Identification of the residues in the helix F/G loop important to catalytic function of membrane-bound prostacyclin synthase.
A topological model of prostaglandin I(2) synthase (PGIS) was created by homology modeling. This model, along with site-specific antibodies and other topology studies, has suggested that the residue(s) within helix F/G loop of PGIS may be involved in forming the substrate access channel and located in a position that influences the membrane-bound PGIS catalytic function (1). To test this hypothesis, we have explored an approach to identify the residues of the helix F/G loop important to enzyme activity of the membrane-bound PGIS by a combination of 2-D NMR experiment and mutagenesis methods. Using the distance measured from the model as a guide, the helix F/G loop was mimicked in a synthetic peptide by introducing a spacer to maintain a distance of about 7 A between the N- and the C-termini (PGIS residues 208 and 230). The peptide was used to interact with the enzyme substrate analogue, U46619. High-resolution 2-D NMR experiments were performed to determine the contacts between the peptide and U46619. The interaction between the constrained F/G loop peptide and U46619 was confirmed by the observation of the conformational changes of the peptide and U46619 using the comparison of the cross-peaks between the NOESY spectra of U46619 with the peptide, without the peptide, and the peptide alone. Through the combination of the 2-D NMR experiments, completed (1)H NMR assignments of the F/G loop segment in the presence and absence of U46619 were obtained, and these data were used to predict the contact residues (Leu214 and Pro215) of the F/G loop with PGIS substrate. The predicted influence of residues on enzyme catalytic activity in membrane-bound environments was confirmed by the mutagenesis of the F/G loop residues of human PGIS. These observations support that the F/G loop is involved in forming the substrate access channel for membrane-bound PGIS and suggests that the NMR experiment-based mutagenesis approach may be applied to study structure and function relationships for other proteins.
Deng
Hui
H
The Vascular Biology Research Center and Division of Hematology, Department of Internal Medicine, The University of Texas Health Science Center, Houston, Texas 77030, USA.
Wu
Jiaxin
J
So
Shui-Ping
SP
Ruan
Ke-He
KH
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D020134:Catalytic Domain / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000201:enzymology; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D013379:Substrate Specificity
D011994:Recombinant Proteins; D012996:Solutions; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase