PDBsum entry 2ke3

Hydrolase

PDB id: 2ke3

Contents

Protein chain

43 a.a. *

* Residue conservation analysis

PDB id:

2ke3

Name:

Hydrolase

Title:

Pc1/3 dcsg sorting domain in chaps

Structure:

Neuroendocrine convertase 1. Chain: a. Fragment: unp residues 711-753. Synonym: nec 1, pc1, prohormone convertase 1, proprotein convertase 1, pc3, furin homolog, propeptide-processing protease. Engineered: yes

Source:

Mus musculus. Mouse. Organism_taxid: 10090. Gene: pcsk1, att-1, nec-1, nec1. Expressed in: escherichia coli. Expression_system_taxid: 562.

NMR struc:

20 models

Authors:

J.D.Dikeakos,P.Di Lello,M.J.Lacombe,R.Ghirlando,P.Legault, T.L.Reudelhuber,J.G.Omichinski

Key ref:

J.D.Dikeakos et al. (2009). Functional and structural characterization of a dense core secretory granule sorting domain from the PC1/3 protease. Proc Natl Acad Sci U S A, 106, 7408-7413. PubMed id: 19376969 DOI: 10.1073/pnas.0809576106

Date:

22-Jan-09 Release date: 14-Apr-09

Protein chain

P63239  (NEC1_MOUSE) -  Neuroendocrine convertase 1 from Mus musculus

Seq: 753 a.a.

Struc: 43 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.21.93 - proprotein convertase 1.
• Reaction: Release of protein hormones, neuropeptides and renin from their precursors, generally by cleavage of -Lys-Arg-|- bonds.

DOI no: 10.1073/pnas.0809576106

Proc Natl Acad Sci U S A 106:7408-7413 (2009)

PubMed id: 19376969

Functional and structural characterization of a dense core secretory granule sorting domain from the PC1/3 protease.

J.D.Dikeakos, P.Di Lello, M.J.Lacombe, R.Ghirlando, P.Legault, T.L.Reudelhuber, J.G.Omichinski.

ABSTRACT

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.

Selected figure(s)

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.

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).

Figures were selected by an automated process.