Nonlocal structural perturbations in a mutant human insulin: sequential resonance assignment and 13C-isotope-aided 2D-NMR studies of [PheB24-->Gly]insulin with implications for receptor recognition.
Insulin's mechanism of receptor binding is not well understood despite extensive study by mutagenesis and X-ray crystallography. Of particular interest are "anomalous" analogues whose bioactivities are not readily rationalized by crystal structures. Here the structure and dynamics of one such analogue (GlyB24-insulin) are investigated by circular dichroism (CD) and isotope-aided 2D-NMR spectroscopy. The mutant insulin retains near-native receptor-binding affinity despite a nonconservative substitution (PheB24-->Gly) in the receptor-binding surface. Relative to native insulin, GlyB24-insulin exhibits reduced dimerization; the monomer (the active species) exhibits partial loss of ordered structure, as indicated by CD studies and motional narrowing of selected 1H-NMR resonance. 2D-NMR studies demonstrate that the B-chain beta-turn (residues B20-23) and beta-strand (residues B24-B28) are destabilized; essentially native alpha-helical secondary structure (residues A3-A8, A13-A18, and B9-B19) is otherwise maintained. 13C-Isotope-edited NOESY studies demonstrate that long-range contacts observed between the B-chain beta-strand and the alpha-helical core in native insulin are absent in the mutant. Implications for the mechanism of insulin's interaction with its receptor are discussed.
Hua
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Shoelson
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Weiss
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MA
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D005998:Glycine / Q000737:chemistry*; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010649:Phenylalanine / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011972:Receptor, Insulin / Q000378:metabolism*; D013329:Structure-Activity Relationship
D007328:Insulin; D010649:Phenylalanine; D011972:Receptor, Insulin; D005998:Glycine