Mapping the functional surface of insulin by design: structure and function of a novel A-chain analogue.
Functional surfaces of a protein are often mapped by combination of X-ray crystallography and mutagenesis. Such studies of insulin have yielded paradoxical results, suggesting that the native state is inactive and reorganizes on receptor binding. Of particular interest is the N-terminal alpha-helix of the A-chain. Does this segment function as an alpha-helix or reorganize as recently proposed in a prohormone-convertase complex? To correlate structure and function, we describe a mapping strategy based on protein design. The solution structure of an engineered monomer ([AspB10, LysB28, ProB29]-human insulin) is determined at neutral pH as a template for synthesis of a novel A-chain analogue. Designed by analogy to a protein-folding intermediate, the analogue lacks the A6-A11 disulphide bridge; the cysteine residues are replaced by serine. Its solution structure is remarkable for segmental unfolding of the N-terminal A-chain alpha-helix (A1 to A8) in an otherwise native subdomain. The structure demonstrates that the overall orientation of the A and B chains is consistent with reorganization of the A-chain's N-terminal segment. Nevertheless, the analogue's low biological activity suggests that this segment, a site of clinical mutation causing diabetes mellitus, functions as a preformed recognition alpha-helix.
Hua
Q X
QX
Center for Molecular Oncology, University of Chicago, IL 60637, USA.
Hu
S Q
SQ
Frank
B H
BH
Jia
W
W
Chu
Y C
YC
Wang
S H
SH
Burke
G T
GT
Katsoyannis
P G
PG
Weiss
M A
MA
D000595:Amino Acid Sequence; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017510:Protein Folding; D012996:Solutions; D013329:Structure-Activity Relationship
D007328:Insulin; D012996:Solutions