Analysis of Mutations in Human Growth Hormone for Interaction with Growth Hormone Receptor and Structural Stability Using Purified Proteins Expressed in E. coli
Presentation Number: SAT 429
Date of Presentation: April 1st, 2017
Shaheena Parween1, Maria Consolata Miletta2, Andrée Eblé3 and Amit V. Pandey*4
1UniVersity Children's Hospital Bern, Bern, Switzerland, 2University Children's Hospital Bern, Bern, SWITZERLAND, 3University Children's Hospital Bern, Bern, Switzerland, 4University Children's Hospital Bern,, Bern, Switzerland
Introduction: Human growth hormone acts via binding to two molecules of growth hormone receptor (GHR) which dimerize and start signaling pathways for downstream effects. Mutations in GH1 gene cause isolated growth hormone deficiency (IGHD) by affecting production, secretion and stability of growth hormone as well as its binding to GHR (1). The P59L mutant of GH1 had been shown earlier to be associated with IGHD, and GH-secretion studies had showed a moderate difference in secretion between GH-P59L and wt-GH (2).
Objective: Most studies reporting IGHD have focused on defects in transcription or secretion of GH protein due to mutations in GH1 gene. Study of protein interactions between hormone and its receptor requires purified proteins to confirm the differences in binding affinities due to mutations.
Methods: We have adopted the analysis of GH1 gene mutations by recombinant production of mutant human GH proteins in bacteria and purifying the proteins by chromatographic methods. Purified GH variants were separated using SDS-PAGE and analyzed by western blotting to confirm their identity with anti-GH antibodies. Purified proteins were further analyzed by receptor binding and conformational stability assays as described (3).
Results: Computational analysis showed that P59 residue is close to first GH binding site of GHR. In silico mutagenesis and molecular dynamics simulations indicated a defective binding of GH-P59L to the GHR. Binding affinity of purified WT GH was found to be 10 fold higher than GH-P59L mutant, providing a definitive link between biochemical assays and known phenotype of the reported patient. Analysis of WT and P59L GH stability by FASTpp assay using thermolysin at different temperatures to study protein degradation revealed no major differences, confirming that IGHD due to P59L mutation in GH1 is due to reduced binding of GH protein with GHR.
Conclusions: Advanced protein chemistry and computational analysis methods can be used to characterize the molecular basis of defects in GH1 gene causing IGHD. Use of purified recombinant proteins allowed us to apply advanced biophysical and biochemical methods for analysis of mutations in human growth hormone.
Nothing to Disclose: SP, MCM, AE, AVP