Toward the active conformation of insulin: stereospecific modulation of a structural switch in the B chain.
How insulin binds to the insulin receptor has long been a subject of speculation. Although the structure of the free hormone has been extensively characterized, a variety of evidence suggests that a conformational change occurs upon receptor binding. Here, we employ chiral mutagenesis, comparison of corresponding d and l amino acid substitutions, to investigate a possible switch in the B-chain. To investigate the interrelation of structure, function, and stability, isomeric analogs have been synthesized in which an invariant glycine in a beta-turn (Gly(B8)) is replaced by d- or l-Ser. The d substitution enhances stability (DeltaDeltaG(u) 0.9 kcal/mol) but impairs receptor binding by 100-fold; by contrast, the l substitution markedly impairs stability (DeltaDeltaG(u) -3.0 kcal/mol) with only 2-fold reduction in receptor binding. Although the isomeric structures each retain a native-like overall fold, the l-Ser(B8) analog exhibits fewer helix-related and long range nuclear Overhauser effects than does the d-Ser(B8) analog or native monomer. Evidence for enhanced conformational fluctuations in the unstable analog is provided by its attenuated CD spectrum. The inverse relationship between stereospecific stabilization and receptor binding strongly suggests that the B7-B10 beta-turn changes conformation on receptor binding.
Hua
Qing-Xin
QX
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Nakagawa
Satoe
S
Hu
Shi-Quan
SQ
Jia
Wenhua
W
Wang
Shuhua
S
Weiss
Michael A
MA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011972:Receptor, Insulin / Q000378:metabolism
D007328:Insulin; D011972:Receptor, Insulin