The Huck Institutes of the Life Sciences

Inhibition of Secreted Phospholipase A2 by Proanthocyanidins: A Comparative Enzymological and in Silico Modeling Study

Lambert JD, Yennawar N, Gu Y, and Elias RJ (2012) J. Agric. Food Chem. 60(30): 7417–7420

Lambert et al. 2012 Figure 4
Comparison of the effects of binding of B-type or A-type PaCs on the three-dimensional structure of group III (honey bee venom) PLAs: (A) the B-type tetramer (represented as sticks in cyan) occupies a closed hydrophobic tunnel in the crystal structure of bee venom PLA2 without significantly altering it; (B) on binding of the A-type (nonlinear) tetramer, the tunnel is rendered open.

Abstract

Secreted phospholipase A2 (PLA2) plays a critical role in mobilizing arachidonic acid in phospholipids. We have previously reported that PLA2 is inhibited by B-type proanthocyanidins (PaCs). To further understand the inhibitory activity of these compounds, we compared the inhibitory potency of B-type PaCs to that of A-type PaCs and modeled them with PLA2using in silico techniques. The B-type trimer and tetramer inhibited PLA2 (IC50 = 16 and 10 µM). The A-type compounds were less potent (18-35% inhibition at 50 µM). The active site of PLA2 lies in a hydrophobic tunnel. Modeling studies revealed that the B-type PaCs occupy this tunnel and are stabilized by a number of van der Waals interactions. The result is reduced substrate access to the active site. The A-type compounds can occupy this tunnel only by shifting the N-terminal loop outward. Our data provide a structural basis to screen additional PaCs for anti-PLA2 activity.

Inhibition of group III (honey bee venom) PLA2 by test proanthocyanidins

Lambert et al. 2012 Figure 2
A-type and B-type trimers (DP = 3) and tetramers (DP = 4) were compared. Activity was normalized to the vehicle-treated controls and expressed as the mean ± standard deviation of at least three independent experiments.

Model of PLA2 with B-type PaCs

Lambert et al. 2012 Figure 3
Based on the transition state analogue crystal structure (A), both the B-type trimer (B) and tetramer (C) were modeled with group III (honey bee venom) PLA2. Key amino acids involved in binding are indicated for both trimer (D) and tetramer (E). Active site residues are indicated in magenta and key nonactive site hydrophobic residues in cyan.

Materials and Methods

In Silico Modeling

An in silico model of PLA2 was developed by analysis of the crystal structure of bee venom PLA2 (PDB code 1poc). The enzyme crystal structure was reported with a transition state analogue bound in proximity to His34 in the active site (Figure 3A). The active site is contained within a tunnel formed by three helices (shown in lime green and blue) and the calcium binding loop near the N-terminus. The tunnel is approximately 20 Å in length and has a maximum diameter of 11 Å.

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