneath the residue: L= Lowe Syndrome, D= Dent 2, O = Both Lowe and Dent[20], J = mutations found in INPP5E in patients with JBTS1[12], T=mutations in SHIP2 associated with Type II Diabetes[63]. A = mutation in SHIP1 found in a patient with AML[66]. S = mutation in SHIP1 found in mice to produce a more severe phenotype than the knockout animal[83], M = Mutation in Synaptojanin 2 found to cause progressive hearing loss in the Mozart mouse[84]. b). Structural analysis of the 5-phosphatases. The region of most variation, which interacts with the acyl chain of PI(4)P when in complex with INPP5B, is indicated by a dashed black circle. The position of a second metal binding site in INPP5B, here a crystallographically identified Ca2+ ion (PDB code 3N9V.pdb, Tresaugues et. al, unpublished), which may have functional significance, is labeled in italics.

Figure 3. Structural analysis of missense mutations in OCRL

a). Patient missense mutations are highlighted in the crystal structures of isolated OCRL domains in complex with binding partners, the PH domain and connecting region is not shown. The affected residues are represented as spheres. The bound PI(4)P diC8 lipid is represented as black/colored sticks on the INPP5B catalytic domain crystal structure (PDB code: 3MTC.pdb). The Mg²⁺ active site metal is a yellow sphere. Lowe syndrome associated mutations tend to cluster in the core folding modulus of the protein or in critical catalytic residues or loops and clearly affect the stability, loop structure, or catalytic activity of the enzyme. In contrast, the majority of mutations associated with a milder, Dent phenotype are surface residues that may not have a completely deleterious affect on the enzyme’s stability or activity. The structure of the ASH-RhoGAP module of OCRL in complex with Rab8A (black, PDB ID: 3QBT) and the F&H motif-containing peptide from Ses1 (yellow, PDB ID: 3QIS) shows the positions of patient missense mutations not present in the catalytic domain. The only clinical mutation known to affect protein-protein interactions and not protein stability (F668V) is indicated. The hydrophobic triad implicated in Rab specificity determination is shown as salmon-colored sticks. b). A closer view of INPP5B in complex with PI(4)P (PDB code: 3MTC) in a different orientation relative to a. Active site residues are shown as sticks, and waters in the active site are not shown for simplicity. Residues implicated in Lowe syndrome or Dent 2 disease are colored as indicated, numbering is for the corresponding residue in human OCRL.

Figure 4. Model of OCRL at the membrane

OCRL and interaction partners are modeled, utilizing currently available structural information. Based on this modeling, all known interaction surfaces can be engaged simultaneously without obvious steric conflict. Coloring is as shown as in previous figures, Cdc42 is teal. The membrane is depicted as a line, the legs and feet of the clathrin triskelion (PDB ID: 3IYV.pdb) as a purple surface. The interactions of the clathrin boxes of OCRL with the clathrin N-terminal domain were modeled after the structure of the clathrin N-terminal domain in complex with β-adaptin 3 (PDB ID: 1C9L, not shown). OCRL (PDB ID: 2KIE, 3MTC, 3QBT, 3QIS, 2QV2) and interaction partners (PDB ID: 3QBT, 1GRN, 2EJ8) are represented as ribbons. Based on structural homology, the interaction between OCRL and RhoGTPases is assumed to be similar to those seen for other RhoGAPs. The clathrin adaptor AP2 is not shown for simplicity.

Patient mutations in INPP5E are highlighted on the structure of INPP5E (PDB ID: 2xsw.pdb, Tresaugues et. al, unpublished), represented as light orange/atom colored sticks. PI(4)P is modeled to show the active site using the structure of INPP5B in complex with PI(4)P as a model (PDB ID: 3MTC).