Biogenesis of the secretory granule: chromogranin A coiled-coil structure results in unusual physical properties and suggests a mechanism for granule core condensation.
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.
Mosley
Coleman A
CA
Bioinformatics Graduate Program, University of California at San Diego, La Jolla, CA 92093-0838, USA.
Taupenot
Laurent
L
Biswas
Nilima
N
Taulane
Joseph P
JP
Olson
Norman H
NH
Vaingankar
Sucheta M
SM
Wen
Gen
G
Schork
Nicholas J
NJ
Ziegler
Michael G
MG
Mahata
Sushil K
SK
O'Connor
Daniel T
DT
D000465:Algorithms; D000818:Animals; D002395:Catecholamines / Q000737:chemistry* / Q000378:metabolism; D002837:Chromaffin Granules / Q000502:physiology / Q000648:ultrastructure*; D053379:Chromogranin A / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D005089:Exocytosis; D006801:Humans; D051379:Mice; D017348:Microscopy, Electron, Scanning Transmission; D008954:Models, Biological; D008956:Models, Chemical; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D034741:RNA, Small Interfering; D022142:Secretory Vesicles / Q000502:physiology / Q000648:ultrastructure*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship
D002395:Catecholamines; D053379:Chromogranin A; D010446:Peptide Fragments; D034741:RNA, Small Interfering; C109084:chromogranin A (344-364)