Structural bioinformatics studies of glutamate transporters and their AlphaFold2 predicted water-soluble QTY variants and uncovering the natural mutations of L->Q, I->T, F->Y and Q->L, T->I and Y->F.

Glutamate transporters play key roles in nervous physiology by modulating excitatory neurotransmitter levels, when malfunctioning, involving in a wide range of neurological and physiological disorders. However, integral transmembrane proteins including the glutamate transporters remain notoriously difficult to study, due to their localization within the cell membrane. Here we present the structural bioinformatics studies of glutamate transporters and their water-soluble variants generated through QTY-code, a protein design strategy based on systematic amino acid substitutions. These include 2 structures determined by X-ray crystallography, cryo-EM, and 6 predicted by AlphaFold2, and their predicted water-soluble QTY variants. In the native structures of glutamate transporters, transmembrane helices contain hydrophobic amino acids such as leucine (L), isoleucine (I), and phenylalanine (F). To design water-soluble variants, these hydrophobic amino acids are systematically replaced by hydrophilic amino acids, namely glutamine (Q), threonine (T) and tyrosine (Y). The QTY variants exhibited water-solubility, with four having identical isoelectric focusing points (pI) and the other four having very similar pI. We present the superposed structures of the native glutamate transporters and their water-soluble QTY variants. The superposed structures displayed remarkable similarity with RMSD 0.528Å-2.456Å, despite significant protein transmembrane sequence differences (41.1%->53.8%). Additionally, we examined the differences of hydrophobicity patches between the native glutamate transporters and their QTY variants. Upon closer inspection, we discovered multiple natural variations of L->Q, I->T, F->Y and Q->L, T->I, Y->F in these transporters. Some of these natural variations were benign and the remaining were reported in specific neurological disorders. We further investigated the characteristics of hydrophobic to hydrophilic substitutions in glutamate transporters, utilizing variant analysis and evolutionary profiling. Our structural bioinformatics studies not only provided insight into the differences between the hydrophobic helices and hydrophilic helices in the glutamate transporters, but they are also expected to stimulate further study of other water-soluble transmembrane proteins.

Structural bioinformatics studies of serotonin, dopamine and norepinephrine transporters and their AlphaFold2 predicted water-soluble QTY variants and uncovering the natural mutations of L->Q, I->T, F->Y and Q->L, T->I and Y->F.

Monoamine transporters including transporters for serotonin, dopamine, and norepinephrine play key roles in monoaminergic synaptic signaling, involving in the molecular etiology of a wide range of neurological and physiological disorders. Despite being crucial drug targets, the study of transmembrane proteins remains challenging due to their localization within the cell membrane. To address this, we present the structural bioinformatics studies of 7 monoamine transporters and their water-soluble variants designed using the QTY code, by systematically replacing the hydrophobic amino acids leucine (L), valine (V), isoleucine (I) and phenylalanine (F) with hydrophilic amino acids (glutamine (Q), threonine (T) and tyrosine (Y). The resulting QTY variants, despite significant protein transmembrane sequence differences (44.27%-51.85%), showed similar isoelectric points (pI) and molecular weights. While their hydrophobic surfaces significantly reduced, this change resulted in a minimal structural alteration. Quantitatively, Alphafold2 predicted QTY variant structures displayed remarkable similarity with RMSD 0.492Å-1.619Å. Accompanied by the structural similarities of substituted amino acids in the context of 1.5Å electron density maps, our study revealed multiple QTY and reverse QTY variations in genomic databases. We further analyzed their phenotypical and topological characteristics. By extending evolutionary game theory to the molecular foundations of biology, we provided insights into the evolutionary dynamics of chemically distinct alpha-helices, their usage in different chemotherapeutic applications, and open possibilities of diagnostic medicine. Our study rationalizes that QTY variants of monoamine transporters may not only become distinct tools for medical, structural, and evolutionary research, but these transporters may also emerge as contemporary therapeutic targets, providing a new approach to treatment for several conditions.