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

• Published in: Biochemistry (Easton) 2009-06, Vol.46 (38)
• Main Authors: Mosley, C.A., Taupenot, L., Biswas, N., Taulane, J.P., Olson, N.H., Vaingankar, S.M., Wen, G., Schork, N.J., Ziegler, M.G., Mahata, S.K., O'Connor, D.T.
• Format: Article
• Language: English
• Subjects: ABLATION; BASIC BIOLOGICAL SCIENCES; CATECHOLAMINES; DENSITY; DICHROISM; ELECTRON DENSITY; ELECTRON MICROSCOPY; FILTRATION; GELS; GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; GENES; HORMONES; INHIBITION; MASS; MASS SPECTROSCOPY; MOLECULAR WEIGHT; NUCLEATION; Other,BIO, CHEM; PERIODICITY; PHYSICAL PROPERTIES; PROTEINS; SCATTERING; SOLUTIONS; TROPOMYOSIN; VARIATIONS
• ISSN: 0006-2960; 1520-4995

The secretory pro-hormone chromogranin A (CHGA) is densely packed into storage granules along with catecholamines, playing a catalytic role in granule biogenesis. 3-Dimensional structural data on CHGA are lacking. We found a superfamily structural homology for CHGA in the tropomyosin family of 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 approximately 6-7 such domains spanning CHGA. We provide additional physiochemical evidence (chromatographic, spectral, microscopic) consistent with this unusual structure. Alpha-helical secondary structure (at up to approximately 45%) was confirmed by circular dichroism. CHGA molecular mass was estimated by MALDI-TOF mass spectrometry at approximately 50 kDa and by denaturing gel filtration at approximately 50-61 kDa, while its native Stokes radius was approximately 84.8 A, as compared to an expected approximately 30 A; the increase gave rise to an apparent native molecular weight of approximately 578 kDa, also consistent with the extended conformation of a coiled-coil. Small-angle X-ray scattering (SAXS) on CHGA in solution best fit an elongated cylindrical conformation in the monodisperse region with a radius of gyration of the rod cross-section (Rt) of approximately 52 A, compatible with a coiled-coil in the hydrated, aqueous state, or a multimeric coiled-coil. Electron microscopy with negative staining revealed an extended, filamentous CHGA structure with a diameter of approximately 94 +/- 4.5 A. Extended, coiled-coil conformation is likely to permit protein 'packing' in the secretory granule at approximately 50% higher density than a globular/spherical conformation. Natural allelic variation in the catestatin region was predicted to disrupt the coiled-coil. Chromaffin granule ultrastructure revealed a approximately 108 +/- 6.3 A periodicity of electron density, suggesting nucleation of a binding complex by the CHGA core. Inhibition of CHGA expression, by siRNA, disrupted regulated secretory protein traffic by approximately 65%, while targeted ablation of the CHGA gene in the mouse reduced chromaffin granule cotransmitter concentrations by approximately 40-80%. These results suggest new roles for secretory protein tertiary structure in hormone and transmitter storage, with implications for secretory cargo condensation (or dense core 'packing' structure) within the regulated pathway.