Stability and global fold of the mouse prohormone convertase 1 pro-domain.
We have purified the mouse prohormone convertase 1 (PC1) pro-domain expressed in Escherichia coli cells and demonstrated, using a number of biophysical methods, that this domain is an independent folding unit with a T(m) of 39 degrees C at a protein concentration of 20 microM and pH 7.0. This differs significantly from similar pro-domains in bacteria and human furin, which are unfolded at 25 degrees C and require the catalytic domain in order to be structured [Bryan et al. (1995) Biochemistry 34, 10310-10318; Bhattacharjya et al. (2000) J. Biomol. NMR 16, 275-276]. Using heteronuclear NMR spectroscopy, we have determined the backbone (1)H, (13)C, and (15)N assignments for the pro-domain of PC1. On the basis of (1)H/(13)C chemical shift indices, NOE analysis, and hydrogen exchange measurements, the pro-domain is shown to consist of a four-stranded beta-sheet and two alpha-helices. The results presented here show that both the bacterial pro-domain in complex with subtilisin and the uncomplexed mouse PC1 pro-domain have very similar overall folds despite a lack of sequence homology. The structural data help to explain the location of the secondary processing sites in the pro-domains of the PC family, and a consensus sequence for binding to the catalytic domain is proposed.
Tangrea
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Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville 20850, USA.
Alexander
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Bryan
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Eisenstein
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E
Toedt
J
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Orban
J
J
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology; D002942:Circular Dichroism; D003903:Deuterium / Q000737:chemistry; D004795:Enzyme Stability; D051379:Mice; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D045664:Proprotein Convertase 1; D043484:Proprotein Convertases; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011522:Protons; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D014461:Ultracentrifugation
D010446:Peptide Fragments; D011498:Protein Precursors; D011522:Protons; D003903:Deuterium; D043484:Proprotein Convertases; D013381:Subtilisins; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D016282:Aspartic Acid Endopeptidases