. 2007 Oct 17;8:397. doi: 10.1186/1471-2105-8-397

Table 2.

Structural assessment of the functional site predictions.¹

LeuT_Aa	rGAT1	rSERT	hDAT	Structural location	Description	Experimental evidence	Predicted by²
(a.) Substrate/Na⁺-binding sites

G20	G59⁺	G94⁺	G75	TM1a	Na⁺binding site	[2–95]	P,S,R,E
N21	Y60⁺	Y95⁺	F76⁺	TM1a	Leu binding site	[94–99]	None
A22	A61	A96	A77	TM1a	Leu & Na⁺binding sites	None	F,R
V23	I62⁺	V97	V78	Unwound TM1	Na⁺binding site	[95]	F
G24	G63⁺	D98⁺	D79⁺	Unwound TM1	Leu binding site	[95, 97, 100, 101]	F,R,E,D
L25	L64⁺	L99⁺	L80⁺	TM1b	Leu binding site	[92, 95, 102]	P,F,S,R,E
G26	G65⁺	G100⁺	A81	TM1b	Leu binding site	[92, 95]	P,F,S,R
N27	N66⁺	N101⁺	N82	TM1b	Na⁺binding site	[92, 95]	P,F,S,R,E
E62	E101⁺	E136⁺	E117⁺	TM2	Stabilizes TM6 unwound region	[55–58]	P,F,S,R,E
V104	L136^-	I173⁺	V152⁺	TM3	Leu binding site	[93, 96, 103–105]	None
Y108	Y140⁺	Y176⁺	Y156⁺	TM3	Leu binding site	[106–108]	F,S,R
F252	F293	F334	C319⁺	TM6a	Leu binding site	[109]	P,F,S,R
F253	F294⁺	F335⁺	F320⁺	TM6a	Leu binding site	[93, 98, 103, 110]	P,F,S
T254	S295⁺	S336	S321⁺	TM6a	Leu & Na⁺binding sites	[108, 110, 111]	P,F,E
S256	G297	G338	G323⁺	Unwound TM6	Leu binding site	[112]	P,F,E
L257	L298⁺	P339⁺	V324	Unwound TM6	Leu binding site	[110, 113]	P
G258	G299	G340	G325⁺	Unwound TM6	Leu binding site	[98]	P
F259	L300	F341⁺	F326^-	Unwound TM6	Leu binding site	[93, 98, 103]	P
G260	G301	G342	G327⁺	Unwound TM6	Leu binding site	[112]	P,S,E
N286	N327⁺	N368	N353	TM7	Na⁺binding site	[111, 114]	P,F,R
A351	L392	L434	L418	TM8	Na⁺binding site	None	F,R,E,D
T354	D395⁺	D437	D42⁺	TM8	Na⁺binding site	[111, 115]	P,F,R,E,D
S355	S396⁺	S438⁺	S422	TM8	Leu & Na⁺binding sites	[93, 103, 111]	P,F,S,D
I359	T400	G442⁺	G426	TM8	Leu binding site	[93, 103]	None

(b.) Cytoplasmic gate

R5	R44⁺	R70	R60⁺		Cytoplasmic gate	[116]	S
W8	W47⁺	W82	W63⁺		Cytoplasmic gate	[58, 116]	S
S267	S308	S349	S334		Cytoplasmic gate	None	S,R,E
Y268	Y309^-	Y350	Y335⁺		Cytoplasmic gate	[57, 106, 115]	S,E
D369	D410	D452	D436⁺		Cytoplasmic gate	[58, 115]	S,E

(b.) Extracellular/periplasmic gate

R30	R69⁺	R104⁺	R85⁺	TM1b	Extracellular gate	[57, 92, 95, 97, 117]	P,F,S,R,E
Y47	Y86⁺	Y121	Y102⁺	TM2	Extracellular gate	[106, 108]	P,F,S,R,E
Q250	Q291⁺	Q332	Q317⁺	TM6a	Extracellular gate	[57, 108, 110]	P,F,S,R,E
E290	S331⁺	S372	L355	TM7	Extracellular gate	[111, 114]	P
D404	D451	E493⁺	D476⁺	TM10	Extracellular gate	[58, 104]	P,S

¹The importance of the functional site benchmark has been clearly established by Yamashita et al. from the LeuT_Aastructure [10]. Residue numbers correspond to those of LeuT_Aa, rGAT1, rSERT, and hDAT (columns 1–4, respectively). Residues shown in bold with + are those whose functional role is supported by experimental data. Residues shown in bold with - are those whose functional role is not supported by experimental data. Our literature search failed to find mutagenesis evidence for residues without boldface. The criterion for positive experimental confirmation was a minimum of two-fold reduction in substrate uptake rate and/or affinity of the single site mutant compared to WT.²Six unique bioinformatic approaches were utilized in order to predict functional sites within the NSS family. The methods employed are: P = phylogenetic motif, F = false positive expectation, S = site conservation, R = Rate4Site, E = evolutionary trace, and D = SDPpred.