PDBsum entry 2k2j

Hydrolase, signaling protein

PDB id: 2k2j

Contents

Protein chain

117 a.a. *

* Residue conservation analysis

PDB id:

2k2j

Name:

Hydrolase, signaling protein

Title:

Nmr solution structure of the split ph domain from phospholipasE C gamma 2

Structure:

1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2. Chain: a. Fragment: ph domains (unp residues 471-514 and 850-913). Synonym: phosphoinositide phospholipasE C, plc-gamma-2, phospholipasE C-gamma-2, plc-iv. Engineered: yes

Source:

Homo sapiens. Human. Gene: plcg2. Expressed in: escherichia coli.

NMR struc:

20 models

Authors:

R.Harris,T.D.Bunney,M.Katan,P.C.Driscoll

Key ref:

C.Walliser et al. (2008). Rac Regulates Its Effector Phospholipase C{gamma}2 through Interaction with a Split Pleckstrin Homology Domain. J Biol Chem, 283, 30351-30362. PubMed id: 18728011 DOI: 10.1074/jbc.M803316200

Date:

02-Apr-08 Release date: 09-Sep-08

Protein chain

P16885  (PLCG2_HUMAN) -  1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2 from Homo sapiens

Seq: 1265 a.a.

Struc: 117 a.a.*

* PDB and UniProt seqs differ at 38 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.1.4.11 - phosphoinositide phospholipase C.
• Pathway: myo-Inositol Phosphate Metabolism
• Reaction: a 1,2-diacyl-sn-glycero-3-phospho-(1D-myo-inositol-4,5-bisphosphate) + H2O = 1D-myo-inositol 1,4,5-trisphosphate + a 1,2-diacyl-sn-glycerol + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1074/jbc.M803316200

J Biol Chem 283:30351-30362 (2008)

PubMed id: 18728011

Rac Regulates Its Effector Phospholipase C{gamma}2 through Interaction with a Split Pleckstrin Homology Domain.

C.Walliser, M.Retlich, R.Harris, K.L.Everett, M.B.Josephs, P.Vatter, D.Esposito, P.C.Driscoll, M.Katan, P.Gierschik, T.D.Bunney.

ABSTRACT

Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLCgamma isoforms, only PLCgamma(2) has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLCgamma(2) required for activation. Based on reconstitution experiments with isolated PLCgamma variants and Rac2, we show that an unusual pleckstrin homology (PH) domain, designated as the split PH domain (spPH), is both necessary and sufficient to effect activation of PLCgamma(2) by Rac2. We also demonstrate that Rac2 directly binds to PLCgamma(2) as well as to the isolated spPH of this isoform. Furthermore, through the use of NMR spectroscopy and mutational analysis, we determine the structure of spPH, define the structural features of spPH required for Rac interaction, and identify critical amino acid residues at the interaction interface. We further discuss parallels and differences between PLCgamma(1) and PLCgamma(2) and the implications of our findings for their respective signaling roles.

Selected figure(s)

Figure 3.
The role of the N-terminal and split PH domains of PLCγ[2] in cellular activation by Rac2. Left, COS-7 cells were transfected with increasing amounts per well of vector encoding wild-type or mutant PLCγ isozymes. The total amount of DNA was maintained constant in each transfection by adding empty vector. The empty vector (control) (A and B) and the vectors encoding PLCγ2-222, PLCγ2-212, PLCγ2-221, and PLCγ2-211 (B) were used only at 1000 ng/well, since there were only minimal changes in inositol phosphate production even at this high amount of vector DNA. Under these conditions, the inositol phosphate formation in B was as follows: control, 223 ± 30 cpm; PLCγ2-222, 436 ± 67 cpm; PLCγ2-212, 390 ± 59 cpm; PLCγ2-221, 348 ± 54 cpm; PLCγ2-211, 360 ± 6 cpm (mean ± S.D. of triplicate determinations). [^3H]Inositol phosphate accumulation was measured as described under “Experimental Procedures.” Right, COS-7 cells were cotransfected as indicated with empty vector (control) and/or vectors encoding Rac2, Rac2^G12V, or either wild-type or mutant PLCγ isozymes. The amounts of vectors encoding the PLCγ isozymes were adjusted according to their basal activities shown in the left panels (PLCγ1-111 and PLCγ1-211, 300 ng/well; PLCγ1-112, 100 ng/well; PLCγ1-121 and PLCγ1-122, 10 ng/well; all other vectors, 1000 ng per well). The total amount of DNA was maintained constant in each transfection by adding empty vector. In additional experiments (results not shown), we found that expression of Rac2^G12V also caused only a minor (≤1.9-fold) stimulation of inositol phosphate formation in cells cotransfected with 1000 ng/well of vector encoding PLCγ1-111 or PLCγ1-211.

Figure 5.
Surface charge distribution of PLCγ[1] and PLCγ[2] spPHs. Surface electrostatic potentials representations of these two spPHs were computed with PyMol (top, PLCγ[2] spPH; bottom, PLCγ[1] spPH). Electrostatic potentials are represented as positive (blue), negative (red), and neutral (white) charges. The large loop that links the two parts of the spPHs (which is present in the published NMR structure of the PLCγ[1] spPH) is not shown. The N view notation refers to the surface derived from the amino acid residues from the N-terminal half of the domain, and the C view refers to those residues derived from the C-terminal part.

The above figures are reprinted from an Open Access publication published by the ASBMB: J Biol Chem (2008, 283, 30351-30362) copyright 2008.

Figures were selected by an automated process.