Trinidad, D. | Search bioRxiv

bioRxiv2023

The full spectrum of OCT1 (SLC22A1) mutations bridges transporter biophysics to drug pharmacogenomics

Membrane transporters play a fundamental role in the tissue distribution of endogenous compounds and xenobiotics and are major determinants of efficacy and side effects profiles. Polymorphisms within these drug transporters result in inter-individual variation in drug response, with some patients not responding to the recommended dosage of drug whereas others experience catastrophic side effects. For example, variants within the major hepatic Human organic cation transporter OCT1 (SLC22A1) can change endogenous organic cations and many prescription drug levels. To understand how variants mechanistically impact drug uptake, we systematically study how all known and possible single missense and single amino acid deletion variants impact expression and substrate uptake of OCT1. We find that human variants primarily disrupt function via folding rather than substrate uptake. Our study revealed that the major determinants of folding reside in the first 300 amino acids, including the first 6 transmembrane domains and the extracellular domain (ECD) with a stabilizing and highly conserved stabilizing helical motif making key interactions between the ECD and transmembrane domains. Using the functional data combined with computational approaches, we determine and validate a structure-function model of OCT1s conformational ensemble without experimental structures. Using this model and molecular dynamic simulations of key mutants, we determine biophysical mechanisms for how specific human variants alter transport phenotypes. We identify differences in frequencies of reduced function alleles across populations with East Asians vs European populations having the lowest and highest frequency of reduced function variants, respectively. Mining human population databases reveals that reduced function alleles of OCT1 identified in this study associate significantly with high LDL cholesterol levels. Our general approach broadly applied could transform the landscape of precision medicine by producing a mechanistic basis for understanding the effects of human mutations on disease and drug response.

biophysics↗

bioRxiv2022

Deep Insertion, Deletion, and Missense Mutation Libraries for Exploring Protein Variation in Evolution, Disease, and Biology

Insertions and deletions (indels) are a major source of genetic variation in evolution and the cause of nearly 30% of Mendelian disease. Despite their importance, indels are left out of nearly every systematic mutational scan to date due to technical challenges associated with making indel-containing libraries, limiting our understanding of indels in disease, biology, and evolution. Here we present a library generation method, DIMPLE, that generates deletions, insertions, and missense at similar frequencies within any gene. To benchmark DIMPLE, we generated libraries within four genes (Kir2.1, VatD, TRPV1, and OPRM1) of varying length and evolutionary origin. DIMPLE produces libraries that are near complete, low cost, and low bias. We measured how missense mutations and indels of varying length impact the potassium channel Kir2.1 surface expression. Across all Kir2.1s secondary structure, deletions are more disruptive than insertions, beta sheets are extremely sensitive to large deletions, and flexible loops allow insertions far more frequently than deletions. DIMPLEs low bias, ease of use, and low cost will enable high throughput probing of the importance of indels in disease and evolution.

genomics↗