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.
The Vascular Biology Research Center and Division of Hematology, Department of Internal Medicine, The University of Texas Health Science Center, Houston, Texas 77030, USA.
Hui
H
Deng
Jiaxin
J
Wu
Shui-Ping
SP
So
Ke-He
KH
Ruan
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