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PDBsum entry 2ke3
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References listed in PDB file
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Key reference
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Title
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Functional and structural characterization of a dense core secretory granule sorting domain from the pc1/3 protease.
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Authors
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J.D.Dikeakos,
P.Di lello,
M.J.Lacombe,
R.Ghirlando,
P.Legault,
T.L.Reudelhuber,
J.G.Omichinski.
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Ref.
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Proc Natl Acad Sci U S A, 2009,
106,
7408-7413.
[DOI no: ]
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PubMed id
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Abstract
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Several peptide hormones are initially synthesized as inactive precursors. It is
only on entry of these prohormones and their processing proteases into dense
core secretory granules (DCSGs) that the precursors are cleaved to generate
their active forms. Prohormone convertase (PC)1/3 is a processing protease that
is targeted to DCSGs. The signal for targeting PC1/3 to DCSGs resides in its
carboxy-terminal tail (PC1/3(617-753)), where 3 regions (PC1/3(617-625),
PC1/3(665-682), and PC1/3(711-753)) are known to aid in sorting and membrane
association. In this article, we have determined a high-resolution structure of
the extreme carboxy-terminal sorting domain, PC1/3(711-753) in micelles by NMR
spectroscopy. PC1/3(711-753) contains 2 alpha helices located between residues
722-728 and 738-750. Functional assays demonstrate that the second helix
(PC1/3(738-750)) is necessary and sufficient to target a constitutively secreted
protein to granules, and that L(745) anchors a hydrophobic patch that is
critical for sorting. Also, we demonstrate that calcium binding by the second
helix of PC1/3(711-753) promotes aggregation of the domain via the hydrophobic
patch centered on L(745). These results provide a structure-function analysis of
a DCSG-sorting domain, and reveal the importance of a hydrophobic patch and
calcium binding in controlling the sorting of proteins containing alpha helices
to DCSGs.
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Figure 1.
Structure of PC1/3[711–753] in CHAPS. The 20 lowest-energy
conformers were superimposed using the backbone atoms C′,
C^α, and N of the first helix between residue S^722 and residue
F^728 (A) and the second helix between residue D^738 and residue
N^750 (B). Ribbon (C and E) and helical wheel (D and F)
representations of the 2 alpha helices in the PC1/3[711–753]
DCSG-sorting domain. Hydrophobic side chains are shown in the
ribbon representations, and hydrophobic amino acids are
highlighted in orange in the helical wheels while hydrophilic
amino acids are represented in blue.
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Figure 2.
PC1/3[711–753] interacts with calcium. (A) Overlay of the
2D ^1H-^15N HSQC spectra of ^15N-labeled PC1/3[711–753] in the
free form (black) and in the presence of 10 mM CaCl[2] (red).
Spectra were recorded in 20 mM d-11 Tris (pH = 6.5) at 26.6
°C with a protein concentration of 1.0 mM in 20 mM CHAPS.
Examples of shifted signals are circled. (B) Histogram of the
variations Δδ[(ppm)] = [(0.17ΔN[H])^2 + (ΔH[N]) ^2]^1/2 (39).
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