Journal of Biological Chemistry
Volume 278, Issue 15, 11 April 2003, Pages 12913-12919
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PROTEIN STRUCTURE AND FOLDING
Domain Motions and Quaternary Packing of Phosphofructokinase-2 from Escherichia coli Studied by Small Angle X-ray Scattering and Homology Modeling*

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The binding of MgATP and fructose-6-phosphate to phosphofructokinase-2 from Escherichia coli induces conformational changes that result in significant differences in the x-ray-scattering profiles compared with the unligated form of the enzyme. When fructose- 6-phosphate binds to the active site of the enzyme, the pair distribution function exhibits lower values at higher distances, indicating a more compact structure. Upon binding of MgATP to the allosteric site of the enzyme, the intensity at lower angles increases as a consequence of tetramer formation, but differences along higher angles also suggest changes at the tertiary structure level. We have used homology modeling to build the native dimeric form of phosphofructokinase-2 and fitted the experimental scattering curves by using rigid body movements of the domains in the model, similar to those observed in known homologous structures. The best fit with the experimental data of the unbound protein was achieved with open conformations of the domains in the model, whereas domain closure improves the agreement with the scattering of the enzyme-fructose-6-phosphate complex. Using the same approach, we utilized the scattering curve of the phosphofructokinase-2-MgATP complex to model the arrangement and conformation of dimers in the tetramer. We observed that, along with tetramerization, binding of MgATP to the allosteric site induces domain closure. Additionally, we used the scattering data to restore the low resolution structure of phosphofructokinase-2 (free and bound forms) by an ab initio procedure. Based on these findings, a proposal is made to account for the inhibitory effect of MgATP on the enzymatic activity.

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Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M212137200

*

This work was supported by Fondo Nacional de Desarrollo Cientı́fico y Tecnológico, Chile Grant 1010645 and Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil Grants 99/09471-7, 01/07798-0, and 98/14138-2. We also thank Programa de Apoio a Núcleos de Excelência (Ministerio de Ciência e Tecnologia, Brazil), Brazil and National Synchrotron Light Laboratory (Campinas, Brazil) for financial support.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.