Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure (Review)

Figures & data

Figure 1.  Ribbon representation of the crystal structure for MFS transporters. (A) E. coli GlpT. (B) E. coli LacY. (C) O. formigens OxlT. (D) E. coli EmrD.

Table I.  Summary of the different programs used to enforce constraints, modeling and refinement for eukaryotic MFS models based on crystal structures of prokaryotic GlpT or LacY.

Homology model	Constraints	Modeling program	Refinement
Human Glut1	Alignment, experimentally derived loops and glycosylation sites	Nest	MODELLER
Human G6PT	Predicted topology (7 programs, see text), alignment (T-COFFEE), experimentally derived N- and C- termini	MODELLER	MODELLER
Rat OCT1	Alignment (ClustalW) and topology prediction (TMHMM)	Quanta 2000	CHARMM
Rabbit OCT2	Homologous sequence search (PSI-BLAST) and sequence-based structural models (3D-PSSM) alignment (ClustalW)	ICM 3.0	SYBYL 6.9
S. cerevisiae Pho84	Sequence-based and structurally-based alignments (MetaServer)	MODELLER	MODELLER

Figure 2.  Ribbon representation for models of eukaryotic MFS transporters. (A) Human Glut1. E. coli GlpT. (B) human G6PT. (C) Rat OCT1. (D) Rabbit OCT2. (E) S. cerevisiae Pho84. For a comparison with the structure of GlpT, see Figure 1A.

Table II.  Reported sequence identities and sequence similarities of modeled eukaryotic MFS transporter proteins with GlpT sequence *Modeled using LacY. ^#Not reported in publication. Sequence identity and similarity calculations were carried out using EMBOSS Pairwise Alignment Algorithms with an open gap penalty 10, a gap extension penalty of 0.2 and BLOSUM62 matrix.

Homology model	% Sequence identity with GlpT	% Sequence similarity with GlpT	Sequence identity with LacY^#	Sequence similarity with LacY^#
Human Glut1	19.6^#	25.2^#	12.8	21.0
Human G6PT	27.0	54.0	16.8	21.6
Rat Oct1*	12.4	28.8	17.4	26.5
Rabbit Oct2	13.8	41.5	13.9	12.0
S. cerevisiae Pho84	15.6^#	24.5^#	15.5	24.5
E. coli GlpT*	–	–	14.5	23.8

Figure 3.  (A) Wall-eyed stereo image ribbon representation of the structural alignment between the GlpT crystal structure (green) and GlpT model (magenta) based on LacY crystal structure. (B) Wall-eyed stereo image of GlpT active site showing Arg 45 on transmembrane helix 1 and Arg269 on transmembrane helix 7 are in different locations on the GlpT crystal structure (green) and GlpT model (magenta) based on LacY crystal structure. Helices 2, 4 and 11 have been removed in order to visualize the Arg residues in the central cavity.

Table III.  R.m.s.d. from structural-based Cα alignment using the program ALIGN (Cohen 1997). Numbers in parenthesis represent the Cα pairs in the alignment.*Modeled with LacY. ^$Alignment with all mainchain atoms with atom name match required.

Homology model	Cα R.m.s.d. with GlpT crystal structure (Å)	Cα R.m.s.d with LacY crystal structure (Å)
LacY (Crystal)	3.0 Å (342)	–
Human Glut1	1.47 Å (397)	2.7 Å (347)
Human G6PT	1.00 Å (392)	2.9 Å (343)
Rat Oct1*	2.91 (302)	0.40 (304)
Rabbit Oct2	1.16 Å (415)	2.9 Å (337)
S. cerevisiae Pho84	0.27 Å (371)	3.0 Å (342)
E. coli GlpT*	2.82 (335) ^$2.75 (1347)	0.58 (373)

Table IV.  WHATCHECK assessment of X-ray and modeled transporter structures. Structure Z-scores, positive is better than average while negative is below average. *Modeled with LacY.

	WHATCHECK Structure Z-scores
X-ray structures	2nd generation packing quality	Ramachandran plot appearance	Chi-1/chi-2 rotamer normality	Backbone conformation
E. coli GlpT	0.7	−2.2	−2.2	−7.6
E. coli LacY	0.3	−4.6	−1.4	−5.9
E. coli EmrD	−3.075	−5.684	−5.323	−17.021
Modeled structures
Human Glut1	−2.016	−0.799	−2.736	−9.461
Human G6PT	−2.081	2.672	−2.459	−7.084
Rat Oct1*	−2.511	−6.094	−3.879	−9.398
Rabbit Oct2	−2.873	−5.115	−0.545	−13.842
S. cerevisiae Pho84	−4.124	−4.475	−2.516	−12.465
E. coli GlpT model*	−2.968	0.916	−2.200	−5.295

Cohen GH. ALIGN: a program to superimpose protein coordinates, accounting for insertions and deletions. J Appl Cryst 1997; 30: 1160–1161

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