Structural and functional characterization of human microsomal prostaglandin E synthase-1 by computational modeling and site-directed mutagenesis

doi:10.1016/j.bmc.2006.01.010

Bioorganic & Medicinal Chemistry

Volume 14, Issue 10, 15 May 2006, Pages 3553-3562

https://doi.org/10.1016/j.bmc.2006.01.010 Get rights and content

Abstract

Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) has recently been recognized as a novel, promising drug target for inflammation-related diseases. Functional and pathological studies on this enzyme further stimulate to understand its structure and the structure–function relationships. Using an approach of the combined structure prediction, molecular docking, site-directed mutagenesis, and enzymatic activity assay, we have developed the first three-dimensional (3D) model of the substrate-binding domain (SBD) of mPGES-1 and its binding with substrates prostaglandin H2 (PGH2) and glutathione (GSH). In light of the 3D model, key amino acid residues have been identified for the substrate binding and the obtained experimental activity data have confirmed the computationally determined substrate–enzyme binding mode. Both the computational and experimental results show that Y130 plays a vital role in the binding with PGH2 and, probably, in the catalytic reaction process. R110 and T114 interact intensively with the carboxyl tail of PGH2, whereas Q36 and Q134 only enhance the PGH2-binding affinity. The modeled binding structure indicates that substrate PGH2 interacts with GSH through hydrogen binding between the peroxy group of PGH2 and the –SH group of GSH. The –SH group of GSH is expected to attack the peroxy group of PGH2, initializing the catalytic reaction transforming PGH2 to prostaglandin E2 (PGE2). The overall agreement between the calculated and experimental results demonstrates that the predicted 3D model could be valuable in future rational design of potent inhibitors of mPGES-1 as the next-generation inflammation-related therapeutic.

Graphical abstract

Introduction

Prostaglandin (PG) E2 is produced by a variety of cells and tissues, and exhibits potent diverse bioactivities.¹ Its production is mediated by three enzymatic reactions involving phospholipase A2 (PLA2), cyclooxygenase (COX), and PGE2 synthase (PGES). In this biosynthetic pathway, arachidonic acid (AA) releases from membrane phospholipids by cytosolic or secretory PLA2 and is converted to prostaglandin H2 (PGH2) by COXs. PGH2 is then isomerized to prostaglandin E2 (PGE2) by terminal PGES enzymes.² PGES enzymes, that lie downstream of COXs, occur in three forms in mammalian cells.3, 4, 5, 6 Among them, the microsomal and membrane-bound synthase (namely mPGES-1) has received much more attention and established as a novel drug target in the areas of inflammation, tumorigenesis, and bone disorders. Hence, mPGES-1 is involved in a number of diseases including arthritis, burn injury and pain diseases, atherosis, cancer, and even the exacerbation of Alzheimer’s disease.2, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 Recently reported studies have led to well characterization of its inducible distribution, expression, enzymatic kinetics, and biological and pathological functions.18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 The expression of mPGES-1 is up-regulated by proinflammatory stimuli and down-regulated by anti-inflammatory glucocorticoids, often in accordance with that of COX-2.3, 4, 7, 19, 25, 26 The protein mPGES-1 has been identified as the central switch during immune-induced pyresis,²⁰ and deletion of mPGES-1 would reduce inducible and basal PGE2 production and alter the gastric prostanoid profile.24, 26 Compared to its upstream enzymes, inhibition of mPGES-1 does not block normal functions of other PGs and, therefore, lacks the unexpected side effects produced by the inhibition of COXs, making it more attractive for the development of potential therapeutics, especially for the treatment of inflammation-related diseases.3, 29, 30 However, no clinically useful inhibitor of mPGES-1 has been identified. So far, only two compounds, that is, the COX-2 inhibitor NS-398 (Chart 1) and 5-lipoxygenase-activating protein (FLAP) inhibitor MK-886 (Chart 1), have been found to be able to inhibit mPGES-1 with the IC₅₀ values at micromole level.2, 3, 4, 29 It is highly desirable to develop more potent and selective inhibitors of mPGES-1 based on the structure and function of the enzyme for development of the next-generation therapeutics.

Initially, mPGES-1 was discovered as recombinant human microsomal glutathione-S-transferase (GST)-1-like 1 (MGST1-L1) and recognized as a member of membrane-associated proteins involved in eicosanoid and glutathione (GSH) metabolism (MAPEG) superfamily.2, 7, 31, 32 It shows significant homology with other MAPEG proteins, especially with the nearest subfamily member MGST1. Hydropathy analysis suggests that all the MAPEG proteins have similar three-dimensional (3D) and membrane-spanning topographic properties.21, 31, 32, 33, 34 Site-directed mutagenesis revealed that R110 has an essential role in the catalytic function of mPGES-1, whereas the mutation on either R51 or R70 did not affect the activity.2, 3 Unfortunately, further structure–function investigation is restrained by the lack of the detailed 3D structure of this membrane-bound protein, which makes very difficult the structure-based design of drugs targeting mPGES-1. A two-dimensional (2D) electron projection map (with a resolution of 10 Å) of mPGES-1 revealed a trimer structure²¹ which is very similar to that of MGST1, but the resolution of 10 Å is insufficient for the purpose of building a 3D model of mPGES-1. Nevertheless, the observed MGST1 trimer had a resolution of 3 Å for the 2D electron projection map and a resolution of 6 Å for the 3D map of MGST1 structure. The available structural information35, 36, 37, 38, 39 has provided a stepping stone forward for building a 3D model of mPGES-1 structure with help from using modern molecular modeling techniques.

In order to understand the molecular mechanism of the substrate binding, an ‘ab initio’ structure prediction approach has been developed in the present study to build a 3D model of the substrate-binding domain (SBD) of mPGES-1 by making use of the structural information available for both mPGES-1 and MGST1 of the MAPEG superfamily. Based on the 3D model of the SBD, key residues that are crucial for the substrate binding have been identified through further structural analysis and molecular docking. Site-directed mutagenesis and catalytic activity assay have been performed to validate the predicted 3D SBD model of the wild-type mPGES-1 and its mutants. The overall agreement between the computational and experimental results demonstrates some important structural features of the SBD of mPGES-1 and its binding with the substrates, providing a rational basis for future structure-based drug design.

Section snippets

Structural models of the SBD of mPGES-1

The amino acid sequence alignment of mPGES-1 with MGST1 (Fig. 1) shows that four regions with high homology (>70%) can be assigned to four α-helices. These are α-helix A from sequence position #11 to #38, α-helix B from #78 to #93, α-helix C from #96 to #114, and α-helix D from #126 to #147. The longest loop between α-helix A and α-helix B contains typically conserved motifs. According to the geometric parameters used for the α-helices (Table 1), the explored 144,784 conformations derived from

Discussion

Structural determination of membrane-spanning proteins is still exceedingly difficult by experimental methods such as X-ray diffraction and NMR. As a stimulating drug target, detailed information about the mPGES-1 structure and the relationship with its functions are sorely needed. In the present study, this need is partially satisfied by performing computational 3D structure predictions of the SBD of mPGES-1 and its binding with the substrates PGH2 and GSH, followed by wet experimental tests

‘Ab initio’ structure prediction

The sequence alignment was generated by using ClusterW with the Blosum scoring function.44, 45 The best alignment was selected according to not only the alignment score, but also the reciprocal position of the conserved residues. These included the conserved FANPED motif at positions #44 to #49, VERXXRAH motif from position #65 to #72, and R110. There was a gap of four residues from #55 to #58. The total homology is 73%, with the sequence identity of 38.8%. The membrane-spanning regions were

Acknowledgments

The research was supported in part by the Center for Computational Sciences (CCS) and the College of Pharmacy at University of Kentucky and by NIH Grant HL046.

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Structural and functional characterization of human microsomal prostaglandin E synthase-1 by computational modeling and site-directed mutagenesis

Abstract

Graphical abstract

Introduction

Section snippets

Structural models of the SBD of mPGES-1

Discussion

‘Ab initio’ structure prediction

Acknowledgments

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