Mechanistic Study of Human Glucose Transport Mediated by GLUT1.

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Mechanistic Study of Human Glucose Transport Mediated by GLUT1 Xuegang Fu, Gang Zhang, Ran Liu, Jing Wei, Daisy Zhang-Negrerie, Xiaodong Jian, and Qingzhi Gao J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.5b00597 • Publication Date (Web): 28 Jan 2016 Downloaded from http://pubs.acs.org on February 1, 2016

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Journal of Chemical Information and Modeling

Mechanistic Study of Human Glucose Transport Mediated by GLUT1

1

2

Xuegang Fu1, Gang Zhang1, Ran Liu1, Jing Wei1*, Daisy Zhang-Negrerie3, Xiaodong Jian4, Qingzhi

3

Gao1,2*

4

1

5 6

Technology, Tianjin University, Tianjin, 300072, P. R. China 2

7

Tianjin University Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, 300072, P. R. China

8

3

9

4

10

Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of Pharmaceutical Science and

Concordia International School, 999 Mingyue Rd. Shanghai, 201206, P. R. China National Supercomputing Center in Tianjin, TEDA Service Outsourcing Industrial Park, Binhai New Area, Tianjin, 300457, P. R. China

11 12

ABSTRACT: The glucose transporter 1 (GLUT1) belongs to the major facilitator superfamily (MFS)

13

and is responsible for the constant uptake of glucose. However, the molecular mechanism of sugar

14

transport remains obscure. In this study, homology modeling and molecular dynamics (MD) simulations

15

in lipid bilayers were performed to investigate the combination of the alternate and multisite transport

16

mechanism of glucose with GLUT1 in atomic detail. To explore the substrate recognition mechanism,

17

the outward-open state human GLUT1 homology model was generated based on the template of xylose

18

transporter XylE (PDB ID: 4GBZ), which shares up to 29% sequence identity and 49% similarity with

19

GLUT1. Through the MD simulation study of glucose across lipid bilayer with both the outward-open

20

GLUT1 and the GLUT1 inward-open crystal structure, we investigated six different conformational

21

states and identified four key binding sites in both exofacial and endofacial loops that are essential for

22

glucose recognition and transport. The study further revealed that four flexible gates consisting of

23

W65/Y292/Y293-M420/TM10b-W388 might play important roles in the transport cycle. The study

ACS Paragon Plus Environment 1


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showed that some side chains close to the central ligand binding site underwent larger position changes.

2

These conformational interchanges formed gated networks within an S-shaped central channel that

3

permitted staged ligand diffusion across the transporter. This study provides new inroads for the

4

understanding of GLUT1 ligand recognition paradigm and configurational features which are important

5

for molecular, structural, and physiological research of the MFS members, especially for

6

GLUT1-targeted drug design and discovery.

7 8

KEYWORDS: GLUT1, outward-open state, transport mechanism, modeling, molecular dynamics

9 10

INTRODUCTION

11

The major facilitator superfamily (MFS) is a large gathering of membrane transporter proteins found

12

in bacteria and human bodies. MFS transports numerous substrates such as sugars, peptides, vitamins,

13

amino acids, enzyme cofactors, different organic and inorganic ions as well as drugs.1 Glucose

14

transporters (GLUTs) belong to MFS and are encoded by the 2A solute carrier family (SLC2A) genes.2

15

The human GLUT proteins are comprised of fourteen isoforms and are categorized into three classes:

16

Class 1 (GLUTs 1-4, 14), Class 2 (GLUTs 5, 7, 9 and 11), and Class 3 (GLUTs 6, 8, 10, 12 and 13).

17

GLUTs have varied distributions in different cell types and mediate the transport of several hexoses in

18

eukaryotic cells with the exception of GLUT13. Due to GLUTs’ main function as to keep constant

19

availability of glucose for metabolism through the movement of glucose between intracellular and

20

extracellular compartments, they are found to be involved in a broad spectrum of diseases including

21

cancer and diabetes.3

22

Warburg described a phenomenon in which a myriad of cancers exhibit increased glucose uptake and

23

aerobic glycolysis.4 Various studies have demonstrated that the inhibition of some specific subtypes of

24

GLUTs leads to reduction of cancer-cell proliferation and inducing apoptosis of cancer cells.5-9 GLUT1

25

is the first glucose transporter isoform having been purified, and it is the most extensively studied among

26

all MFS proteins. GLUT1 shares a topology structure consisting of 12 conserved transmembrane


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segments (TMs) with other MFS family proteins, which are separated into two discrete units with TMs

2

1-6 defined as the N-domain and TMs 7-12 called C-domain. The missense mutations of GLUT1 may

3

lead to an autosomal dominant genetic disease called GLUT1 deficiency syndrome (GLUT1DS).10 It has

4

also been demonstrated that GLUT1 is associated with the energy supply of the brain through

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blood-brain barrier11 and is an important prognostic indicator for tumor genesis.12-14 Similar to the

6

strategy of

7

diagnostic agent delivery has been recently achieved by engineering glycoconjugates to be transportable

8

substrates.15-19 Although considerable efforts have been put into unraveling molecular mechanisms

9

underlying the process of glucose transport, detailed conformational dynamics of the translocation

10

18

F-FDG-mediated PET cancer diagnosis, successful GLUT1-targeted tumor drug or

pathway as well as the spatio-temporal regulation of the substrate are still not well known.

11

Studies on nucleobase symporter and bacterial lactose transporter have demonstrated that the transport

12

mechanism of MFS may be represented by the alternating access model which combines the

13

“rocker-switch” movement20 and the “gated pore” mechanism.21 According to the alternating access

14

model, a transporter alternately exposes binding sites accessible from both sides of the membrane.22, 23

15

During this transition, a transporter must undergo several conformational shifts involving at least three

16

states: the outward-open, occluded, and inward-open states. At present, the existing MFS structural

17

conformations have provided direct evidence supporting the alternating access transport mechanism for

18

many integral membrane proteins.24-29

19

Based on cell biology and cytochalasin-B-inhibitor-binding study, Naftalin and Carruthers put

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forward one complementary proposal for sugar transportation, defined as “geminate exchange” model

21

where sugar can bind to coexistent exo- or endofacial binding sites and dissociate into a connecting

22

central cavity.23, 30 The most recent investigation from Naftalin’s group disclosed some more important

23

observations on xylose transport suggesting that a combination of the alternating access and fixed

24

multisite models may provide a means to adequately explain and understand the reality of MFS

25

mediated sugar transport.31, 32

26

About ten years ago some research groups reported a series of homology models and attempted to

27

deduce the glucose transport mechanism of GLUT1.33-35 However, due to the lack of the GLUT crystal



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structure back then these homology models were obtained only from those templates with available

2

crystal structures, albeit of low sequence identities (LacY and GlpT: 13% and 12.9% to GLUT1 based

3

on their primary sequences).

4

In 2012, Yan and coworkers reported the first outward-open crystal structure for xylose symporter

5

XylE (PDB ID: 4GBZ), which, as an Escherichia coli homology of GLUT1-4, shared up to 29%

6

sequence identity and about 49% similarity with GLUT1.29 In 2014, the great effort from Yan’s group

7

led to a second success in obtaining the first inward-open crystal structure of GLUT1 (PDB ID: 4PYP).36

8

Recently, Park focused his efforts on the transport of glucose by GLUT1 with the outward-facing

9

conformational model, in which he mainly investigated the migration behavior of glucose from the

10

central cavity to the deep inner position and the releasing process.6

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To obtain more precise insights into the molecular basis and reveal the whole transport process of

12

glucose from substrate recognition to the cytoplasmic delivery, both exofacial and endofacial models for

13

GLUT1 are needed and understanding of the fundamental aspects for glucose binding and transport are

14

important. In this article, to perform molecular dynamics of the glucose transport system, we generated

15

the outward-open state model of GLUT1 using crystal structure of XylE as an initial homology template

16

which was cocrystallized with D-glucose in an outward-facing conformation. For the inward-open state

17

model, we adopted the crystal structure reported by Yan’s group for study of endofacial glucose

18

transportation mechanism. In order to thoroughly explore the glucose transport mechanism in atomic

19

detail, each model was used to investigate how the glucose binding varied between different stages

20

which involved in both the outward- and inward-open transportation states. A molecular dynamics

21

simulation

22

1-palmitoyl-2-oleylhosphatidylcholine (POPC) lipid bilayer was performed to monitor the movement of

23

the ligand, the mobility of the tunnel helical transmembrane segments (TMs), and the fluctuations of the

24

distal side chains in the key binding site and central transmembrane zone.

of

up

to

60-ns

with

the

CHARMM27

force

ﬁeld

in

solvated

25

Through this study, we identified all the possible key binding sites in both exofacial and endofacial

26

surfaces that are essential for substrate recognition and translocation. The results demonstrated that some


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key subsets of the residues either formed interaction network to stabilize substrate-binding or arranged

2

as flexible gates for regulating substrate translocation. Furthermore, molecular flipping and rotation of

3

the glucose in an S-shaped GLUT1 transport trajectory were suspected to also play important roles in the

4

transport process.

5 6

METHODS

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To systematically investigate the transport process of glucose, we decided to establish six different

8

stages for GLUT1, each of which may represent a key regulatory node in the transport consequences: the

9

substrate-free (apo) outward-open state: Sout-apo; the partially occluded outward-open (exofacial) state:

10

Sout-ex; the occluded outward-open (central) state: Sout-cen; and correspondingly, the occluded

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inward-open (central) state: Sin-cen; the partially occluded inward-open (endofacial) state: Sin-endo; and the

12

substrate-free (apo) inward-open state: Sin-apo.

13

According to the aforementioned key states, the corresponding potential binding pockets were also

14

defined as follows: Pout-ex, represents the exofacial binding pocket; Pout-cen/Pin-cen, the binding pockets in

15

the middle of the transporter; and Pin-endo, the endofacial binding pocket. All of these abbreviations will

16

be used consistently in the Figures.

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Protein Sources. The outward-facing GLUT1 (Sout-apo, Sout-ex, Sout-cen) were constructed based on the

18

X-ray crystal structure of D-glucose-occluded XlyE (PDB ID: 4GBZ) as the newest available homology

19

template,29 which shares sequence identities of 29% and similarities of 49% with GLUT1. As a useful

20

online tool, ProBiS-CHARMMing37,38 also provided the same result suggesting XlyE as the best

21

template for GLUT1 homology modeling (see supporting information Figure S1). The alignments were

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done with ClustalW,39 100 homology models of the exofacial GLUT1 were generated by using

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Modeller9v740 with default parameters, and the best model was chosen based on the stereochemical

24

quality assessed by PROCHECK.41

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For the inward-open conformations (Sin-apo, Sin-endo, Sin-cen), we adopted the latest crystal structure of

26

the

inward-facing

human

GLUT1

(PDB

ID:

4PYP),

which

was

bound

27

n-nonyl-β-D-glucopyranoside36 (from RCSB Protein Data Bank at http://www.rcsb.org/pdb/).

with

28

Ligands Positioning. To perform MD simulation, six key models were generated from

29

abovementioned proteins as the initial position. Four models (Sout-ex, Sout-cen, Sin-endo, Sin-cen) were then



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ligated with glucose according to the following methods: 1. The generated best homology model for the

2

outward-open state of GLUT1 was adopted as Sout-apo. 2. To find the key substrate recognition binding

3

site in the outward-open GLUT1 state (since in the template XlyE, D-glucose was bound in the central

4

binding cavity), a pocket finding analysis based on the Sout-apo state model was carried out with Q-site

5

Finder42. The binding site detection result was further confirmed to be identical by utilizing another very

6

useful online too of ProBiS-CHARMMing.37,38 D-glucose was then docked into the binding site by using

7

AutoDcok 4.0 software43 to generate the initial partially-occluded GLUT1 state: Sout-ex. 3. The initial

8

binding position of D-glucose in the central cavity with Sout-cen state was obtained from superimposition

9

of the substrate-free outward-open GLUT1 homology model with the X-ray structure of XlyE (PDB ID:

10

4GBZ, with D-glucose bound in the central cavity), and then D-glucose was transposed to GLUT1 by

11

removing of the XlyE protein. 4. Sin-apo was obtained by simply removing the ligand from the crystal

12

structure of GLUT1/n-nonyl-β-D-glucopyranoside (β-NG) complex. 5. The Sin-cen state was prepared by

13

replacing the n-nonyl side chain of the β-NG molecule in the complex of 4PYP structure with a

14

hydrogen atom. 6. The initial Sin-endo model was obtained with the same method applied in preparing the

15

Sout-ex state using Q-site Finder and AutoDock 4.0 software except that the protein was changed to the

16

apo inward-facing conformation (Sin-apo).

17

Molecular Docking. Docking of the glucose into the binding sites of GLUT1 was performed using

18

the AutoDock4.0 program. Docking files were prepared using AutoDock Tools 1.5.6 software. Polar

19

hydrogen atoms were added, non-polar hydrogen atoms were merged and Gasteiger charges and

20

salvation parameters were assigned by default. Lamarckian genetic algorithm (LGA) was applied for all

21

docking calculations. A population size of 300 and 25,000,000 energy evaluations were used for 100

22

search runs. The grid dimensions were 46×46×46 points with grid spacing of 0.375Å centered on the

23

labels, which are the markers from Q-site Finder indicating the positions of the found pockets. The pose

24

with the lowest energy was selected for the further MD simulation.

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MD Simulations. To mimic the physiological environment for the proteins, the six different states

26

of the GLUT1 proteins were put in the center of a 1-palmitoyl-2-oleylhosphatidylcholine (POPC) lipid

27

bilayer which was comprised of about 209 POPC molecules. Subsequently, these systems of protein

28

membrane were solvated in a water cubic box (solvation layer of ~10 Å thickness with the periodic box

29

size ~120 Å x 120 Å x 100 Å) and were added 17 chloride ions to preserve the electric neutrality using

30

VMD v1.91.44

31

A 60-ns-long MD simulation was performed on the six solvated initial systems with the CHARMM27

32

force ﬁeld after 25000 steps of minimization using the NAMD 2.9 package.45 The long-range

33

electrostatic forces were controlled by the smooth particle-mesh Ewald (PME) method.46 The


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nonbonded cutoff was set to 12 Å. The bonds formed by hydrogen atoms were constrained by the

2

SHAKE algorithm.47 The systems were heated from 0 K to 310 K. Efficient pressure control was

3

achieved by applying the Langevin piston method, with the target pressure set to 1 atm. The

4

integration-time step was set to 2 fs. VMD v1.91 was use for trajectory analysis.

5

RESULTS AND DISCUSSION

6

Theoretical Model of Outward-open GLUT1. At present, GLUT1 has only yielded the

7

inward-open conformation (PDB ID: 4PYP). Therefore, the outward-open conformation of the

8

alternating access model has been obtained by automated comparative modeling. Traditional homology

9

modeling is considered to be the most accurate method based on the fundamental observation that if the

10

sequences of two proteins are related, their tertiary structures would persistently exhibit the same fold,

11

and this connection provides the logic base for predicting the 3D protein structures.48

12

The amino acid sequence alignment of GLUT1 with E. coli XylE protein29 showed that they share

13

massive conserved residues within the transmembrane segments (TMs) (Figure S2). According to the

14

abovementioned method, 100 homology models for the outward-open state of GLUT1 were successfully

15

generated and their stereo-chemical quality of the backbone conformations were assessed by the

16

Ramachandran’s plot assessment. For the best model, 94.5%, 4.5% and 1.0% of the residues were

17

respectively assigned as the “most favored”, “additionally allowed” and “generously allowed” regions.

18

Besides, no residue was found in the “disallowed” region (Figure S4). It indicated that the backbone

19

conformation and the non-bonded interactions of the obtained homology model structure fitted well

20

within the range of a model of high quality. This model was selected for further minimization and

21

molecular simulation studies.

22

Our outward-open structural model of GLUT1 had the notable difference with the previously

23

reported GLUT1 models.33, 34 The constructed model contained four intracellular α-helices in the inner

24

region of the protein (shown in Figure 1: IC1 ~ IC4), which were absent in previous models. This

25

difference resulted from the characteristic structures of the template proteins, in which the corresponding

26

segment of this cytoplasmic domain either showed a loop structure or was lost, such as the lactose

27

transport protein LacY (PDB ID: 1PV6)49 and the glycerol-3-phosphate transporter GlpT (PDB ID:

28

1PW4).28 Hence, the homology models from these templates were provided as the loop-like structure for

29

this intracellular helix domain (e.g. PDB ID: 1SUK and GLUT1 model in ref. 34) (see also Figure S5).

30

According to the inward-open GLUT1 crystal structure and the potential uniqueness of such intracellular

31

helical bundle (ICHB) in some other sugar transporters,36 our exofacial GLUT1 model is expected to be

32

more precise and useful for the studies of the transport mechanism. Comparisons of our results with the



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Ramachandran plot distribution for the generated model with the template XylE and the previous

2

reported model also supported this conclusion (Table S1).

3 4

As shown in Figure 1, in the overall 3D structure of the outward-open GLUT1 model, TMs 4, 5, 7 and 11 form a rather compact core domain which possesses distinct topology and function.

5 6

Figure 1. The homology model of the outward-open state of GLUT1. The general structure is shown on the left, the

7

magenta part is the N domain of GLUT1, the cyan part, the C domain, and the yellow part, the loop connecting the N

8

and C domains (intracellular helical bundles：ICHB). The top right is the extracellular view, where the transmembrane

9

segments are labeled with numbers; the bottom right is the cytoplasmic view, where the transmembrane segments and

10

intracellular helical bundles are labeled with numbers.

11

Molecular Dynamics Simulation of the Six Key Conformational States in the Solvated Lipid

12

Bilayers. After the initial systems containing all six key state models were set up as aforementioned in

13

the Methods Section (Figure 1), a series of energy minimizations and 60-ns simulations were carried out.

14

The root mean-square deviation (RMSD) fluctuations of the backbone atoms, in reference to the initial

15

structures, showed that the systems all reached equilibrium after 10 ns of simulation (Chart S1). To

16

determine the involvement of water molecules in each key conformational state during the whole

17

simulation process, a statistic analysis has been performed, and the trajectories of the last 5 ns period

18

were collected to detect and further analyze for specific internal water molecules which may play

19

important role in glucose transport (Chart S2). During the simulation process, all structures underwent

20

relatively small conformational changes. The clearest indicator of the structural stability was the

21

observation that the α-helicity of the two six-TM-helix-bundle were very well maintained in the core ACS Paragon Plus Environment 8

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1

domains for all models. As shown in Figure 2, a simple measure of the conformational stability of the

2

simulation results were provided by RMSD of the Cα atoms in the helical domains from the

3

corresponding starting structures. The averaged RMSD changes before and after the MD simulation was

4

relatively small was also indicative for the structural stability of all built key conformational state

5

models.

6 7 8 9 10 11

Figure 2. Comparisons of the structures of the six states extracted after the MD simulation with the starting conformations. The parts in cyan and magenta represent the starting structures and those after the MD simulation, respectively. The RMSD refers to the Cα position after the MD simulation with respect to that in the starting structure of the same state; values are: 1.288 Å for Sout-apo, 1.317 Å for Sout-ex, 1.269 Å for Sout-cen, 1.120 Å for Sin-cen, 1.112 Å for Sin-endo, and 1.177 Å for Sin-apo.

12

Identification of the Four Key Binding Sites (Pout-ex, Pout-cen, Pin-cen, Pin-endo).

13

Pout-ex: As described in the Method Section, results from the pocket-finding and molecular-docking

14

studies revealed that a putative glucose binding pocket was located in the outer vestibule of GLUT1.

15

This region was about 5 Å away from the entrance of the channel, and about 10 Å away from the



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reported central cavity. Based on this glucose binding location searching result, the glucose was firstly

2

docked into this pocket and the conformation from docking analysis was adopted as the initial structure

3

in the MD simulation.

4

Analysis of the whole simulation trajectory for the Sout-ex system revealed that the glucose down

5

shifted ~4 Å from the docking site (the initial position) towards the central cavity during the

6

minimization, and the conformation of the complex reached equilibrium after 4 ns into the MD

7

simulation. This result indicated that this stabilized binding position of the glucose in the equilibrium

8

conformation of the complex could be considered as the true exofacial binding site Pout-ex (shown in

9

figure 3A, pocket-1), which might be critical for glucose and substrate recognition. The Pout-ex site was

10

composed of T30, I33, N34, V69, A70, F72, S73, R126, N288, Y292, Y293, N415, and M420 (Figure

11

3B). This pocket had contrasting surface features and was characterized as an amphipathic binding site

12

surrounded by three N-domain transmembrane segments (TM1, TM2 and TM4) and two C-domain TMs

13

(TM7 and TM11). In this ligand recognition pocket, the residues T30 on TM1, V69 on TM2, R126 on

14

TM4, and N415 on TM11 formed four intermolecular hydrogen bonds with the glucose displaying major

15

forces stabilizing the substrate. Both R126 and N415 further formed their H-bond networks respectively

16

with N29 on TM1 and N288 on TM11, which were surmised to play a crucial role in stabilizing ligand

17

binding and the transporter tertiary structure. Several published reports have demonstrated with

18

experimental evidences that the mutations of the conserved arginine-126 on TM4 by other amino acids

19

resulted in low glucose-transport capacity and led to the GLUT1 deficiency syndrome disease.50-53

20

Observations from our study, in accord with previous research findings, strongly support the conclusions

21

drawn for the glucose recognition binding pocket Pout-ex we have identified. We propose that the other

22

residue N415 on TM11 also be an essential component for glucose recognition and play a substantial

23

role in substrate binding and transport.

24

Besides the strong hydrogen bonds mentioned above, many weak hydrogen bonds were also detected

25

and their impact was not ignorable. Statistical analysis estimated that there were on average five water

26

molecules in the Pout-ex site, which were not stationary but mobile to exchange with other water

27

molecules during the simulation (Chart S2). The water molecules formed temporary hydrogen-bonds


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1

with the glucose as well as with the surrounding residues to enhance the hydrogen-bond network (Figure

2

S6).

3

Pout-cen: For the central binding site in the outward-open occluded state (Sout-cen), the system arrived at

4

equilibrium after 8 nanoseconds of simulation. The glucose moved about 3 Å to the cytoplasmic side

5

from the starting position. In this simulation, the D-glucose underwent about 30° clockwise flipping

6

from the position of the D-glucose in the xylose transporter template (PDB ID: 4GBZ), the similar

7

flipping phenomena was also observed during transport process in the previous study.6 Similar to what

8

was observed during the glucose recognition binding site determination, there was a central binding

9

pocket identified from the MD trajectory analysis (Pout-cen, shown in figure 3A, pocket-2). Pout-cen was

10

surrounded by the N domain TM5, C domain TM7, 8 and 10. Residues Q161 NE2 in TM5, Q283 NE2

11

in TM7, and E380 OE2 in TM10 formed direct hydrogen-bond interactions with O6, O4, and the

12

ethereal oxygen atoms of the glucose, and the conformation was further stabilized by the surrounding

13

residues in TM7 and TM11 (upfront in Figure 3C, not shown). In the central binding state, Q161 and

14

E380 were regarded as important residues during the transport of glucose has also been addressed in the

15

former study.6

16

Pin-cen: In the crystal-structure-based inward-open occluded state (Sin-cen) system, after the MD

17

minimization, the D-glucose downshifted about 1.4 Å from the starting position towards the cytoplasmic

18

side to be stabilized in a different binding pocket: Pin-cen (Figure 3A, pocket-3). The system reached

19

equilibrium after a 90° rotation of the molecule during the 7th ~8th ns period. In the original inward

20

GLUT1 crystal structure, the long n-nonyl chain of the β-NG ligand stretched into a deep channel

21

toward the cytoplasmic direction in order to avoid steric clashes. Compared with β-NG, glucose has

22

smaller molecular volume and more flexibility, and as a result, D-glucose was stabilized in a binding site

23

differed from that for β-NG and with a totally different interaction model. In the Sin-cen complex, direct

24

hydrogen bonding between Y292(TM7) and N415(TM11) with O1, N411(TM7) with O2 and O3 of the

25

glucose were observed. Eventually, the conformation of the glucose was further stabilized by the

26

circumjacent H-bond network between TM7 and TM2. Figure 3D illustrates the binding mode of the

27

glucose in the central cavity binding site Pin-cen of the inward-open GLUT1.



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Page 12 of 31

1

Pin-endo: Similar to the Sout-ex system, the pocket-finding and docking studies also gave a putative

2

endofacial binding site, and indicated that the glucose was first docked into this location being 12 Å

3

away from the exit of the channel set as the starting point for the Sin-endo state in the MD simulation.

4

Detection of the whole simulation trajectory of the Sin-endo system revealed that the glucose moved

5

upward about 4 Å toward the central cavity from the docking site during minimization, and the

6

conformation of the complex reached equilibrium after 5 ns of MD simulation. Therefore the second

7

endofacial binding site Pin-endo in addition to the inward-open central binding pocket was identified

8

(Figure 3A, pocket-4), and it was found to be surrounded mostly by the C termini residues:

9

L278/Q279/S281/Q282/Q283 in TM7, G384/P387/W388 in TM10, and A407/S410/N411 in TM11

10

(Figure 3E). The residues Q282, Q283 in TM7, A407 and N411 in TM11 formed direct hydrogen bonds

11

with the glucose. However, the hydrogen bond network was weak, with only one hydrogen bond formed

12

between G384 (TM10) and Q282.

13 14

Figure 3. Glucose binding pockets found in GLUT1 and the hydrogen bonds formed during the transport process. The

15

four binding pockets are shown on the left. Pocket 1 is the ligand recognition pocket: Pout-ex; pocket 2, Pout-cen; pocket 3,

16

Pin-cen; and pocket 4, Pin-endo. On the right, B, C, D and E show the direct hydrogen bonds formed with the glucose and

17

the surrounding hydrogen bond networks for each pocket. The helices in magenta are parts of the N domain TMs, in


Page 13 of 31

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1

cyan, parts of the C domain TMs. The ball and stick model in blue and red represent the glucose molecule, and the

2

black straight lines depict the hydrogen bonds.

3

The Alternate and Multisite Transport Model for GLUT1. Based on the analyses of the observed

4

results from the MD simulations for different structural states, we propose a mechanistic model, consisting of

5

a combination of alternate access and multisite binding, for transport cycle of glucose in GLUT1 (Figure 4).

6

Although there has been no successful crystal structure captured for the outward facing conformations

7

for GLUT1, our simulation results suggest that glucose uptake process started with the ligand being

8

firstly recognized in an exclusive substrate recognition binding site in the outward-open conformer

9

rather than being directly bound to the central zone.

10

As illustrated in Figure 4, the whole process of glucose uptake involves six important states that

11

control: (1) ligand recognition on both the inner and outer vestibules of the transporter, and (2)

12

accommodation of ligands in the two binding sites in the central cavity where the glucose binds and

13

dissociates to diffuse in- or outwards.

14

In addition, during ligand transport, conformational interchanges of the transmembrane helices

15

produced flexible gated networks within the branched central channel that maintained the bound ligand

16

in the alternate sides and permitted the staged ligand diffusion across the transporter. Detailed

17

description from one state to another will be discussed below.

18 19

Figure 4. Proposed mechanism for glucose transportation of GLUT1. In this model, the substrates were recognized,

20

transported and released into or out of the cell through six states: Sout-apo, the substrate-free (apo) outward-open state; ACS Paragon Plus Environment 13


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Page 14 of 31

1

Sout-exo, the partially occluded outward-open (exofacial) state for glucose recognition; Sout-cen, the occluded

2

outward-open (central) state for binding of the glucose in the central zone; Sin-cen, the occluded inward-open (central)

3

state which separately existing in the central zone for ligand translocation; Sin-endo, the partially occluded inward-open

4

(endofacial) state; Sin-apo, the substrate-free (apo) inward-open state. The transport activity involved conformational

5

interchanges of the transmembrane helices which formed several gates to control staged ligand diffusion across the

6

transporter. Those key gates are shown in red lines cooperating with the corresponding key residues in the different

7

states.

8

The Glucose Was Firstly Recognized by Pout-ex. From early investigations on xylose-related

9

transport, the conventional single-alternating-site model predicts that ligand-binding (e.g. xylose) occurs

10

exclusively in a central site in both the open-outward and the inward-facing conformations. However, as

11

reported by Naftalin et al, xylose dockings with moderately high affinity were actually observed at

12

several shared sites in all the inward- and outward-facing xylose transport proteins.23,32,35 In this study,

13

we determined one binding site (Pout-ex) situated within a gated external vestibule that differed from the

14

reported central cavity binding pocket. We suggest that Pout-ex play a crucial role in substrate recognition

15

during the transport cycle. As shown in Figure 5A, our simulation revealed that during glucose

16

recognition by the Pout-ex binding site, relatively small conformational changes of the core

17

transmembrane helices occurred between the ligand-free (Sout-apo) and the glucose-bound (Sout-ex) states.

18

However, one significant movement from residue W65 on TM2 involved the indole ring forming an

19

entrance cap above the ligand by twisting about 90 degrees towards the tunnel cavity. This

20

side-chain-based structural protrusion formed by tryptophan would likely create a gate to prevent access

21

by additional substrate as well as protect the properly bound ligand from escaping. In addition to the

22

previous discussion about Pout-ex, this observation emphasizes again the indispensable role of the Pout-ex

23

pocket as a substrate recognition site for glucose transportation as well as novel substrate- or

24

inhibitor-design.

25 26

Transport from Ligand Recognition to the Central Site. As glucose moved down to the central

27

binding cavity (Pout-cen) about 6 Å away from the exofacial ligand recognition pocket (Pout-ex), several


Page 15 of 31

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1

local regions in the central transmembrane zone underwent large conformational changes on both

2

skeleton carbon Cα positions and side chains (Figure 5B). Namely, after glucose was accommodated in

3

the central binding site, the side chains of M420 (TM11) and Y292/Y293 (TM7) in the Sout-cen state

4

underwent big shift toward the inner cavity. The mercaptomethyl group in M420 and the phenol group

5

in Y292/Y293 cooperatively pushed the glucose towards the central pocket via van der Waals forces.

6

The similar observation has been confirmed by Dr. Park from his original study that Y292 together with

7

F26 show a large translocation during the MD simulation and may play a key role as a keeper for

8

water-gate opening or closing for glucose binding.6 Conformational interchanges on M420/Y292/Y293

9

seemed to be the main driving force for facilitating the diffusion of glucose, which also led to the

10

formation of the secondary gated network within the branched central channel that shielded the substrate

11

from the extracellular matrix. Besides the van der Waals interaction, the newly formed hydrogen bond

12

network, as an additional driving force, made the glucose rotate about 90o during this transportation (see

13

Figure 3 from B to C). During this translocation, the glucose moved from the recognition site Pout-ex,

14

which was in the N domain, to the central binding site Pout-cen that was predominantly surrounded by the

15

C domain helices. TMs 4, 7 and 11 were implicated in the exofacial binding sites in the outward-open

16

conformation, which was consistent with the earlier structural and mutational analysis results reported

17

by Mueckler.54 In addition, key residues N288, Y292 and N415 as three highly conservative amino acids

18

were involved in both of the predicted glucose binding sites of Pout-ex and Pout-cen, suggesting that they all

19

play an important role in the ligand recognition and transport activities, and this was also supported by

20

the significant reduction of GLUT1 transport activity observed in the native cysteine residues (N288 and

21

N415) after site-directed mutagenesis55,56 and in agreement with the previous report.6



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Page 16 of 31

1 2

Figure 5. Conformation changes of GLUT1 between states during the transportation. A: overlap of the Sout-apo (yellow)

3

and Sout-ex (green). B: overlap of the Sout-ex(green) and Sout-cen(purple).

4 5

Figure 6. Conformation changes of GLUT1 between states during the transportation. A: overlap of Sout-cen (cyan) and

6

Sin-cen (orange). B: detailed transport process of glucose from the Sout-cen (purple) to Sin-cen (cyan). C: overlap of Sin-cen

7

(orange) and Sin-endo (blue).

8

Glucose Transportation in the Central Cavity. Sugar binding in the central cavity likely triggered

9

a conformational change resulting in the inward-open conformation. A rigid-body rotation of the N and

10

C subdomains was observed between the outward- and inward-open states. Although there was only a


Page 17 of 31

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1

slight shift in distance for the glucose from the outward-open central binding site to the inward-open

2

central cavity (about 2 Å between Pout-cen and Pin-cen), all six C-terminal TMs underwent a dramatic

3

change of state transformation - from Sin-cen to Sin-endo – during the process The residues especially

4

L278-Y292 in the middle of the TM7 underwent a kink, causing a conformational change of TM7. In the

5

side chains, residues Q282 and Y292 underwent the biggest movement of about 6.5 Å towards the

6

tunnel axis. This conformational interchange produced another gate that facilitated staged ligand

7

diffusion across the central cavity toward the endofacial pocket. Therefore, residues Q282 and Q283

8

exerted significant impact on the substrate binding in the different states, which was also confirmed by

9

the mutagenesis analyses.57 Parallel to previously reported results on xylose transporter (XylE),32 TM10

10

in the central transmembrane zone, as the destabilizing ICH, travelled about 5 Å in the horizontal

11

direction toward the channel, and this movement led its subdomain, TM10b, to approach from the lower

12

part of the binding cavity. This large angular displacement of TM10b resulted in the opening up of the

13

gate formed by W388 which could be important for the downward diffusion of the glucose. More

14

detailed analysis further revealed that the benzene ring of F291 in TM7 functioned as a bat to strike the

15

glucose transit to the Pin-cen binding site (Figure 6A). Meanwhile, the hydrogen bonds formed with the

16

surrounding residues were broken which helped smooth the glucose translocation. The glucose

17

experienced a 180o rotation and new hydrogen bond networks were formed in the new inward-open

18

binding cavity Pin-cen (Figure 6B). During the translocation of glucose in the central binding pocket,

19

slight rearrangement of residues Q161 and W388 underneath the sugar was also observed from the MD

20

simulation study of Dr. Park.6 Compare to his result, as dipicted in Figure 6B, our simulation observed

21

the same residue involvement but with more dynamic conformational changes on these two residues.

22

Glucose Inner Binding and Recognition. The minimized overall Sin-endo structure was similar to the

23

partially occluded inward-open state of XlyE template (PDB ID: 4JA3) with RMSD being 1.3 Å over

24

155 residues. In the lower part of the binding site in this inward-open state, MD simulation showed that

25

a segment of L278-Y292 in the middle of TM7 partially lost its α-helix nature and functioned as a kink.

26

The dramatic change on the TM7 structure sufficiently enlarged the Pin-endo pocket which consequently

27

contributed to the lowering of the binding affinity for ligand dissociation from the binding pocket. More



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Page 18 of 31

1

interestingly, the C-terminal sub-segment TM10b of TM10 was interrupted by a conservative

2

382

3

GLUT1 (Figure 6C). This change has also been observed in the crystal structure of 4JA3,58 but from the

4

current MD study, we were not able to figure out more detailed dynamics on this part in terms of the

5

glucose diffusion and cytoplasmic release.

GPGP385 kink and moved towards the N subdomain to partially block the endofacial entrance of

6

MD simulation also showed that the shift of TM10b led to a new salt-bridge interaction between E393

7

and R153, which stabilized the barrier effect of TM10b. Involvement of R153 in the endofacial glucose

8

binding site Pin-endo was in accord with the results of missense mutation in the GLUT1 transporter and

9

may explain the reason why the GLUT1 DS was suggested to be related to R153C/H.52,53

10 11

CONCLUSION

12

The combination of the alternating access and multisite transport mechanistic model was proposed for

13

GLUT1 mediated glucose transport. Six key conformational states of GLUT1 were complementary to

14

the conformational structures of the transportation cycle. Four binding sites and several flexible gates

15

were demonstrated for glucose transport in GLUT1. There were extra binding sites on both outer and

16

inner vestibules of the transporter and they were considered to play critical roles in the regulation of

17

ligand recognition. Notably, the identified exofacial binding pocket (Pout-ex), supported by the GLUT1

18

mutagenesis study results, may be significant for substrate recognition during glucose uptake, therefore

19

very useful for drug design and GLUT1-related pharmaceutical research. Two sequential glucose

20

binding sites were identified within the central cavity that formed the neck of the “S-shaped” transport

21

channel and were presumed to play key roles in facilitating ligand transport and diffusion.

22

MD simulation study identified important conformational changes based on position data within the

23

cavity and transmembrane helices and these changes could cause staged diffusive transport between

24

adjacent sites. The destabilized side chains and amino acid sequences close to the binding sites may

25

promote glucose transport by forming flexible gates in the channel to facilitate substrate diffusion. The

26

intracellular helical bundles (ICHB) loop domain connecting the N and C terminus may also hold key


Page 19 of 31

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1

functions in affecting glucose transport and cytoplasmic release but need further investigations with

2

interdisciplinary methods.

3

ASSOCIATED CONTENT

4

Supporting Information

5

Detailed information on sequence alignment, model evaluation, water involvement at the biding sites,

6

MD fluctuation analyses and related explanations and discussions. This material is available free of

7

charge via the Internet at http://pubs.acs.org.

8 9 10 11 12 13 14 15

AUTHOR INFORMATION Corresponding Authors *Phone: +86-13512479137. Fax: +86-22-27892050 *E-mail: [email protected] Notes: The authors declare no competing financial interest.

16

ACKNOWLEDGMENTS

17

We thank Prof. Jianping Lin (College of Pharmacy, Nankai University) for helpful discussions and

18

suggestions during the study. This research is supported by grants from the Tianjin Municipal Applied

19

Basic and Key Research Scheme of China (11JCYBJC14400, 12ZCDZSY11500, 13JCZD27500) and

20

the National Basic Research Program of China (2015CB856500).

21 22



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50. Pascual, J. M.; Wang, D.; Yang, R.; Shi, L.; Yang, H.; De Vivo, D. C. Structural Signatures and Membrane Helix 4 in GLUT1: Inferences from Human Blood-Brain Glucose Transport Mutants. J. Biol. Chem. 2008, 283, 16732–16742. 51. Rotstein, M.; Engelstad, K.; Yang, H.; Wang, D.; Levy, B.; Chung, W. K.; De Vivo, D. C. Glut1 Deficiency: Inheritance Pattern Determined by Haploinsufficiency. Ann. Neurol. 2010, 68, 955–958. 52. Brockmann, K.; Wang, D.; Korenke, C. G.; von Moers, A.; Ho, Y. Y.; Pascual, J. M.; Kuang, K.; Yang, H.; Ma, L.; Kranz-Eble, P.; Fischbarg, J.; Hanefeld, F.; De Vivo, D. C. Autosomal Dominant Glut-1 Deficiency Syndrome and Familial Epilepsy. Annals of Neurology 2001, 50, 476–485. 53. Juan, M. P.; Ronald, L.; vanHeertum.; Wang, D.; Kristin, E.; Darryl, C. D. Imaging the Metabolic Footprint of Glut1 Deficiency on the Brain. Ann. Neurol. 2002, 52, 458–464. 54. Hruz, P. W.; Mueckler, M. M. Structural Analysis of the GLUT1 Facilitative Glucose Transporter (review). Mol. Membr. Biol. 2001, 18, 183–193. 55. Olsowski, A.; Monden, I.; Krause, G.; Keller, K. Cysteine Scanning Mutagenesis of Helices 2 and 7 in GLUT1 Identifies an Exofacial Cleft in both Transmembrane Segments. Biochemistry 2000, 39, 2469–2474. 56. Hruz, P. W.; Mueckler, M. M. Cysteine-scanning Mutagenesis of Transmembrane Segment 11 of the GLUT1 Facilitative Glucose Transporter. Biochemistry 2000, 39, 9367–9372.

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57. Weber, Y. G.; Storch, A.; Wuttke, T. V.; Brockmann, K.; Kempfle, J.; Maljevic, S.; Margari, L.; Kamm C.; Schneider S. A.; Huber S. M.; Pekrun A.; Roebling R.; Seebohm G.; Koka S.; Lang C.; Kraft E.; Blazevic D.; Salvo-Vargas A.; Fauler M.; Mottaghy F. M.; Münchau A.; Edwards M. J.; Presicci A.; Margari F.; Gasser T.; Lang F.; Bhatia K. P.; Lehmann-Horn F.; Lerche H. GLUT1 Mutations are a Cause of Paroxysmal Exertion-induced Dyskinesias and Induce Hemolytic Anemia by a Cation Leak. J. Clin. Invest. 2008, 118, 2157–2168. 58. Quistgaard, E. M.; Löw, C.; Moberg, P.; Trésaugues, L.; Nordlund, P. Structural Basis for Substrate Transport in the GLUT-homology Family of Monosaccharide Transporters. Nat. Struct. Mol. Biol. 2013, 20, 766–768.



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for Table of Contents use only

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Mechanistic Study of Human Glucose Transport Mediated by GLUT1

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Xuegang Fu1, Gang Zhang1, Ran Liu1, Jing Wei1*, Daisy Zhang-Negrerie3, Xiaodong Jian4, Qingzhi

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Gao1,2*

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7


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332x132mm (96 x 96 DPI)

ACS Paragon Plus Environment


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The homology model of the outward-open state of GLUT1. The general structure is shown on the left, the magenta part is the N domain of GLUT1, the cyan part, the C domain, and the yellow part, the loop connecting the N and C domains (intracellular helical bundles：ICHB). The top right is the extracelluar view, where the transmembrane segments are labeled with numbers; the bottom right is the cytoplasmic view, where the transmembrane segments and intracellular helical bundles are labeled with numbers. 208x165mm (96 x 96 DPI)


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Comparisons of the structures of the six states extracted after the MD simulation with the starting conformations. The parts in cyan and magenta represent the starting structures and those after the MD simulation, respectively. The RMSD refers to the Cα position after the MD simulation with respect to that in the starting structure of the same state; values are: 1.288 Å for Sout-apo, 1.317 Å for Sout-ex, 1.269 Å for Soutcen, 1.120 Å for Sin-cen, 1.112 Å for Sin-endo, and 1.177 Å for Sin-apo. 335x256mm (96 x 96 DPI)



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Glucose binding pockets found in GLUT1 and the hydrogen bonds formed during the transport process. The four binding pockets are shown on the left. Pocket 1 is the ligand recognition pocket: Pout-ex; pocket 2, Poutcen; pocket 3, Pin-cen; and pocket 4, Pin-endo. On the right, B, C, D and E show the direct hydrogen bonds formed with the glucose and the surrounding hydrogen bond networks for each pocket. The helices in magenta are parts of the N domain TMs, in cyan, parts of the C domain TMs. The ball and stick model in blue and red represent the glucose molecule, and the black straight lines depict the hydrogen bonds. 416x230mm (96 x 96 DPI)


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Proposed mechanism for glucose transportation of GLUT1. In this model, the substrates were recognized, transported and released into or out of the cell through six states: Sout-apo, the substrate-free (apo) outwardopen state; Sout-ex, the partially occluded outward-open (exofacial) state for glucose recognition; Sout-cen, the occluded outward-open (central) state for binding of the glucose in the central zone; Sin-cen, the occluded inward-open (central) state which separately existing in the central zone for ligand translocation; Sin-endo, the partially occluded inward-open (endofacial) state; Sin-apo, the substrate-free (apo) inward-open state. The transport activity involved conformational interchanges of the transmembrane helices which formed several gates to control staged ligand diffusion across the transporter. Those key gates are shown in red lines cooperating with the corresponding key residues in the different states. 551x340mm (96 x 96 DPI)



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Conformation changes of GLUT1 between states during the transportation. A: overlap of the Sout-apo (yellow) and Sout-ex (green). B: overlap of the Sout-ex(green) and Sout-cen(purple). 353x151mm (96 x 96 DPI)


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Conformation changes of GLUT1 between states during the transportation. A: overlap of Sout-cen (cyan) and Sin-cen (orange). B: detailed transport process of glucose from the Sout-cen (purple) to Sin-cen (cyan). C: overlap of Sin-cen (orange) and Sin-endo (blue). 329x157mm (96 x 96 DPI)


Regulation of glucose transport and GLUT1 glucose transporter expression by O2 in muscle cells in culture.

GDM-Induced Macrosomia Is Reversed by Cav-1 via AMPK-Mediated Fatty Acid Transport and GLUT1-Mediated Glucose Transport in Placenta.

Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site.

Molecular Dynamics Simulations of the Human Glucose Transporter GLUT1.

Crystal structure of the human glucose transporter GLUT1.

A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome.

Metal ion- and phosphate-mediated transport of glucose by insulin.

Inhibition of human GLUT1 and GLUT5 by plant carbohydrate products; insights into transport specificity.

Regulation of human trophoblast GLUT1 glucose transporter by insulin-like growth factor I (IGF-I).

Mechanistic study of silver-mediated furan formation by oxidative coupling.

Inhibition of glucose transport in human erythrocytes by ubiquinone Q0.

GLUT1 axis in ovarian cancer cells.

4-mediated glucose uptake.

GLUT1-mediated glucose uptake plays a crucial role during Plasmodium hepatic infection.

Distinct Akt phosphorylation states are required for insulin regulated Glut4 and Glut1-mediated glucose uptake.

Glucose is a key driver for GLUT1-mediated nanoparticles internalization in breast cancer cells.

High Glucose Causes Human Cardiac Progenitor Cell Dysfunction by Promoting Mitochondrial Fission: Role of a GLUT1 Blocker.

Mechanism of inhibition of human glucose transporter GLUT1 is conserved between cytochalasin B and phenylalanine amides.

Curcumin directly inhibits the transport activity of GLUT1.

Stimulation of glucose transporter (GLUT1) mRNA and protein expression by inhibitors of glycosylation.

Expression of GLUT1 and GLUT2 glucose transporter isoforms in rat islets of Langerhans and their regulation by glucose.

Pathogenic mutations causing glucose transport defects in GLUT1 transporter: The role of intermolecular forces in protein structure-function.

Glucose binding enhances the papain susceptibility of the intracellular loop of the GLUT1 glucose transporter.

The glucose transport activity of GLUT1 is markedly decreased by substitution of a single amino acid with a different charge at residue 415.