Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Improving GPCRs stability and expression using point mutations (#138)

Joao P. L. Velloso 1 2 3 , Douglas Pires 1 2 3 4 , David Ascher 1 2 3 5
  1. School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland, Australia
  2. Systems and Computational Biology, Bio21 Institute, University of Melbourne, Melbourne, Victoria, Australia
  3. Computational Biology and Clinical Informatics, Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia
  4. School of Computing and Information Systems, University of Melbourne, Melbourne, Victoria, Australia
  5. Baker Department of Cardiometabolic Health, University of Melbourne, Melbourne, Victoria, Australia

Despite enormous efforts to structurally characterise GPCRs, experimental elucidation of their 3D structures remains a challenge. This is in part because they are dynamic, metastable membrane proteins.  In order to obtain high-resolution crystals, GPCR engineering is usually required to minimise conformational heterogeneity and maximise crystal contacts and stability (Kobilka and Deupi, 2007). In this study, we explored the use of computational approaches to guide rational GPCR engineering. Initially, we performed extensive manual curation from the literature. In total, we curated 1808 mutations across 46 different GPCRs with experimental measured stability data. This was used to assess the performance of 8 different computational approaches for screening the effects of mutations on protein stability. The maximum accuracy and Matthew’s correlation coefficients across these methods were just 0.65 and 0.30, respectively, despite some of these mutations present in their training data. This highlighted the complexity of using generic computational approaches to model mutations in GPCRs. We next sought to develop a predictor capable of identifying mutations that improve GPCR stability without compromising function. Over 622 features describing both the protein sequence and 3D structure were generated. Applying supervised machine learning, our initial models have achieved a Matthew's correlation coefficient of 0.30 when evaluated against a non-redundant test set. Interpreting the model, the major contribution to performance were sequence-based quantitative measures (substitution probability tables; Pandurangan et al., 2017). Moving forward, we are working on understanding on what mutations the model works well, in order to further improve our predictive performance, and to develop rules to help guide GPCR engineering

  1. Kobilka, Brian K. et al. Conformational complexity of G-protein-coupled receptors.Trends in Pharmacological Sciences, Volume 28, Issue 8, 397 - 406
  2. Pandurangan AP, Ochoa-Montaño B, Ascher DB, Blundell TL. SDM: a server for predicting effects of mutations on protein stability. Nucleic Acids Res. 2017 Jul 3;45(W1):W229-W235. doi: 10.1093/nar/gkx439. PMID: 28525590; PMCID: PMC5793720.