Biogenesis of the Secretory Granule: Chromogranin A Coiled-Coil Structure Results in Unusual Physical Properties and Suggests a Mechanism for Granule Core Condensation

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• 作者：Coleman A. Mosley ; Laurent Taupenot ; Nilima Biswas ; Joseph P. Taulane ; Norman H. Olson ; Sucheta M. Vaingankar ; Gen Wen ; Nicholas J. Schork ; Michael G. Ziegler ; Sushil K. Mahata ; Daniel T. O'Connor
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：September 25, 2007
• 年：2007
• 卷：46
• 期：38
• 页码：10999 - 11012
• 全文大小：649K
• 年卷期：v.46,no.38(September 25, 2007)
• ISSN：1520-4995

文摘

The secretory pro-hormone chromogranin A (CHGA) is densely packed into storage granulesalong with catecholamines, playing a catalytic role in granule biogenesis. 3-Dimensional structural dataon CHGA are lacking. We found a superfamily structural homology for CHGA in the tropomyosin familyof alpha-helical coiled-coils, even in mid-molecule regions where primary sequence identity is only modest.The assignment was confirmed by an independent algorithm, suggesting ~6-7 such domains spanningCHGA. We provide additional physiochemical evidence (chromatographic, spectral, microscopic) consistentwith this unusual structure. Alpha-helical secondary structure (at up to ~45%) was confirmed by circulardichroism. CHGA molecular mass was estimated by MALDI-TOF mass spectrometry at ~50 kDa andby denaturing gel filtration at ~50-61 kDa, while its native Stokes radius was ~84.8 Å, as compared toan expected ~30 Å; the increase gave rise to an apparent native molecular weight of ~578 kDa, alsoconsistent with the extended conformation of a coiled-coil. Small-angle X-ray scattering (SAXS) on CHGAin solution best fit an elongated cylindrical conformation in the monodisperse region with a radius ofgyration of the rod cross-section (R_t) of ~52 Å, compatible with a coiled-coil in the hydrated, aqueousstate, or a multimeric coiled-coil. Electron microscopy with negative staining revealed an extended,filamentous CHGA structure with a diameter of ~94 ± 4.5 Å. Extended, coiled-coil conformation islikely to permit protein "packing" in the secretory granule at ~50% higher density than a globular/sphericalconformation. Natural allelic variation in the catestatin region was predicted to disrupt the coiled-coil.Chromaffin granule ultrastructure revealed a ~108 ± 6.3 Å periodicity of electron density, suggestingnucleation of a binding complex by the CHGA core. Inhibition of CHGA expression, by siRNA, disruptedregulated secretory protein traffic by ~65%, while targeted ablation of the CHGA gene in the mousereduced chromaffin granule cotransmitter concentrations by ~40-80%. These results suggest new rolesfor secretory protein tertiary structure in hormone and transmitter storage, with implications for secretorycargo condensation (or dense core "packing" structure) within the regulated pathway.