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Table 2 Structural assessment of the functional site predictions.1

From: Assessing the ability of sequence-based methods to provide functional insight within membrane integral proteins: a case study analyzing the neurotransmitter/Na+ symporter family

LeuTAa

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

  1. 1 The importance of the functional site benchmark has been clearly established by Yamashita et al. from the LeuTAa structure [10]. Residue numbers correspond to those of LeuTAa, 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.2 Six 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.