RESUMEN
While cryo-electron microscopy (cryo-EM) has revolutionized the structure determination of supramolecular protein complexes that are refractory to structure determination by X-ray crystallography, structure determination by cryo-EM can nonetheless be complicated by excessive conformational flexibility or structural heterogeneity resulting from weak or transient protein-protein association. Since such transient complexes are often critical for function, specialized approaches must be employed for the determination of meaningful structure-function relationships. Here, we outline examples in which transient protein-protein interactions have been visualized successfully by cryo-EM in the biosynthesis of fatty acids, polyketides, and terpenes. These studies demonstrate the utility of chemical crosslinking to stabilize transient protein-protein complexes for cryo-EM structural analysis, as well as the use of partial signal subtraction and localized reconstruction to extract useful structural information out of cryo-EM data collected from inherently dynamic systems. While these approaches do not always yield atomic resolution insights on protein-protein interactions, they nonetheless enable direct experimental observation of complexes in assembly-line biosynthesis that would otherwise be too fleeting for structural analysis.
Asunto(s)
Dominio Catalítico , Microscopía por Crioelectrón/métodos , Enzimas/ultraestructura , Ácidos Grasos/biosíntesis , Complejos Multiproteicos/ultraestructura , Policétidos/metabolismo , Terpenos/metabolismo , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Transferasas Alquil y Aril/ultraestructura , Cristalografía por Rayos X , Enzimas/química , Enzimas/metabolismo , Ácido Graso Sintasas/química , Ácido Graso Sintasas/metabolismo , Ácido Graso Sintasas/ultraestructura , Imagenología Tridimensional/métodos , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/metabolismo , Sintasas Poliquetidas/ultraestructura , Unión Proteica , Reproducibilidad de los ResultadosRESUMEN
Isoprenoid biosynthesis in many bacteria, plant chloroplasts and parasitic protozoa but not in humans proceeds via the mevalonate independent 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. Its penultimate reaction step is catalyzed by (E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase (GcpE/IspG) which transforms 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate (MEcPP) to HMBPP. In this report we present the structure of GcpE of Thermus thermophiles in complex with its product HMBPP at a resolution of 1.65 Å. The GcpE-HMBPP like the GcpE-MEcPP structure is found in a closed, the ligand-free GcpE structure in an open enzyme state. Imposed by the rigid protein scaffold inside the active site funnel, linear HMBPP and circular MEcPP adopt highly similar conformations. The confined space also determines the conformational freedom of transition state intermediates and the design of anti-infective drugs. The apical Fe of the [4Fe-4S] cluster is coordinated to MEcPP in the GcpE-MEcPP complex and to a hydroxyl/water ligand but not to HMBPP in the GcpE-HMBPP complex. The GcpE-HMBPP structure can be attributed to one step in the currently proposed GcpE reaction cycle.
Asunto(s)
Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/ultraestructura , Organofosfatos/química , Terpenos/química , Thermus thermophilus/química , Conformación MolecularRESUMEN
Helicobacter pylori colonizes the human gastric epithelium and causes diseases such as gastritis, peptic ulcers, and stomach cancer. Undecaprenyl pyrophosphate synthase (UPPS), which catalyzes consecutive condensation reactions of farnesyl pyrophosphate with eight isopentenyl pyrophosphate to form lipid carrier for bacterial peptidoglycan biosynthesis, represents a potential target for developing new antibiotics. In this study, we solved the crystal structure of H. pylori UPPS and performed virtual screening of inhibitors from a library of 58,635 compounds. Two hits were found to exhibit differential activities against Helicobacter pylori and Escherichia coli UPPS, giving the possibility of developing antibiotics specially targeting pathogenic H. pylori without killing the intestinal E. coli.