Torres, O. | Search bioRxiv

bioRxiv2024

A key residue of the extracellular gate provides quality control contributing to ABCG substrate specificity

For G-type ATP-binding cassette (ABC) transporters, a hydrophobic "di-leucine motif" as part of a hydrophobic extracellular gate has been described to separate a large substrate-binding cavity from a smaller upper cavity and proposed to act as a valve controlling drug extrusion. Here, we show that an L704F mutation in the hydrophobic extracellular gate of Arabidopsis ABCG36/PDR8/PEN3 uncouples the export of the auxin precursor indole-3-butyric acid (IBA) from that of the defense compound camalexin (CLX). Molecular dynamics simulations reveal an increase in free energy and pulling forces for CLX at both the entrance and exit sites of ABCG36L704F, respectively, providing a mechanistic rationale for the transport discrimination of CLX. Mutagenesis of L704 to tyrosine allows export of structurally related non-ABCG36 substrates, indole-3-acetic acid (IAA) and indole, suggesting an allosteric communication between the extracellular gate and the central substrate binding pocket. In summary, our work supports the conclusion that L704 is a key residue of the extracellular gate that provides a final quality control contributing to ABCG substrate specificity.

biochemistry↗

bioRxiv2023

Lightway access to AlphaMissense data that demonstrates a balanced performance of this missense mutation predictor

Single amino acid substitutions can profoundly affect protein folding, dynamics, and function, leading to potential pathological consequences. The ability to discern between benign and pathogenic substitutions is pivotal for therapeutic interventions and research directions. Given the limitations in experimental examination of these variants, AlphaMissense has emerged as a promising predictor of the pathogenicity of single nucleotide polymorphism variants. In our study, we assessed the efficacy of AlphaMissense across several protein groups, such as mitochondrial, housekeeping, transmembrane proteins, and specific proteins like CFTR, using ClinVar data for validation. Our comprehensive evaluation showed that AlphaMissense delivers outstanding performance, with MCC scores predominantly between 0.6 and 0.74. We observed low performance on the CFTR and disordered, membrane-interacting MemMoRF datasets. However, an enhanced performance with CFTR was shown when benchmarked against the CFTR2 database. Our results also emphasize that quality of AlphaFolds predictions can seriously influence AlphaMissense predictions. Most importantly, AlphaMissenses consistent capability in predicting pathogenicity across diverse protein groups, spanning both transmembrane and soluble domains was found. Moreover, the prediction of likely-pathogenic labels for IBS and CFTR coupling helix residues emphasizes AlphaMissenses potential as a tool for pinpointing functionally significant sites. Additionally, to make AlphaMissense predictions more accessible, we have introduced a user-friendly web resource (https://alphamissense.hegelab.org) to enhance the utility of this valuable tool. Our insights into AlphaMissenses capability, along with this online resource, underscore its potential to significantly aid both research and clinical applications.

bioinformatics↗