RESUMEN
The pyruvate dehydrogenase complex (PDHc) is a key megaenzyme linking glycolysis with the citric acid cycle. In mammalian PDHc, dihydrolipoamide acetyltransferase (E2) and the dihydrolipoamide dehydrogenase-binding protein (E3BP) form a 60-subunit core that associates with the peripheral subunits pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3). The structure and stoichiometry of the fully assembled, mammalian PDHc or its core remained elusive. Here, we demonstrate that the human PDHc core is formed by 48 E2 copies that bind 48 E1 heterotetramers and 12 E3BP copies that bind 12 E3 homodimers. Cryo-electron microscopy, together with native and cross-linking mass spectrometry, confirmed a core model in which 8 E2 homotrimers and 12 E2-E2-E3BP heterotrimers assemble into a pseudoicosahedral particle such that the 12 E3BP molecules form six E3BP-E3BP intertrimer interfaces distributed tetrahedrally within the 60-subunit core. The even distribution of E3 subunits in the peripheral shell of PDHc guarantees maximum enzymatic activity of the megaenzyme.
Asunto(s)
Microscopía por Crioelectrón , Complejo Piruvato Deshidrogenasa , Humanos , Complejo Piruvato Deshidrogenasa/metabolismo , Complejo Piruvato Deshidrogenasa/química , Modelos Moleculares , Dihidrolipoamida Deshidrogenasa/metabolismo , Dihidrolipoamida Deshidrogenasa/química , Multimerización de Proteína , Unión Proteica , Subunidades de Proteína/metabolismo , Subunidades de Proteína/química , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/metabolismo , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/químicaRESUMEN
Type 1 pili are important virulence factors of uropathogenic Escherichia coli that mediate bacterial attachment to epithelial cells in the urinary tract. The pilus rod is comprised of thousands of copies of the main structural subunit FimA and is assembled in vivo by the assembly platform FimD. Although type 1 pilus rods can self-assemble from FimA in vitro, this reaction is slower and produces structures with lower kinetic stability against denaturants compared to in vivo-assembled rods. Our study reveals that FimD-catalysed in vitro-assembled type 1 pilus rods attain a similar stability as pilus rods assembled in vivo. Employing structural, biophysical and biochemical analyses, we show that in vitro assembly reactions lacking FimD produce pilus rods with structural defects, reducing their stability against dissociation. Overall, our results indicate that FimD is not only required for the catalysis of pilus assembly, but also to control the assembly of the most stable quaternary structure.
Asunto(s)
Proteínas de Escherichia coli , Proteínas Fimbrias , Proteínas Fimbrias/genética , Proteínas Fimbrias/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Fimbrias Bacterianas/químicaRESUMEN
The Escherichia coli pyruvate dehydrogenase complex (PDHc) is a ~5 MDa assembly of the catalytic subunits pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). The PDHc core is a cubic complex of eight E2 homotrimers. Homodimers of the peripheral subunits E1 and E3 associate with the core by binding to the peripheral subunit binding domain (PSBD) of E2. Previous reports indicated that 12 E1 dimers and 6 E3 dimers bind to the 24-meric E2 core. Using an assembly arrested E2 homotrimer (E23), we show that two of the three PSBDs in the E23 dimerize, that each PSBD dimer cooperatively binds two E1 dimers, and that E3 dimers only bind to the unpaired PSBD in E23. This mechanism is preserved in wild-type PDHc, with an E1 dimer:E2 monomer:E3 dimer stoichiometry of 16:24:8. The conserved PSBD dimer interface indicates that PSBD dimerization is the previously unrecognized architectural determinant of gammaproteobacterial PDHc megacomplexes.
Asunto(s)
Dihidrolipoamida Deshidrogenasa , Acetiltransferasa de Residuos Dihidrolipoil-Lisina , Escherichia coli , Complejo Piruvato Deshidrogenasa , Dihidrolipoamida Deshidrogenasa/genética , Dihidrolipoamida Deshidrogenasa/química , Dihidrolipoamida Deshidrogenasa/metabolismo , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/química , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/metabolismo , Dimerización , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Complejo Piruvato Deshidrogenasa/química , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
Complexin-1 is a SNARE effector protein that decreases spontaneous neurotransmitter release and enhances evoked release. Complexin binds to the fully assembled four-helical neuronal SNARE core complex as revealed in competing molecular models derived from x-ray crystallography. Presently, it is unclear how complexin binding to the postfusion complex accounts for its effects upon spontaneous and evoked release in vivo. Using a combination of spectroscopic and imaging methods, we characterize in molecular detail how complexin binds to the 1:1 plasma membrane t-SNARE complex of syntaxin-1a and SNAP-25 while simultaneously binding the lipid bilayer at both its N- and C-terminal ends. These interactions are cooperative, and binding to the prefusion acceptor t-SNARE complex is stronger than to the postfusion core complex. This complexin interaction reduces the affinity of synaptobrevin-2 for the 1:1 complex, thereby retarding SNARE assembly and vesicle docking in vitro. The results provide the basis for molecular models that account for the observed clamping effect of complexin beginning with the acceptor t-SNARE complex and the subsequent activation of the clamped complex by Ca2+ and synaptotagmin.