NMR and photo-CIDNP studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition.
The proinsulin-insulin system provides a general model for the proteolytic processing of polypeptide hormones. Two proinsulin-specific endopeptidases have been defined, a type I activity that cleaves the B-chain/C-peptide junction (Arg31-Arg32) and a type II activity that cleaves the C-peptide/A-chain junction (Lys64-Arg65). These endopeptidases are specific for their respective dibasic target sites; not all such dibasic sites are cleaved, however, and studies of mutant proinsulins have demonstrated that additional sequence or structural features are involved in determining substrate specificity. To define structural elements required for endopeptidase recognition, we have undertaken comparative 1H NMR and photochemical dynamic nuclear polarization (photo-CIDNP) studies of human proinsulin, insulin, and split proinsulin analogues as models of prohormone processing intermediates. The overall conformation of proinsulin is observed to be similar to that of insulin, and the connecting peptide is largely unstructured. In the 1H NMR spectrum of proinsulin significant variation is observed in the line widths of insulin-specific amide resonances, reflecting exchange among conformational substates; similar exchange is observed in insulin and is not damped by the connecting peptide. The aromatic 1H NMR resonances of proinsulin are assigned by analogy to the spectrum of insulin, and assignments are verified by chemical modification. Unexpectedly, nonlocal perturbations are observed in the insulin moiety of proinsulin, as monitored by the resonances of internal aromatic groups. Remarkably, these perturbations are reverted by site-specific cleavage of the connecting peptide at the CA junction but not the BC junction. These results suggest that a stable local structure is formed at the CA junction, which influences insulin-specific packing interactions. We propose that this structure (designated the "CA knuckle") provides a recognition element for type II proinsulin endopeptidase.
Weiss
M A
MA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Frank
B H
BH
Khait
I
I
Pekar
A
A
Heiney
R
R
Shoelson
S E
SE
Neuringer
L J
LJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000096:biosynthesis*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011384:Proinsulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
D007328:Insulin; D011384:Proinsulin; D010450:Endopeptidases