322 | Nature | Vol 578 | 13 February 2020
Article
We observed considerable non-protein electron density in the
C-terminal bundle of PfHT1 that corresponded exactly to the sugar-
binding site for d-glucose in human GLUT3^4 (Figs. 1 e, 2a, b, Extended
Data Fig. 4e). Almost all d-glucose hydrogen-bonding residues in human
GLUT3 were conserved and similarly positioned in PfHT1 (Fig. 2a, b,
Extended Data Fig. 1c). d-Glucose in PfHT1 was therefore modelled with
the observed orientation in human GLUT3 and refined with appropriate
stereochemical restraints (Methods). It was, nonetheless, important to
validate the binding pose of d-glucose, especially since a crystallization
lipid interacted with d-glucose in human GLUT3 (Fig. 2b). Forthwith,2 117b54
9NTD CTDICHdomain Omit map: 5 VPfHT 1
GLUT5inward
GLUT3outwardTM7bD-Glucosed CTDNTD CTDICH2ICH1 ICH410a
123 4
567a7b89
10b11 12CTDICH
domainNTD
ExtInt
ICH 3ceab250
Time(s)50 2000420280140100150PfHT 1Empty
D-Glu (pmol)Time (s)(^14) [ 0
C]D
-glucose (nmol
mg
–1 protein)
PfHT 1 Sugar
P. falciparum
PVM
channel
GLUT1/5
Sugar
RBCchannels
(NPPs)
Erythrocyte
PVM
PPM
Fig. 1 | Pf HT1 in d-glucose-bound occluded conformation. a, P. falciparum
infected erythrocyte illustrating the localization of Pf HT1 within the parasitic
plasma membrane and the GLUT-dependent uptake of glucose and fructose.
GLUT1/5, GLUT1 or GLUT5; NPP, new permeability pathway; RBC, red blood cell;
PVM, parasitophorous vacuole membrane; PPM, parasite plasma membrane.
b, Time-dependent uptake of [^14 C]d-glucose (black circles) by Pf HT1
proteoliposomes. Inset, Pf HT1 [^14 C]d-glucose (cyan trace) and non-specific
uptake in empty liposomes (black trace). Error bars represent mean ± s.e.m. of
n = 3 biologically independent experiments. d-Glu, d-glucose. c, Cartoon
representation of the structure of the Pf HT1 d-glucose-bound complex in the
occluded conformation, showing the N-terminal six-transmembrane (6TM)
domain (NTD) (blue), the C-terminal 6TM domain (CTD) (magenta), the
intrahelical domain (ICH) (grey) and d-glucose (stick representation). Pf HT1
has a large intracellular salt-bridge network that stabilizes the occluded
conformation, as seen in structures of related sugar porters in outward-facing
conformations^22 ,^23 (Extended Data Fig. 4c). The structures of sugar porters in
the outward-facing conformation are further stabilized by interactions
between the intracellular helices ICH1, ICH2, ICH3 and ICH4, which collectively
interact with ICH5 to latch the NTD and CTD together^4. ICH5 has previously
been observed only in the outward-facing conformation^4 ,^5. In the occluded
Pf HT1 structure, we were unable to model ICH5 (Extended Data Fig. 4e), which
implies that Pf HT1 is primed for transition into the inward-facing
conformation. Ext, exterior; int, interior. d, Ribbon representation of the CTD
domain of Pf HT1 (magenta), superimposed with TM7 and TM10 gating helices
of outward-facing GLUT3 (RCSB Protein Data Bank code (PDB) 4ZWC, shown in
teal) and inward-facing GLUT5 (PDB 4YB9, shown in light orange) e, Surface
representation of the Pf HT1 structure in the occluded conformation with
d-glucose shown as sticks (left), and the corresponding polder omit map (blue
mesh at 5σ) shown for the d-glucose surrounded by the NTD and CTD helices
(right), coloured as in c.
Competitive uptake of (^14) [C]
D-glucose
(% of
WT
)
0
20
40
60
100
80
120
No
competitorNo
protei
n
D-GlucoseD-Mannose D
-Allos
e
D-Galactose
3-O
-me-
D-glucoseD
-Talos
e
D-Gulos
e
D-Altrose
2-Deoxy-
D-glucose
c
a PfHT 1 D-glucose binding site b
C1
7a
8
123
(^654)
W412
Q169
A404
N341 N311
Q306
Q305 W436
7b
10b
10a
5
11
PfHT1and human GLUT 3
E378
MO lipid
7a
8
7b
10b
10a
5 11
d
WT
Q169
N
Q305
A
N311
A
W4
12A
Q169
A
Q306
A
N341
A
A404E
120
100
80
60
40
20
N341 N311
W412
Q169
Q306
Q305 W436
efPfHT1and GLUT 3
F289V314 C1C^1
W4 36
H168N158
F403
I310
N435
A439N413
7a
8
7b
10b
10a
5
11
PfHT 1 D-glucose binding-site residues
PfHT1 peripheral binding-site residues
Conserved
WT
H168
N
I310
A
V314F F403
A
N435
A
W4
36A
A439N
120
100
80
60
40
20
0
D-Glucose
PfHT1sugarcompetition D-Glucose D-Fructose
0
Relative
% of WT transport
A404
D-Fructose
Non-conserved
Relative
% of WT transport
D-Glucose D-Galactose D-Gulose
HO O
HO OHOH
OH
O
HO OHOH
HOOH
O
OHOH
HOOH
OH
Fig. 2 | Molecular recognition of d-glucose by Pf HT1. a, Cartoon
representation of the Pf HT1 sugar-binding site with d-glucose (yellow sticks)
and the interacting residues labelled; residues Q169, Q305, Q306, N311 and
N341 were determined to be essential for sugar transport, as shown in d.
Putative hydrogen bonds are indicated with dotted lines. b, Sugar-binding site
comparison between Pf HT1 (yellow sticks) and human GLUT3 (PDB 4ZW9)
(grey, conserved; cyan, non-conserved). The crystallization lipid monoolein
(MO) interacting with TM7b in human GLUT3 is shown in cyan, and the Pf HT1
A404 residue that corresponds to E378 in human GLUT3 is shown in dark blue.
Putative hydrogen bonds are indicated with dotted lines. c, The competitive
uptake of [^14 C]d-glucose by Pf HT1 proteoliposomes in the absence (white bar)
and presence of non-labelled d-glucose epimers and homologues (black bars);
note, competitive uptake cannot distinguish between transported and non-
transported sugars and non-specific uptake in empty liposomes (red bar). Data
are mean ± s.e.m. of n = 3 biologically independent experiments. d, Transport
activity of Pf HT1 mutants for residues in d-glucose-binding site, for [^14 C]
d-glucose (black bars) and [^14 C]d-fructose (white bars). Data are mean ± s.e.m.
of n = 3 biologically independent experiments. e, Comparison of peripheral
d-glucose-binding site between Pf HT1 (yellow sticks) and human GLUT3 (grey,
conserved; cyan, non-conserved), and monoolein lipid interacting with human
GLUT3 in cyan. f, Transport activity of Pf HT1 mutants for residues in the
peripheral binding site (in which the residue is substituted with alanine or the
equivalent residue in human GLUT3) for [^14 C]d-glucose (black bars) and [^14 C]
d-fructose (white bars). Data are mean ± s.e.m. of n = 3 biologically independent
experiments.