Mutations at the dimer, hexamer, and receptor-binding surfaces of insulin independently affect insulin-insulin and insulin-receptor interactions
| Autor: | Zi Xian Lu, Steven E. Shoelson, Michael A. Weiss, Claire S. Lynch, Lina Parlautan |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Magnetic Resonance Spectroscopy
Macromolecular Substances Stereochemistry Phenylalanine medicine.medical_treatment Molecular Sequence Data Protein design Random hexamer Biochemistry Structure-Activity Relationship medicine Humans Insulin Structure–activity relationship Amino Acid Sequence Binding site chemistry.chemical_classification Binding Sites biology Circular Dichroism Receptor Insulin Protein tertiary structure Amino acid Insulin receptor chemistry Mutation biology.protein |
| Zdroj: | Biochemistry. 31:1757-1767 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00121a025 |
| Popis: | Mutagenesis of the dimer- and hexamer-forming surfaces of insulin yields analogues with reduced tendencies to aggregate and dramatically altered pharmacokinetic properties. We recently showed that one such analogue, HisB10----Asp, ProB28----Lys, LysB29----Pro human insulin (DKP-insulin), has enhanced affinity for the insulin receptor and is useful for studying the structure of the insulin monomer under physiologic solvent conditions [Weiss, M. A., Hua, Q. X., Lynch, C. S., Frank, B. H., & Shoelson, S. E. (1991) Biochemistry 30, 7373-7389]. DKP-insulin retains native secondary and tertiary structure in solution and may therefore provide an appropriate baseline for further studies of related analogues containing additional substitutions within the receptor-binding surface of insulin. To test this, we prepared a family of DKP analogues having potency-altering substitutions at the B24 and B25 positions using a streamlined approach to enzymatic semisynthesis which negates the need for amino-group protection. For comparison, similar analogues of native human insulin were prepared by standard semisynthetic methods. The DKP analogues show a reduced tendency to self-associate, as indicated by 1H-NMR resonance line widths. In addition, CD spectra indicate that (with one exception) the native insulin fold is retained in each analogue; the exception, PheB24----Gly, induces similar perturbations in both native insulin and DKP-insulin backgrounds. Notably, analogous substitutions exhibit parallel trends in receptor-binding potency over a wide range of affinities: D-PheB24 greater than unsubstituted greater than GlyB24 greater than SerB24 greater than AlaB25 greater than LeuB25 greater than SerB25, whether the substitution was in a native human or DKP-insulin background. Such "template independence" reflects an absence of functional interactions between the B24 and B25 sites and additional substitutions in DKP-insulin and demonstrates that mutations in discrete surfaces of insulin have independent effects on protein structure and function. In particular, the respective receptor-recognition (PheB24, PheB25), hexamer-forming (HisB10), and dimer-forming (ProB28, LysB29) surfaces of insulin may be regarded as independent targets for protein design. DKP-insulin provides an appropriate biophysical model for defining structure-function relationships in a monomeric template. |
| Databáze: | OpenAIRE |
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