Chapter 3

Results and Discussion

35

Part I

Homology Modeling of GLUT4

36

3.1 Homology Modeling of GLUT4 3.1.1 Template Identification

Identification of the best template for modeling is the initial and important step in the

homology modeling approach. For this, we employed a homology search in the PDB

database using BLAST and PSI-BLAST to identify potential templates. Significant templates

were not obtained by this approach and the next level search was done with fold recognition

servers that use the sequence-structure comparison to detect the distant homologs. The fold

recognition servers identified three MFS family members namely GlpT [120], LacY [121],

and EmrD [122] from E. coli as potential templates for the GLUT4 modeling. Despite having

very low sequence similarity, the fold recognition servers identified the same fold within

GLUT4 and the three proteins belong to MFS family. The three templates had the same

percentage of sequence similarity (35-38%) with GLUT4. However, we selected GlpT as a

template for GLUT4 modeling because it has a eukaryotic homolog and has a hydrophilic

cavity suitable for the transport of a polar molecule. GlpT crystal structure has been widely

used for modeling members of MFS family including human GLUT1 [123], glucose 6-

phosphate transporter [233], human organic anion transporter [234], rabbit organic cation

transporter-2 (OCT2) [235], yeast inorganic phosphate:proton symporter (Pho84) [236] and

human vesicular glutamate transporter [237].

The initial sequence alignment for structure prediction was taken from the fold

recognition program. The structure based sequence alignment from the fold recognition

server provides a better way of identifying structural features from the template and in this

way secondary structure details are copied from the template. GLUT4 has shown only 18.3%

sequence identity with GlpT, but the secondary structure predictions are in good agreement

with that of the template. The alignment was further modified by including information from

the secondary structure prediction, transmembrane prediction, disordered region prediction

and multiple sequence alignment with the other Class I GLUTs. We have included the

biochemical information available from GLUT1 in order to correct the alignment. The initial

alignment was manually corrected iteratively to satisfy the experimental evidence. A multiple

sequence alignment of GLUT1, GLUT4, and the template is shown in Fig. 3.1. Mueckler et

al. have already proposed a helical arrangement of the transmembrane segments around the

37

glucose transport channel for GLUT1 [88]. Using the information from GLUT1, a helical

arrangement of the transmembrane domains for GLUT4 is constructed and is shown in

Fig.3.2.

Figure 3.1. Multiple sequence alignment of GLUT1, GLUT4 and GlpT from E. coli. The transmembrane helices of GLUT1 and GlpT are represented in box. The residues identified to be important for glucose transport from GLUT1 experimental studies are highlighted in grey background. Consensus secondary structure prediction for GLUT4 is shown (H, helix and C, coil) below the alignment. The GLUT4 disordered regions are marked as bold italic

38

characters. The predicted ATP binding domains in GLUT1 are marked as ‘-‘ symbol and glycosylation site as ‘G’ above the alignment. Other important sites are marked, FQQI motif in ‘&’ symbol, QLS site in ‘*’ symbol and GRR motif in ‘^’ symbol above the alignment.

Figure 3.2. Helical wheel arrangement for the 12 transmembrane segments of GLUT4 based on biochemical studies of GLUT1. 3.1.2 GLUT4 Model Building

The 3D model of GLUT4 was generated using the HM program Modeller9v2 based

39

on the corrected sequence alignment between GLUT4 and GlpT. The predicted homology

model is shown in Fig. 3.3 a. The proposed model has two half helical bundle domains, D1

(helices1-6) and D2 (helices 6-12) which bear a pseudo two fold symmetry between each

other and is represented in two color scheme.

Figure 3.3. (a) Cartoon representation of the GLUT4 model generated. The two half helix bundle domains D1 and D2 colored in grey and green color respectively. The N and C terminal end regions and the long cytoplasmic loop regions are colored in red. (b) 12 transmembrane helices and residues involved in glucose transport are emphasized. Residues interacting with glucose are represented in green stick format. Residues playing a role in glucose transport are represented in orange stick format. 3.1.3 Model Validation

We selected the best model by analyzing the stereochemical quality check using

PROCHECK and overall quality by ERRAT server. The Ramachandran plot analysis of the

GLUT4 model using PROCHECK showed 86.6% residues in the allowed region and 0.7%

residues in the disallowed region. The residues found in the disallowed region belong to loop

regions, N terminus, and C terminus of GLUT4 and did not show a significant role in

GLUT4 transport activity, which is well in agreement with the data available from

40

biochemical studies. ERRAT has also given a good overall quality assessment score and can

be seen in table 3.1.

Table 3.1. Quality assessment report using PROCHECK and ERRAT for the GLUT4 model, GLUT1 and GlpT (Glycerol-3-phosphate transporter). Score represented in bracket corresponding to PROCHECK validation report of the residues in the loop region.

Structure Residues in the Ramchandran plot allowed region (%)

Residues in the Ramachandran plot disallowed region (%)

Errat Score

GLUT4 model 86.6 (79.6) 0.7(1.6) 82.653

1SUK(GLUT1) 86.7 0.0 76.546

1PW4 85.4 0.0 85.176

3.1.4 Structural Features of the Model

The model of GLUT4 has an inward facing orientation and is agreeing with the

conformation available for the crystal structure of GlpT, the template used for modeling. The

N and C half bundles of helices are oriented around the glucose permeation pore. Since

limited biochemical studies are available on GLUT4, we have resorted to the experimental

data available on GLUT1 to validate our model. It is interesting to note that many residues

that are playing a key role in glucose transport mechanism are conserved in both GLUT1 and

GLUT4. These residues can affect the glucose transport process in different ways. They can

be involved in providing conformational flexibility to the protein or can have a role in helix

packing and lipid binding. The helical arrangement of the transmembrane domains has shown

two fold symmetry between the N- and C-terminal halves (Fig. 3.3 b), a major characteristic

of the MFS family. The transmembrane domains, 1, 2, 4, 5, 7, 8, 10, 11 line the central

transport cavity and 3, 6, 9, and 12 form the outer helices. The orientation of the helices in

our model is in accordance with the one proposed by Mueckler et al. [88, 89]. Experimental

studies with GLUT1 have identified functional and positional role for certain amino acid

residues and is shown in table 3.2. These residues were found to be conserved in GLUT4 as

well and include Q177, Q298, V181, Y309, T326, N333, T334, and W428. They line the

transport cavity and interact with glucose and these residues are represented in green color

41

stick form as shown in Fig. 3.3 b. The transmembrane helix arrangement at the extracellular

and cytoplasmic end is shown in Fig. 3.4. At the extracellular end the channel is in a closed

state which can be observed by the closed interface of helices 1 and 7 (Fig. 3.4 a) whereas at

the cytoplasmic end, the channel is open (Fig. 3.4 b).

42

Table 3.2. Residues involved in glucose transport (based on the experimental studies using GLUT1)

Mutant in GLUT1

Corresponding Residue in GLUT4

Position in structure

Role Reference

Mutant in GLUT1

L21,G22,S23, Q25,G27

Corresponding Residue in GLUT4

L33,G34,S35, Q37,G39

TM1 Glucose transport. [83]

Position in structure

Role Reference

F72,G79,S80 F88,S95,S96 TM2 Glucose transport.

[84, 238]

L21,G22,S23, Q25,G27

L33,G34,S35, Q37,G39

TM1 Glucose transport. [83]

Glucose transport.

[84, 238] F72,G79,S80 F88,S95,S96 TM2

[87] [87]M96 M96 M112 M112 TM3 TM3 Lies in direct apposition to an adjacent inner helix and may be involved in helical movements.

Lies in direct apposition to an adjacent inner helix and may be involved in helical movements.

Y143 E146

Y159 Y143 E162 E146 Y159 TM4 Role in the transport cycle.

Role in conformation of the transporter. [69, 85]TM4 Role in the transport cycle.

Role in conformation of the transporter. [69, 85]

E162 Q161 Q161 Q177 TM5 Critical for transport activity and forms

part of the exofacial substrate-binding site. [61] Q177 TM5 Critical for transport activity and forms

part of the exofacial substrate-binding site. [61]

V165 V165 V181 TM5 Lies near the exofacial substrate-binding site or directly in the sugar permeation pathway.

[66] V181 TM5 Lies near the exofacial substrate-binding site or directly in the sugar permeation pathway.

[66]

Q282 Q282 Q298 TM7 Part of the outside ligand-binding site. [59] Q298 TM7 Part of the outside ligand-binding site. [59]

Q279,Q283 Q295,Q299 TM7 Glucose Transport. [84] Y292 Q279,Q283

Y308 TM7 Irreplaceable. [238] Q295,Q299 TM7 Glucose Transport. [84]

Y293 Y292

Y309 TM7 Forms part of the hydrophobic

Patch involved in substrate interaction. [67]

Y308 TM7 Irreplaceable. [238] Y293

G286,N288 G302,N304 TM7 Glucose transport. [84]

Y309 TM7 Forms part of the hydrophobic Patch involved in substrate interaction.

[67]

T310,N317, T318

T326,N333, T334

TM8 Predicted to lie within the aqueous translocation pathway.

G286,N288 G302,N304 [64]

TM7 Glucose transport. [84] [64]T310,N317,

T318 T326,N333, T334

TM8 Predicted to lie within the aqueous translocation pathway.

P385

P401 TM10 Role in transport. Give conformational flexibility to the molecule.

[86]

W412 W428 TM11 Located in or close to the inner glucose binding site. Role in transport.

[62]

N411,N415,F422

N427,N431,F438

TM11

P385

P401 TM10 Role in transport. Give conformational flexibility to the molecule.

[86]

W412 W428 TM11 Located in or close to the inner glucose binding site. Role in transport. Role in glucose transport activity. [65]

S294, T295 S310,T311 Loop 7-8 Role in conformational change of the transporter during transport.

[62]

N411,N415,F422

N427,N431,F438

TM11 Role in glucose transport activity.

[238]

[65]

S294, T295 S310,T311 Loop 7-8 Role in conformational change of the transporter during transport.

[238]

R92 R153 E329 R333,R334 E393,R400

R108 R169 E345 R349,R350

R92 R153 E329 R333,R334

E409,R416

Loop 2-3 Loop4-5 Loop 8-9 Loop 8-9 Loop10-11

Role in substrate Induced conformational change. Role in conformation of transporter. Required for helical rearrangement. Role in substrate Induced conformational change Required for helical rearrangement.

[70]

E393,R400

R108 R169 E345 R349,R350 E409,R416

Loop 2-3 Loop4-5

Role in substrate Induced conformational change.

[70]

Role in conformation of transporter. Loop 8-9 Loop 8-9

Required for helical rearrangement. Role in substrate Induced conformational change

Loop10-11 Required for helical rearrangement.

43

(a) (b)

Figure 3.4. Transmembrane helix arrangement at the (a) extracellular and (b) cytoplasmic end of the GLUT4. Transmembrane segments are shown in cylindrical helix representation. Loop regions are avoided for clarity. 3.1.5 Comparative Analysis of GLUT1 and GLUT4 Model

GLUT1 has the highest sequence similarity with GLUT4 (63.3%) compared to any

other GLUT family proteins. Experimental studies with GLUT1 have identified certain key

residues important for glucose transport and these residues were found to be conserved in

GLUT4 also (Fig. 3.1). Superimposed structure of GLUT4 with the previously generated

model of GLUT1 [123] is shown in Fig. 3.5 a, and these two structures showed 6.224 Å

RMSD. The N-terminus, C-terminus, and the loop regions are highly variable and explain the

differences in the regulation, substrate specificity, and transport kinetics between these

transporters. GLUT1 and GLUT4 have the same substrate transport mechanism but they

differ in their substrate affinities. It has been shown that GLUT4 has a lower Km compared to

GLUT1 [22]. These transporters also show differences in their tissue distribution and

regulation. GLUT1 is ubiquitously expressed and predominantly present in the plasma

membrane, where as GLUT4 shows tissue specific expression and is seen in specific

intracellular compartment and gets translocated to the plasma membrane in an insulin

dependent manner. It is the GLUT4 molecule that plays a major role in maintaining the

normal blood glucose level.

44

Figure 3.5. (a) Superimposed structure of GLUT1 and GLUT4. GLUT1 in green color and GLUT4 in red color. (b) Cavities identified by the CASTp cavity prediction program. (c) Electrostatic potential map of the GLUT4 model. Blue indicates the positively charged region whereas red indicates the negatively charged segment. 3.1.6 Electrostatic Potential and Cavity Analysis

The electrostatic potential map generated using APBS was used to analyze the

electrostatic property of GLUT4 and is shown in Fig. 3.5 c. In the electrostatic potential map,

the central portion of the protein

n acidic cluster motif in the C-

termina

a contiguous hydrophobic patch was seen covering

orresponding to the transmembrane segment. GLUT4 has ac

l region and this was found to be negatively charged in our electrostatic potential

map. Cavity analysis was done in order to identify the potential structural pockets and

cavities in GLUT4. We have used CASTp and Pocket Finder for cavity analysis and the

result is shown in Fig. 3.5 b. We have compared the cavities predicted by these programs and

the potential cavities were considered for further analysis. The cavities identified and their

volumes are presented in Table 3.3. GLUT1 cavity measurement based on the GLUT1 model

45

(17) is also shown in parallel for comparison. Cavity1 forms the central glucose translocation

channel and is lined by residues important for glucose transport. Cavity1 (Fig. 3.5b) in the

model is formed when the transporter is oriented towards the cytoplasm. The transporter in

this mode may be in the substrate releasing state. In GLUT1 model, there is a cavity

comprising ATP binding domain and the electrostatic potential data analysis showed a

positive charge distribution suggesting that this region might bind ATP. A similar cavity

(Cavity 2) is identified in GLUT4 and the electrostatic potential of this cavity does not favor

ATP binding. Liu et al. have studied the role of three predicted ATP binding domains in

GLUT1 (Table 3.4). Domain I is homologous to the Walker binding motif A. domain II & III

are homologous to the Walker binding motif B [72]. It has been shown that ATP can bind

GLUT1 and induce significant conformational change, which in turn plays a key role in the

regulation of glucose transport. From the experimental data, the domain III is considered as

the bonafide ATP binding domain in GLUT1 and this domain is fully conserved in GLUT4

also. From the electrostatic potential analysis of GLUT4 model, we identified a positively

charged segment corresponding to domain III. Since the domain III showed 100% identity at

amino acid level with GLUT1 and this region showed positive charge distribution in our

electrostatic potential map analysis, it is highly likely that domain III could be the ATP

binding domain in GLUT4. Cavity 3, identified in the model was found to be adjacent to ATP

binding domain III. Cavity 4 and 5 open to the external face and can be considered as a

continuation of the central channel. The volume measures of these cavities could vary upon

conformational changes in the molecule.

Table 3.3. Cavities identified in GLUT4 and GLUT1 using CASTp and Pocket Finder and corresponding volume measures are in Å units.

Cavity Volume Measures

Cavity (GLUT4) (GLUT4) (GLUT1)

CASTp Pocket Finder CASTp

Cavity1 7226.67 3433 3334

Cavity2 3 338.01

265.69

-

247

344.5

224.89 Cavity3

46

Cavity4 842.27 - 453.61

0 Cavity5 1057.9 77 569.83

Table 3.4. A ng do ntified in G T1 and correspon ions in GLUT4.

ATP b

domain

Position in

GLUT1

Sequence in

GLUT1

Position in

GLUT4

equence in

GLUT4

TP bindi mains ide LU ding reg

inding S

Domain1 111-118 GFSKLGKS 128-134 GLANAAAS

Domain II

Domain III

225-229

332-338

KSVLK

GRRTLHL

242-245

348-354

KSLK

GRRTLHL

3.1 g stud

Various biochemical studies with GLUT1 have identified the amino acid residues

in h glu alysis lining using o model

evealed that most of these residues are highly conserved in GLUT4 as well. Studies have

cytochalasin B (CytB) and genistein interact with both GLUT1 and

LUT

3.1.7.1

ransport. Docking data with glucose have shown a positive interaction

ith the residues Q177, Q298 in the QLS site and N333 in the transport channel (Fig. 3.6 a,

When glucose enters the glucose permeation pore, it forms hydrogen bond

.7 Dockin ies

teracting wit cose. An of channel residues ur GLUT4

r

shown that molecules like

G 4, however the residues involved in these interaction were not identified. ATP is a

known regulator of the glucose transport function of GLUT1 and GLUT4 though a direct

binding and residues involved in ATP binding are not shown in the case of GLUT4. To

further validate our GLUT4 model, we have carried out docking studies with these molecules

and the analysis revealed their direct interaction with GLUT4 and each of these interactions

is explained below.

Glucose

Studies have shown that the highly conserved residues lining the central channel are

important for glucose t

w

3.6 b and 3.6 c).

47

with the residue in the exofacial binding site and subsequent interactions with other residues

in the transport pore facilitates the migration of glucose. The QLS site in TM7, which is a

conserved motif at the exofacial substrate binding site acts as a selectivity filter for the

passage of glucose [60]. The QLS site is conserved in GLUT1, GLUT3 and GLUT4. An

additional QLA motif present in TM5 has shown to be important for glucose transport and is

conserved in GLUT4 and GLUT2 (QLG in GLUT1 and GLUT3) [61]. These two motifs are

positioned at the center of the channel. We have identified both hydrophilic and hydrophobic

residues lining the glucose transport channel and this pattern of residues facilitates the

transport of glucose, a molecule which is hydrophilic due to its -OH groups and hydrophobic

owing to the pyranose ring.

Figure 3.6. Binding of glucose to GLUT4 (a) Glucose interacts with N333 (b) Q298 at the QLS site and (c) Q177. 3.1.7.2 ATP

Another important motif identified in this molecule is the GRR motif, found between

op 8-9 is part of an ATP binding site in GLUT1 (ATP binding domain III). This motif was

nserved in GLUT1, GLUT3 and GLUT4. Compared to GLUT1 which has three

ATP bin

loop 2-3 and loop 8-9. Based on the experimental studies, it was found that the GRR motif in

lo

found to be co

ding sites, GLUT4 has only one potential ATP binding site and is found to be highly

conserved. The electrostatic potential analysis has identified a positively charged segment in

GLUT4 which corresponds to this potential ATP binding site and our docking data with ATP

also favored a positive interaction at this site. These data suggest that the GRRTLHL motif

(domain III) in GLUT4 can function as a potential ATP binding domain. We could obtain two

docking poses for ATP (ATPA and ATPB) at this ATP binding site and are shown in Fig. 3.7 a

48

and 3.7 b. The residues R350, T351, H353, V468, E409, T474, R475 and R416 interacts

with ATP in the first binding pose (Fig. 3.7 a) and the residues, R169, R350, E409, T471

and R474 interact with the second binding pose of ATP (Fig. 3.7 b). The two binding poses of

ATP show a reverse orientation with each other. Molecular dynamic studies with these two

binding poses of ATP suggested the ATPA as bonafide mode of binding.

(a)

(b)

49

Figure 3.7. Docking poses for ATP at the ATP binding domain III of GLUT4 (a) ATPA (b) TPB.

.1.7.3 Cytochalasin B (CytB)

CytB is a well known inhibitor of glucose transport and it inhibits the glucose uptake

negative control in glucose uptake experiments. Docking studies have revealed that CytB

ments 8, 9, 10 and 11 and interact with residues R169, E409,

R472 a

igure 3.8. CytochalasinB docking position at the ATP binding site.

3.1.7.4 Genistein

A 3

by directly interacting with the GLUT4 at the cytoplasmic side [239, 240]. It is widely used as

a

binds at a region close to TM seg

nd R474. It is interesting to note here that Inuki et al. using biochemical approaches

have also suggested a similar region as CytB binding site in GLUT1 [63]. Further analysis has

shown that CytB binds to a position near the ATP binding domain III (Fig. 3.8). This

observation is agreeing very well with the findings from biochemical studies showing an ATP

dependent decrease in the affinities of CytB [241].

F

50

Genistein is an isoflavone derivative and is known to inhibit glucose transport. It act

pocytes with an IC50 of

0µM. Genistein also inhibits the binding of CytB to GLUT1 [242] as well as the protein-

ity [241]. Experimental studies suggest that genistein interacts with ATP

inding

e 3.9. Interactions of Genistein in the ATP binding site of GLUT4.

Our model also suggests that CytB and genistein bind to the same ATP binding site

cated at the cytoplasmic part of GLUT4 and is consistent with the biochemical data

e presence of ATP

[241]. A P induces conformational changes in GLUT1 which enhances the substrate affinity

while r

[243]. The presence of a similar motif supports the possibility of ATP playing a role in the

as a direct inhibitor of insulin-induced glucose uptake in 3T3-L1 adi

2

tyrosine kinase activ

b domain III of GLUT1 [79, 241] and our docking data verified this observation and is

shown in Fig. 3.9. Genistein interacts with residues R169, H353, L352, R467 and R350 via

hydrogen bonds. The two -OH groups in the A ring of genistein contribute to the interactions

with the ATP binding site residues. The B ring -OH group is also anchored through H-bonding

with R169 residue.

Figur

lo

suggesting a decreased affinity fo Genistein and CytB for GLTU4 in th

T

educing its net transport capacity [73]. Alanine scanning mutagenesis of loop 8-9

residues (E329, R332, and R333) have shown to produce ATP insensitive GLUT1 transporters

51

regulation of GLUT4 biological function. Though the functional form of GLUT4 on the

plasma membrane has not been elucidated, GLUT1 is found to occur as a tetramer which

binds ATP [74]. No studies have been reported so far demonstrating the direct ATP binding

and its role in the GLUT4 mediated glucose transport. However, ATP has been shown to have

a negative effect on translocation of GLUT4 by mediating its intracellular sequestration [244].

A cytosolic protein GTBP70 has been shown to bind GLUT4 and plays a role in its

translocation. Presence of high amount of intracellular ATP is found to displace GTBP70 from

GLUT4 and thereby reducing its translocation [81]. Our electrostatic potential map analysis

has revealed a positively charged region at ATP binding domain and docking studies with ATP

have shown the binding of ATP at this region. It is possible that at high intracellular

concentration, ATP may directly bind to GLUT4 and affect its translocation and intrinsic

transport activity. Based on the model we are currently looking at the regulatory role of ATP

on the biological activity of GLUT4.

3.1.7.5 Kaempferitrin

Kaempferitrin (kaempferol 3,7-dirhamnoside) is a glycosylated flavonoid obtained

from the leaf extract of Bauhinia acuminata. Biochemcial data from our laboratory have

suggested that kaempferitrin inhibits insulin stimulated GLUT4 translocation and glucose

ptake. During the glucose uptake study, when kaempferitrin was added along with glucose,

mpetitive inhibition in glucose uptake [245]. This observation has

sugges

u

our results showed a co

ted a possibility that kaempferitrin may directily interacts with GLUT4. To test this

hypothesis, a docking study was carried out with kaempferitrin and GLUT4 model. We

obtained different docking poses of kaempferitrin in the glucose transport pore of GLUT4

with favorable energies. Further analysis revealed that kaempferitrin forms hydrogen bonds

with amino acid residues such as G43, N176, Q177, Q298, S301, N333, and N431 in the

glucose transport channel. Among these residues, Q177, Q298, N333 were reported to be

important for glucose transport [60, 61, 64]. Fig. 3.10 a and 3.10 b shows a favorable mode of

interaction with GLUT4 and the important residues involve in the interaction with

kaempferitrin. These results suggest that kaempferitrin directly interacts with QLS site, a

conserved motif in high affinity glucose transporters, which is critical for the binding and

transport of D-glucose through GLUT4 [60].

52

Figure 3.10. Interaction of kaempferitrin with GLUT4. (a) A view of kaempferitrin docked in the glucose transport channel. (b) Enlarged image showing amino acid residues interacting with kaempferitrin docked in the glucose transportation pore. 3.1.8 Conclusions

The 3D structure of the GLUT4, a class I facilitated glucose transporter, was

generated based on the homology with the GlpT and from the biochemical data available

from a closely related glucose transporter, GLUT1. We have validated this model employing

various strategies including docking studies with the known GLUT4 substrate and inhibitors.

he exact structural details from the homology models because of the It is challenging to get t

lack of availability of high resolution templates, specifically in the case of membrane

proteins. However, the proposed GLUT4 model can give insight into the structural features

until crystal structure of any GLUT protein becomes available. The GLUT4 model generated

could serve as a platform for screening various ligands that could modulate glucose transport.

53