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Refined structures of placental alkaline phosphatase show a consistent pattern of interactions at the peripheral site.

ABSTRACT: In order to gain deeper insights into the functional sites of human placental alkaline phosphatase, the structures of the enzyme with the putative regulators L-Phe, pNPP and 5'-AMP [Llinas et al. (2005), J. Mol. Biol. 350, 441-451] were re-refined. Significant variations in ligand positioning and identity were found compared with the previous report. The multiple corrections to the model improved the phases and the electron-density maps, allowing the modeling of omitted side chains and multiple disordered residues. These improvements led to a change in the position of L-Phe at the peripheral binding site, which appeared to be reversed. The structure with pNPP contained only p-nitrophenol in three distinct sites, while the structure with 5'-AMP contained the p-nitrophenyl group in two of the sites instead of 5'-AMP. Comparison of the re-refined models shows a consistent pattern of interactions at the peripheral site.

SUBMITTER: Stec B

PROVIDER: S-EPMC2917279 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Refined structures of placental alkaline phosphatase show a consistent pattern of interactions at the peripheral site.

Stec Boguslaw B Cheltsov Anton A Millán José Luis JL

Acta crystallographica. Section F, Structural biology and crystallization communications 20100727 Pt 8

In order to gain deeper insights into the functional sites of human placental alkaline phosphatase, the structures of the enzyme with the putative regulators L-Phe, pNPP and 5'-AMP [Llinas et al. (2005), J. Mol. Biol. 350, 441-451] were re-refined. Significant variations in ligand positioning and identity were found compared with the previous report. The multiple corrections to the model improved the phases and the electron-density maps, allowing the modeling of omitted side chains and multiple ...[more]

PMID: 20693656

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Project description:BACKGROUND: The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP) is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP). The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. RESULTS: Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP) and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP. CONCLUSION: The results demonstrate the biochemical modulation of scFv binding. Also, the scFvs bound to the active site and denied the access to the substrate. The selection strategy could generate specific anti-enzyme antibodies to PLAP that can potentially be used for targeting, for modulating enzyme activity in in vitro and in vivo and as probes for the active site. This strategy also has a general application in selecting antibodies from combinatorial libraries to closely related molecules and conformations.

Project description:Alkaline phosphatase is an enzyme with a typical alpha/beta hydrolase fold. The conformational stability of the human placental alkaline phosphatase was examined with the chemical denaturant urea. The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme. None of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, could stabilize one of the unfolding intermediates, which is not a salt effect. Some of the unfolding intermediates were also observed in circular dichroism spectroscopy, which clearly indicates steady loss of helical structure during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure changes, suggesting that the substrate-binding region is more resistant to chemical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N <--> I(1) <--> I(2) <--> I(3) <--> I(4) <--> I(5) <--> D. These discrete intermediates are stable at urea concentrations of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The stepwise unfolding process was interpreted by the folding energy landscape in terms of the unique structure of the enzyme. The rigid central beta-strand domain is surrounded by the peripheral alpha-helical and coil structures, which are marginally stable toward a chemical denaturant.

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Refined structures of placental alkaline phosphatase show a consistent pattern of interactions at the peripheral site.

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Refined structures of placental alkaline phosphatase show a consistent pattern of interactions at the peripheral site.

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