Stimulation of phospholipase Cbeta by membrane interactions, interdomain movement, and G protein binding--how many ways can you activate an enzyme?

Abstract

Signaling proteins are usually composed of one or more conserved structural domains. These domains are usually regulatory in nature by binding to specific activators or effectors, or species that regulate cellular location, etc. Inositol-specific mammalian phospholipase C (PLC) enzymes are multidomain proteins whose activities are controlled by regulators, such as G proteins, as well as membrane interactions. One of these domains has been found to bind membranes, regulators, and activate the catalytic region. The recently solved structure of a major region of PLC-beta2 together with the structure of PLC-delta1 and a wealth of biochemical studies poises the system towards an understanding of the mechanism through which their regulations occurs.

Figure 1. Cartoon on the G protein-PLCβ signaling system

Figure 2. Organization of PLC-β and –δ domains

Top – Schematic diagram showing the domains organization of PLC-β and –δ. Bottom - The structure of the human PLC-β2 represented in ribbon (PDB entry : 2FJU) showing the organization of the different domains.

Figure 3. Comparison of the structure of the PH domains of the PLC-δ1 and –β2

Top left – The structure of rat PH-δ1 (PDB entry :1MAI [5]) showing the hydrogen-bonds between the Ins(1,4,5)P3, water molecules and the K30, K32, W36, R40, E54, S55, R56, K57 and T107 residues are represented in green. Top right - The structure of the human PH-β2 (PDB entry : 2FJU [11]) showing the long β5/β6 loop (highlighted in green. Bottom - The structural alignment of PH-δ1 and PH-β2 sequences showing the absence of the Ins(1,4,5)P₃ binding residues and the longer β5/β6 loop of PH-β2.

Figure 4. Catalytic X/Y domain of the PLC-δ1 and –β2

Top left - The catalytic site of the rat PLC-δ1 (PDB entry: 1DJX [18]) showing the side-chains of residues involved in binding to Ins(1,4,5)P₃ and to Ca²⁺ and in the hydrolysis reaction (represented in stick). The Ca²⁺ ion is represented as a green sphere. The domain surface surrounding the catalytic site is shown in white with the hydrophobic ridge in yellow. The indicated hydrophobic residues of the ridge are those proven by substitution for Ala to give a PLC-δ1 less sensitivity to surface pressure. [13]. Top right – The catalytic site of the human PLC-β2 (PDB entry : 2FJU [11]). Ins(1,4,5)P₃ and Ca²⁺ are not present in the crystal. The residues of the active site, all strictly conserved between PLC-δ1 and PLC-β2, are represented. The segment of the X/Y linker resolved in the structure, occluding the active site, is represented as a meshed blue surface. Middle – Schematic of the reaction catalyzed by PLCs. Bottom - The sequence of the X/Y linkers in PLC-δ1 and PLC-β2.

Figure 5. PLC-β2 binding sites on Gβγ

The region corresponding to the signal transfer regions [66, 67] are represented in light green whereas the general PLC-β2 binding domain are in dark blue [–67]. Additional regions found to be important for the Gβγ -activation of PLC-β2 (strands 2d and 6d) are in light blue [68]. The second-binding site for PLC-β2 found in the N-terminal region of Gβ are in magenta [71]. The Gγ subunit is colored in dark grey. The numeration of blade of the β-propeller is indicated. Inset: surface of the Gβγ subunit with the same color used for the representation in ribbon (PDB entry : 1OMW corresponding to Gβ1γ2)

Figure 6. A model of Gβγ activation of PLC-β2

We propose that the PH domain could have an inhibitory effect on the X/Y domain through the residues 71–88 (β5/β6 loop) by maintaining the catalytic domain in a non- productive orientation. Gβγ could, by binding to the PH and X/Y domain, induce a slight conformational change allowing the X/Y domain to be correctly positioned at the membrane surface and hydrolyze the substrate.