Alanine Scanning Mutagenesis of Insulin

Claus Kristensen,Thomas Kjeldsen,Finn C Wiberg,Lauge Schäffer,Morten Hach,Svend Havelund,Joseph Bass,Donald F Steiner,Asser S Andersen

doi:10.1074/jbc.272.20.12978

Claus Kristensen, Thomas Kjeldsen + Show 7 more

Open Access

https://doi.org/10.1074/jbc.272.20.12978

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Abstract

Alanine scanning mutagenesis has been used to identify specific side chains of insulin which strongly influence binding to the insulin receptor. A total of 21 new insulin analog constructs were made, and in addition 7 high pressure liquid chromatography-purified analogs were tested, covering alanine substitutions in positions B1, B2, B3, B4, B8, B9, B10, B11, B12, B13, B16, B17, B18, B20, B21, B22, B26, A4, A8, A9, A12, A13, A14, A15, A16, A17, A19, and A21. Binding data on the analogs revealed that the alanine mutations that were most disruptive for binding were at positions TyrA19, GlyB8, LeuB11, and GluB13, resulting in decreases in affinity of 1,000-, 33-, 14-, and 8-fold, respectively, relative to wild-type insulin. In contrast, alanine substitutions at positions GlyB20, ArgB22, and SerA9 resulted in an increase in affinity for the insulin receptor. The most striking finding is that B20Ala insulin retains high affinity binding to the receptor. GlyB20 is conserved in insulins from different species, and in the structure of the B-chain it appears to be essential for the shift from the alpha-helix B8-B19 to the beta-turn B20-B22. Thus, replacing GlyB20 with alanine most likely modifies the structure of the B-chain in this region, but this structural change appears to enhance binding to the insulin receptor.

Highlights

Alanine scanning mutagenesis has been used to identify specific side chains of insulin which strongly influence binding to the insulin receptor
Alanine Constructs and Expression in Yeast—For the alanine scanning mutagenesis we made 21 new insulin analog constructs with alanine substitutions in positions B1, B3, B4, B9, B10, B11, B12, B13, B16, B20, B21, B22, A4, A9, A12, A13, A14, A15, A16, A17, and A19 (Fig. 1)
Most of the alanine substitutions result in some decrease in yield, probably because the yeast expression system is optimized for expressing the wild-type insulin precursor

Summary

Introduction

Alanine scanning mutagenesis has been used to identify specific side chains of insulin which strongly influence binding to the insulin receptor. Until the structure of bound insulin and the side chains that are involved in binding is identified by co-crystallization of the receptor and ligand, more information about the binding domain on insulin can be obtained using mutational approaches. The binding domain of the insulin molecule has been studied by investigating receptor binding of a number of insulins from different animal species as well as chemically modified and more recently genetically engineered insulins (2– 4). These studies have provided experimental support for a model in which invariant residues from both A and B chains form a surface that binds to the insulin receptor. A biphasic binding reaction involving this second binding site could explain the negative cooperativity phenomenon (8) as well as discrepancies between receptor binding data and metabolic potencies found in hystricomorph insulins and synthetic analogs with mutations at these positions (9, 10)

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