NMR and photo-CIDNP studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition

Abstract

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.

Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells.

“…Thus, we propose that proinsulin enters IGs in the soluble phase in pancreatic B-cells. These data are entirely consistent with earlier biophysical studies indicating that randomly coiled C-peptides interfere with close-packing of soluble proinsulin hexamers (Steiner, 1973;Weiss et al, 1990), thereby rendering proinsulin incapable of forming large, insoluble complexes in an aqueous environment above pH 3 (Fullerton et al, 1970;Grant et al, 1972). While we do not preclude the possibility that some proteins in some cell types might undergo complex Figure 9.…”

Structural requirements for processing of pro‐adipokinetic hormone I

“…Ultimately, understanding prohormone processing-site recognition will require the application of biophysical methods to obtain complete solution structures of prohormones, but to date, a complete prohormone structure has been obtained only for proinsulin (Weiss et al, 1990). Here, a previously undescribed secondary structural element, called the C-A knuckle, was observed at the Lys-Arg processing site.…”

Sorting and processing of secretory proteins

“…A potentially important structural domain in proinsulin, the CA knuckle, has been defined based upon n.m.r. studies of the molecule [306]. This domain may also be implicated in cleavage at the C-peptide/A-chain junction, but this has yet to be tested directly.…”