Fig 1.
Sequence and protein alignments of the natural and QTY variants of seven monoamine transporters.
The specific alignments are as follows: a NET vs NETQTY, b DAT vs DATQTY, c SERT vs SERTQTY, d VMAT1 vs VMAT1QTY, e VMAT2 vs VMAT2QTY, f VAChT vs VAChTQTY, and g VPAT vs VPATQTY. Molecular weight and isoelectric point (pI) are listed for both the natural and QTY variants. The transmembrane variation percentage and total variation percentage between the natural and QTY variants are also provided. TM alpha-helices (in blue) displayed above the protein sequences and QTY amino acid substitution changes colored in red. Additional color coding is used to distinguish intracellular regions (yellow line), transmembrane helices (blue wave) and extracellular regions (pinkish line). Single-letter abbreviations for the amino acid residues: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lle; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
Table 1.
Characteristics of the 7 monoamine transporters and their water-soluble QTY variants.
Fig 2.
Superposition of cryo-EM structure SERTCryoEM and AlphaFold2 predicted QTY water-soluble variant SERTQTY.
The native SERT (PDB ID: 6VRH) structure is obtained from the Protein Data Bank. N- and C-termini are labelled. The SERTCryoEM (magenta) is superposed with AlphaFold2 predicted water-soluble variant SERTQTY (cyan). The root mean square deviation (RMSD) between the two structures is 1.619Å. N- and C termini and large loops that are not resolved in the experimental structures are removed for clarity of direct comparisons.
Fig 3.
Superposed 7 native monoamine transporters (MATs) and their QTY variants that were predicted by AlphaFold2.
Native MAT structures are colored in green, and their corresponding water-soluble QTY variants in cyan. RMSD values, expressed in Å (in parentheses), are provided for the following comparisons: a NET vs NETQTY (0.701Å), b DAT vs DATQTY (1.120Å), c SERT vs SERTQTY (0.492Å), d VMAT1 vs VMAT1QTY (1.084Å), e VMAT2 vs VMAT2QTY (1.515Å), f VAChT vs VAChTQTY (0.603Å), g VPAT vs VPATQTY (0.696Å). For clarity, N- and C- termini and large loops are deleted. Detailed information can be found in Table 1.
Fig 4.
Hydrophobic surface of the cryo-EM structure and the designed QTY variant of the human serotonin transporter (SERT).
Following the substitution of hydrophobic residues L, I, V, and F with Q, T, and Y, the surfaces showed increased hydrophilicity. The originally hydrophobic surface (brownish) of the native transporter transitioned to a predominantly cyan color, indicating a significant reduction in the hydrophobic surface area along the transmembrane helices for the QTY variant: a SERTCryoEM vs b SERTQTY. For clarity, N- and C- termini and large loops are deleted.
Fig 5.
Hydrophobic surface alterations of seven AlphaFold2-predicted native monoamine transporters and their designed QTY variants.
Following the substitution of hydrophobic residues L, I, V, and F with Q, T, and Y, the surfaces showed increased hydrophilicity. The originally hydrophobic surface (brownish) of the native transporters transitioned to a predominantly cyan color, indicating a significant reduction in the hydrophobic surface area along the transmembrane helices for the QTY variants: a NET vs NETQTY, b DAT vs DATQTY, c SERTQTY, d VMAT1 vs VMAT1QTY, e VMAT2 vs VMAT2QTY, f VAChT vs VAChTQTY, g VPAT vs VPATQTY. For clarity, N- and C- termini and large loops are deleted.
Fig 6.
Natural mutations of QTY-code and rQTY-code.
Native structures are represented in green, while the predicted effects of QTY and reverse QTY mutations are illustrated with colored residues: blue = benign, orange = possibly damaging with low confidence, red = damaging with high confidence. a NET, b DAT, c SERT, d VMAT1, e VMAT2, f VAChT, and g VPAT. For clarity, N- and C termini and large loops are deleted.
Table 2.
Natural mutations of L->Q, I->T, F->Y in the 7 monoamine transporters (No V->T mutations.
A single base mutation on the second position of the codons).
Table 3.
Natural mutations of Q->L, T->I, Y->F in the 7 monoamine transporters (No T->V mutations.
A single base mutation on the second position of the codons).