Ke-He Ruan

Role of Val509 in Time-dependent Inhibition of Human Prostaglandin H Synthase-2 Cyclooxygenase Activity by Isoform-selective Agents

Prostaglandin H synthase (PGHS), a key enzyme in prostanoid biosynthesis, exists as two isoforms. PGHS-1 is considered a basal enzyme; PGHS-2 is associated with inflammation and cell proliferation. A number of highly selective inhibitors for PGHS-2 cyclooxygenase activity are known. Inhibition by these agents involves an initial reversible binding, followed by a time-dependent transition to a much higher affinity enzyme-inhibitor complex, making these agents potent and poorly reversible PGHS-2 inhibitors. To investigate the PGHS-2 structural features that influence the time-dependent action of the selective inhibitors, we have constructed a three-dimensional model of human PGHS-2 by homologous modeling. Examination of the PGHS-2 model identified Val509 as a cyclooxygenase active site residue, that was not conserved in PGHS-1. Recombinant human PGHS-2 with Val509 mutated to either Ile (the corresponding residue in PGHS-1), Ala, Glu, or Lys was expressed by transient transfection of COS-1 cells to evaluate the effects of the mutations on cyclooxygenase activity and on inhibition by four agents reported to be selective for PGHS-2 (NS398, nimesulide, DuP697, and SC58125). All the recombinant proteins were of the expected mass. The mutants exhibited 45-210% of wild-type cyclooxygenase activity, with Km values for arachidonate of 2.1-7.6 μM (wild-type PGHS-2, 3.8 μM), indicating that changes in position 509 had modest effects on cyclooxygenase catalysis. Each of the agents inhibited wild-type PGHS-2 in a time-dependent fashion, and all but nimesulide did the same for the V509A mutant. In contrast, the V509E and V509I PGHS-2 mutants, like recombinant human PGHS-1, did not show time-dependent inhibition with any of the agents, and the V509K mutant responded in a time-dependent manner only to DuP697. Reversible inhibition was still observed with Val509 mutants that did not show time-dependent inhibition. Thus, the side chain structure at position 509 markedly influenced the ability of PGHS-2 to undergo the time-dependent transition without removing inhibitor or substrate binding. These results indicate that Val509 in PGHS-2 has a major role in the structural transition that underlies time-dependent inhibition by the isoform-selective agents.

Identification of Residues Important for Ligand Binding of Thromboxane A2 Receptor in the Second Extracellular Loop Using the NMR Experiment-guided Mutagenesis Approach

The second extracellular loop (eLP2) of the thromboxane A2 receptor (TP) had been proposed to be involved in ligand binding. Through two-dimensional1H NMR experiments, the overall three-dimensional structure of a constrained synthetic peptide mimicking the eLP2 had been determined by our group (Ruan, K.-H., So, S.-P., Wu, J., Li, D., Huang, A., and Kung, J. (2001) Biochemistry 40, 275–280). To further identify the residues involved in ligand binding, a TP receptor antagonist, SQ29,548 was used to interact with the synthetic peptide. High resolution two-dimensional 1H NMR experiments, NOESY, and TOCSY were performed for the peptide, SQ29,548, and peptide with SQ29,548, respectively. Through completed 1H NMR assignment and by comparing the different spectra, extra peaks were observed on the NOESY spectrum of the peptide with SQ29,548, which implied the contacts between residues of eLP2 at Val176, Leu185, Thr186, and Leu187 with SQ29,548 at position H2, H7, and H8. Site-directed mutagenesis was used to confirm the possible ligand-binding sites on native human TP receptor. Each of the four residues was mutated to the residues either in the same group, with different structure or different charged. The mutated receptors were then tested for their ligand binding activity. The receptor with V176L mutant retained binding activity to SQ29,548. All other mutations resulted in decreased or lost binding activity to SQ29,548. These mutagenesis results supported the prediction from NMR experiments in which Val176, Leu185, Thr186, and Leu187 are the possible residues involved in ligand binding. This information facilitates the understanding of the molecular mechanism of thromboxane A2 binding to the important receptor and its signal transduction.

Aspirin and salicylate bind to immunoglobulin heavy chain binding protein (BiP) and inhibit its ATPase activity in human fibroblasts

Salicylic acid (SA), an endogenous signaling molecule of plants, possesses anti‐inflammatory and anti‐neoplastic actions in human. Its derivative, aspirin, is the most commonly used anti‐inflammatory and analgesic drug. Aspirin and sodium salicylate (sa‐licylates) have been reported to have multiple pharmacological actions. However, it is unclear whether they bind to a cellular protein. Here, we report for the first time the purification from human fibroblasts of a ~78 kDa salicylate binding protein with sequence identity to immunoglobulin heavy chain binding protein (BiP). The Kd values of SA binding to crude extract and to recombinant BiP were 45.2 and 54.6 μM, respectively. BiP is a chaperone protein containing a polypeptide binding site recognizing specific heptapeptide sequence and an ATP binding site. A heptapeptide with the specific sequence displaced SA binding in a concentration‐dependent manner whereas a control heptapep‐tide did not. Salicylates inhibited ATPase activity stimulated by this specific heptapeptide but did not block ATP binding or induce BiP expression. These results indicate that salicylates bind specifically to the polypep‐tide binding site of BiP in human cells that may interfere with folding and transport of proteins important in inflammation.—Deng, W.‐G., Ruan, K.‐H., Du, M., Saunders, M. A., Wu, K. K. Aspirin and salicylate bind to immunoglobulin heavy chain binding protein (BiP) and inhibit its ATPase activity in human fibroblasts. FASEB J. 15, 2463–2470 (2001)

Solution structure and topology of the N-terminal membrane anchor domain of a microsomal cytochrome P450: prostaglandin I2 synthase.

The three-dimensional structure of a synthetic peptide corresponding to the N-terminal membrane anchor domain (residues 1-25) of prostaglandin I(2) synthase (also known as cytochrome P450 8A1), an eicosanoid-synthesizing microsomal cytochrome P450, has been determined by two-dimensional (1)H NMR spectroscopy in trifluoroethanol and dodecylphosphocholine which mimic the hydrophobic membrane environment. A combination of two-dimensional NMR experiments, including NOESY, TOCSY and double-quantum-filtered COSY, was used to obtain complete (1)H NMR assignments for the peptide. Using the NOE data obtained from the assignments and simulated annealing calculations, the N-terminal membrane domain reveals a bent-shaped structure comprised of an initial helix (residues 3-11), followed by a turn (residues 12-16) and a further atypical helix (residues 17-23). The hydrophobic side chains of the helix and turn segments (residues 1-20) are proposed to interact with the hydrocarbon interior of the phospholipid bilayer of the endoplasmic reticulum (ER) membrane. The hydrophilic side chains of residues 21-25 (Arg-Arg-Arg-Thr-Arg) point away from the hydrophobic residues 1-20 and are expected to be exposed to the aqueous environment on the cytoplasmic side of the ER membrane. The distance between residues 1 and 20 is approx. 20 A (1 A=0.1 nm), less than the thickness of a lipid bilayer. This indicates that the N-terminal membrane anchor domain of prostaglandin I(2) synthase does not penetrate the ER membrane.

Prostacyclin Synthase Active Sites IDENTIFICATION BY MOLECULAR MODELING-GUIDED SITE-DIRECTED MUTAGENESIS

Prostacyclin synthase (PGIS), a cytochrome P450 enzyme, catalyzes the biosynthesis of a physiologically important molecule, prostacyclin. In this study we have used a molecular modeling-guided site-directed mutagenesis to predict the active sites in substrate binding pocket and heme environment of PGIS. A three-dimensional model of PGIS was constructed using P450BM-3 crystal structure as the template. Our results indicate that residues Ile67, Val76, Leu384, Pro355, Glu360, and Asp364, which were suggested to be located at one side of lining of the substrate binding pocket, are essential for catalytic activity. This region containing β1-1, β1-2, β1-3, and β1-4 strands is predicted well by the model. At the heme region, Cys441 was confirmed to be the proximal axial ligand of heme iron. The conserved Phe and Arg in P450BM-3 were substituted by Leu112 and Asp439, respectively in PGIS. Alteration of Leu112 to Phe retained the activity, indicating that Leu112 is a functional substitution for Phe. In contrast, mutant Asp439 → Ala exhibited a slight increase in activity. This result implies a difference in the heme region between P450BM-3 and PGIS. Our results also indicate that stability of PGIS expression is not affected by heme site or active site mutations.

Identification of Thromboxane A2 Synthase Active Site Residues by Molecular Modeling-guided Site-directed Mutagenesis

Human thromboxane A2 synthase (TXAS) exhibits spectral characteristics of cytochrome P450 but lacks monooxygenase activity. Its distinctive amino acid sequence makes TXAS the sole member of family 5 in the P450 superfamily. To better understand the structure-function relationship of this unusual P450, we have recently constructed a three-dimensional model for TXAS using P450BM-3 as the template (Ruan, K.-H., Milfeld, K., Kulmacz, R. J., and Wu, K. K. (1994) Protein Eng. 7, 1345-1551) and have identified a potential active site region. The catalytic roles of several putative active site residues were evaluated using selectively mutated recombinant TXAS expressed in COS-1 cells. Mutation of Ala-408 to Glu or Arg-413 to Gly led to a complete loss of enzyme activity despite expression of mutant protein levels equivalent to that of the wild-type TXAS. Mutation of Ala-408 to Gly or Leu retained the enzyme activity at levels of 30 or 40%, respectively. This suggests that Ala-408 provides a hydrophobic environment for substrate binding. Mutation of Arg-413 to Lys or Gln completely abolished the enzyme activity, indicating that this residue is essential to catalytic activity and supports its identification as an active site residue. Mutation of Arg-410 to Gly or Glu-433 to Ala resulted in >50% reduction in the enzyme activity without appreciably altering mutant protein expression, consistent with a more subtle effect of these residues on TXAS catalytic efficiency. Mutation of residues predicted to be involved in binding the heme prosthetic group, including the heme thiolate ligand Cys-480, Arg-478, Phe-127, and Asn-110, each markedly reduced the expressed protein level and abolished enzyme activity. This suggests that proper heme binding is important to synthesis or stability of recombinant TXAS. Mutation of Ile-346, which corresponds to P450cam-Thr-252, an essential amino acid involved in dioxygen bond scission, to Thr increased the enzymatic activity by 40%, suggesting that oxygen bond cleavage is not a rate-limiting step in thromboxane A2 biosynthesis. The present results from site-directed mutagenesis support the overall structure of the TXAS active site predicted by homology modeling and have allowed refinement of the position of bound substrate.

Substrate access channel topology in membrane-bound prostacyclin synthase.

Results from our molecular-modelling and site-directed-mutagenesis studies of prostaglandin I(2) synthase (PGIS) have suggested that the large PGIS cytoplasmic domain is anchored to the endoplasmic reticulum (ER) membrane by the N-terminal segment in a way that orients the substrate access channel opening to face the membrane. To test this hypothesis we have explored the accessibility of the PGIS substrate channel opening to site-specific antibodies. The working three-dimensional PGIS model constructed by protein homology modelling was used to predict surface portions near the substrate access channel opening. Two peptides corresponding to the surface immediately near the opening [residues 66-75 (P66-75) and 95-116 (P95-116)], and two other peptides corresponding to the surface about 10-20 A (1 A=0.1 nm) away from the opening [residues 366-382 (P366-382) and 472-482 (P472-482)] were used to prepare site-specific antibodies. All four antipeptide antibodies specifically recognized the synthetic segments of human PGIS and recombinant PGIS, as shown by binding assays and Western-blot analysis. The site-specific antibodies were used to probe the accessibility of the substrate access channel opening in transiently transfected COS-1 cells expressing recombinant human PGIS, and in spontaneously transformed human endothelial cell line ECV cells expressing endogenous human PGIS. Immunofluorescence staining was performed for cells selectively permeabilized with streptolysin O and for cells whose membranes were permeabilized with detergent. Antibodies to peptides in the immediate vicinity of the substrate channel (P66-75 and P95-116) bound to their targets only after general permeabilization with Triton X-100. In contrast, the two antibodies to peptides further from the channel opening (P366-382 and P472-482) bound to their targets even in cells with intact ER membranes. These observations support our topology model in which the PGIS substrate access channel opening is positioned close to the ER membrane.

Evidence of the residues involved in ligand recognition in the second extracellular loop of the prostacyclin receptor characterized by high resolution 2D NMR techniques

In previous studies, we have determined the solution structure of the second extracellular loop (eLP(2)) of the human thromboxane A(2) receptor (TP) and identified the residues in the eLP(2) domain involved in ligand recognition, by using a combination of approaches including a constrained synthetic peptide, 2D NMR spectroscopy, and recombinant proteins. These findings led us to hypothesize that the specific ligand recognition sites may be localized in the eLP(2) for all the prostanoid receptors. To test this hypothesis, we have investigated the ligand recognition site for another prostanoid receptor, the prostacyclin receptor (IP), which mediates an opposite biological function compared to that of the TP receptor. The identification of the interaction between the IP receptor and its agonist, iloprost, was achieved with a constrained synthetic peptide mimicking the eLP(2) region of the receptor. The IP eLP(2) segment was designed and synthesized to form a constrained loop, using a homocysteine disulfide bond connecting the ends of the peptide, based on the distance predicted from the IP receptor model created by homology modeling using the crystal structure of bovine rhodopsin as a template. The evidence of the constrained IP eLP(2) interaction with iloprost was found by the identification of the conformational changes of the eLP(2) induced by iloprost using fluorescence spectroscopy, and was further confirmed by 1D and 2D 1H NMR experiments. In addition, the IP eLP(2)-induced structure of iloprost in solution was elucidated through a complete assignment of the 2D 1H NMR spectra for iloprost in the presence of the IP eLP(2) segment. In contrast, no ordered structure was observed in the 2D 1H NMR experiments for iloprost alone in solution. These studies not only identified that the eLP(2) segment of the IP receptor is involved in ligand recognition, but also solved the 3D solution structure of the bound-form of iloprost, which could be used to study the receptor-ligand interaction in structural terms.

Purification and characterization of a cyclooxygenase-2 and angiogenesis suppressing factor produced by human fibroblasts

Cyclooxygenase‐2 (COX‐2) is an inducible enzyme that plays an important role in several pathophysiological processes, including inflammation, angiogenesis, and tumorigenesis. We have recently observed that COX‐2 induction is restrained in proliferating fibroblasts. The mechanism by which this occurs is unclear. Here, we report the detection and isolation from the conditioned medium of proliferating fibroblasts a factor that suppressed COX‐2 expression. This factor, which was named cytoguardin, suppressed COX‐2 protein levels induced by phorbol 12myristate 13‐acetate, interleukin‐1β, tumor necrosis factor α, and lipopolysaccharide (LPS) in fibroblasts and LPS‐induced COX‐2 protein levels and promoter activities in human endothelial cells and murine RAW 264.7 cells in a comparable concentration‐dependent manner. It inhibited COX‐2 expression induced by angiogenic factors and endothelial tube formation induced by angiogenic factors and colon cancer cell medium. These findings provide evidence for the control of COX‐2 transcription by an endogenous cellular factor.

Identification of the Substrate Interaction Site in the N-terminal Membrane Anchor Segment of Thromboxane A2Synthase by Determination of Its Substrate Analog Conformational Changes Using High Resolution NMR Technique

The present studies describe an investigation for the interaction of N-terminal membrane anchor domain of thromboxane A2 synthase (TXAS) with its substrate analog in a membrane-bound environment using the two-dimensional NMR technique. TXAS and prostaglandin I2synthase (PGIS), respectively, convert the same substrate, prostaglandin H2 (PGH2), to thromboxane A2 and prostaglandin I2, which have opposite biological functions. Our topology studies have indicated that the N-terminal region of TXAS has a longer N-terminal endoplasmic reticulum (ER) membrane anchor region compared with the same segment proposed for PGIS. The differences in their interaction with the ER membrane may have an important impact to facilitate their common substrate, PGH2, across the membrane into their active sites from the luminal to the cytoplasmic side of the ER. To test this hypothesis, we first investigated the interaction of the TXAS N-terminal membrane anchor domain with its substrate analog. A synthetic peptide corresponding to the N-terminal membrane anchor domain (residues 1–35) of TXAS, which adopted a stable helical structure and exhibited a membrane anchor function in the membrane-bound environment, was used to interact with a stable PGH2 analog, U44069. High resolution two-dimensional NMR experiments, NOESY and TOCSY, were performed to solve the solution structures of U44069 in a membrane-mimicking environment using dodecylphosphocholine micelles. Different U44069conformations were clearly observed in the presence and absence of the TXAS N-terminal membrane anchor domain. Through combination of the two-dimensional NMR experiments, completed 1H NMR assignments of U44069 were obtained, and the data were used to construct three-dimensional structures of U44069 in H2O and dodecylphosphocholine micelles, showing the detailed conformation change upon the interaction with the membrane anchor domain. The observation supported the presence of a substrate interaction site in the N-terminal region. The combination of the structural information ofU44069 and U46619 was able to simulate a solution structure of the unstable TXAS and PGIS substrate, PGH2.