Crystal structure of arginase from plasmodium falciparum and implications for l -arginine depletion in malarial infection

Article abstract of DOI:10.1021/bi100390z

The 2.15 A resolution crystal structure of arginase from Plasmodium falciparum, the parasite that causes cerebral malaria, is reported in complex with the boronic acid inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) (K _d = 11 μM). This is the first crystal structure of a parasitic arginase. Various protein constructs were explored to identify an optimally active enzyme form for inhibition and structural studies and to probe the structure and function of two polypeptide insertions unique to malarial arginase: a 74-residue low-complexity region contained in loop L2 and an 11-residue segment contained in loop L8. Structural studies indicate that the low-complexity region is largely disordered and is oriented away from the trimer interface; its deletion does not significantly compromise enzyme activity. The loop L8 insertion is located at the trimer interface and makes several intra- and intermolecular interactions important for enzyme function. In addition, we also demonstrate that arg- Plasmodium berghei sporozoites show significantly decreased liver infectivity in vivo. Therefore, inhibition of malarial arginase may serve as a possible candidate for antimalarial therapy against liver-stage infection, and ABH may serve as a lead for the development of inhibitors.

Full text of DOI:10.1021/bi100390z

5
600 Biochemistry 2010, 49, 5600–5608
DOI: 10.1021/bi100390z
Crystal Structure of Arginase from Plasmodium falciparum and Implications for
†,‡
L
-Arginine Depletion in Malarial Infection
§
§
^
^
Daniel P. Dowling, Monica Ilies, Kellen L. Olszewski, Silvia Portugal, Maria M. Mota,
,
§
Manuel Llin ꢀa s, and David W. Christianson*
§
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323,
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1013, and Unidade de Mal aꢀ ria, Instituto de
Medicina Molecular, Faculdade de Medicina Universidade de Lisboa, 1649-028 Lisboa, Portugal
^
Received March 13, 2010; Revised Manuscript Received May 25, 2010
˚
ABSTRACT: The 2.15 A resolution crystal structure of arginase from Plasmodium falciparum, the parasite that
causes cerebral malaria, is reported in complex with the boronic acid inhibitor 2(S)-amino-6-boronohexanoic
acid (ABH) (K = 11 μM). This is the first crystal structure of a parasitic arginase. Various protein constructs
d
were explored to identify an optimally active enzyme form for inhibition and structural studies and to probe
the structure and function of two polypeptide insertions unique to malarial arginase: a 74-residue low-
complexity region contained in loop L2 and an 11-residue segment contained in loop L8. Structural studies
indicate that the low-complexity region is largely disordered and is oriented away from the trimer interface; its
deletion does not significantly compromise enzyme activity. The loop L8 insertion is located at the trimer
interface and makes several intra- and intermolecular interactions important for enzyme function. In
addition, we also demonstrate that arg- Plasmodium berghei sporozoites show significantly decreased liver
infectivity in vivo. Therefore, inhibition of malarial arginase may serve as a possible candidate for antimalarial
therapy against liver-stage infection, and ABH may serve as a lead for the development of inhibitors.
Malaria, an infectious disease caused by a parasitic protozoan,
is a serious health threat in developing countries because of its
facile transmission by the Anopheles mosquito (1, 2). Malaria is
currently considered to be eradicated in the United States;
however, 10 species of Anopheles mosquitoes are indigenous to
the United States, suggesting that malaria acquired in endemic
countries can be reintroduced into the United States by human
travel and propagated by mosquitoes. For example, there were
The pathogenic course of the disease is characterized by rapidly
proliferating parasite cells, massive erythrocyte lysis, and ische-
mia (9). Clinical manifestations of malaria include hypoglycemia,
lactic acidosis, hemolytic anemia, hemoglobinuria, and hypoargi-
ninemia (9).
Malaria patients often present with hypoargininemia (10, 11),
and metabolomic studies of P. falciparum during its 48 h intra-
erythrocytic life cycle reveal nearly complete depletion of
levels (12). Consistent with this observation, P. falciparum infection
does not affect the influx kinetics of -arginine in erythrocytes (13).
Low levels of -arginine correlate with decreased levels of immunity
and nitric oxide (NO) production (14). The depletion of
L-arginine
1505 cases of malaria diagnosed in the United States in 2007,
including one transfusion-related case and one fatality (3).
Worldwide statistics are significantly more severe, with 243
million cases of malaria being diagnosed and 863000 deaths
resulting in 2008 (primarily children in sub-Saharan Africa) (4).
Five species of malaria specifically infect humans: Plasmodium
falciparum, Plasmodium vivax, Plasmodium malariae, Plasmo-
dium ovale, and Plasmodium knowlesi; P. falciparum is the most
lethal (5). Because the parasite is capable of developing resistance
to drug therapy, combination drug therapies are generally
required to circumvent this problem (6, 7).
L
L
1
L-arginine
in culture is achieved by an arginase from the malarial parasite,
which catalyzes the hydrolysis of the side chain guanidinium
group to form L-ornithine and urea (12). Increased arginase
activity characterizes the alternative immune response, which
downregulates inflammation and tissue damage while upregulat-
ing angiogenesis and tissue repair mechanisms (15). Such condi-
tions favor parasite growth through suppressed T cell and
inflammatory responses to pathogens and increased concentra-
tions of L-ornithine-derived polyamines, which facilitate cellular
proliferation (15).
Increased exogenous arginase activity also characterizes infec-
tions by the parasitic protozoan Leishmania major and the
Malarial infection drastically alters host metabolism since the
parasite co-opts essential nutrients for its own survival at the
expense of a human host; moreover, the parasite introduces waste
products and toxins into the circulatory system of the host (8).
†
This work was supported by National Institutes of Health (NIH)
Grant GM49758 (D.W.C.), an NIH Director’s New Innovator Award
1
Abbreviations: ABH, 2(S)-amino-6-boronohexanoic acid; BME,
(1DP2OD001315-01 to M.L.), the Burroughs Wellcome Fund (M.L.), a
β-mercaptoethanol; hAI and hAII, human arginases I and II, respectively;
dN-PFA, PFA beginning at residue K22 bearing an N-terminal His tag;
L2S, PFA construct containing a 70-residue deletion in the L2 loop; LCR,
low-complexity region; MPD, 2-methyl-2,4-pentanediol; NO, nitric oxide;
PFA, P. falciparum arginase; PMSF, phenylmethanesulfonyl fluoride;
rmsd, root-mean-square deviation; TAME, tosyl-arginine methyl ester;
TCEP, tris(2-carboxyethyl)phosphine hydrochloride.
National Science Foundation Graduate Research Fellowship (K.L.O.),
the Howard Hughes Medical Institute (M.M.M.), and the Funda c- ~a o
para a Ci ^e ncia e Tecnologia (FCT, Portugal) (S.P. and M.M.M.).
‡
The atomic coordinates of the P. falciparum arginase-ABH complex
have been deposited in the Protein Data Bank as entry 3MMR.
*To whom correspondence should be addressed. Telephone: (215)
898-5714. Fax: (215) 573-2201. E-mail: chris@sas.upenn.edu.
pubs.acs.org/Biochemistry
Published on Web 06/09/2010
r 2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 26, 2010 5601
2
(S)-amino-6-boronohexanoic acid (ABH) (29) reveals a binding
mode comparable to that observed in the hAI-ABH com-
plex (30) despite significantly weaker inhibitor binding affinity.
Structural comparisons among mammalian, bacterial, and para-
sitic arginases reveal intriguing differences in the stabilization of
oligomeric structure, and possible functions for the two poly-
peptide insertions in PFA are explored. Finally, the study of arg-
P. berghei sporozoites confirms the importance of arginase
activity in the mechanism of infection, suggesting that inhibition
of malarial arginase may serve as a possible candidate for
antimalarial therapy against liver-stage infection.
MATERIALS AND METHODS
F
IGURE 1: Topology diagram of human arginase I (hAI) and
P. falciparum arginase (PFA). The positions of metal ligands are
indicated by black spheres. Amino acid insertions within loops L2
and L8 of PFA relative to hAI are indicated by red dashed lines; the
large ∼70-residue insertion in the L2 loop is an asparagine-rich low-
complexity region (LCR).
P. falciparum Arginase Constructs. The previously re-
ported P. falciparum arginase construct (21, 22) utilized the entire
P. falciparum arginase gene with a C-terminal streptavidin
affinity tag (PFA-Strep). Here, we report the preparation and
analysis of three new arginase constructs: full-length PFA bear-
ing a C-terminal histidine (His) tag (PFA-C6H), an N-terminal
truncation beginning with K22 and bearing an N-terminal His
tag (dN-PFA), and a similar N-terminal truncation with residues
N84-D157 of loop L2 deleted (dN-PFA-L2S). Additionally, the
H381A variant and the L8 chimera (in which loop L8 is replaced
with the shorter hAI sequence) were generated from the dN-PFA
plasmid. Restriction enzymes and ligase were purchased from
New England Biosciences. Taq Hifi Polymerase was purchased
from Invitrogen. All cell lines were purchased from Stratagene.
The PFA-C6H plasmid was constructed by first amplifying the
P. falciparum arginase coding sequence from parasite genomic
bacterium Helicobacter pylori (16-18). Consequently, the arg-
variants of these organisms do not deplete host L-arginine levels
and are accordingly more susceptible to the NO-dependent
immune response. Decreased -arginine levels have been ob-
served in malaria patients, and supplemental -arginine improves
L
L
endothelial function in patients with moderate to severe malar-
ia (11, 19). Arginase activity is also implicated in slowing the
recovery of malaria patients during treatment (20). Considering
that current patient outcomes within the first 48 h of therapy do
not change between quinine and artesunate treatment, it is
proposed that treating endothelial dysfunction by targeting
P. falciparum arginase with inhibitors within this time frame
may represent a possible adjuvant therapy (20).
0
DNA (3D7 strain) with primers designed to incorporate a 5 NheI
0
site in place of the start codon and a 5 XhoI site in place of the
The P. falciparum arginase gene has been cloned and char-
acterized (21, 22). The amino acid sequence of arginase from
P. falciparum is 28 and 27% identical with those of human
arginases I (hAI) and II (hAII), respectively. Like the human
arginases, P. falciparum arginase (PFA) is a binuclear manganese
metalloenzyme that exists as a trimer with optimal activity at
basic pH (21). Unlike the human arginases, PFA contains a large,
stop codon [primer 1, GCT AGC TTG GAT ACT ATA GAA
AGT TAC ATC; primer 2, CTC GAG CAC TAT ATC GTA
TCC TAA CAC (restriction sites underlined)]. This amplicon
was cloned into the pET24a plasmid to give a recombinant gene
with an N-terminal Met-Ala-Ser in place of the initial Met and a
C-terminal Leu-Glu-6His tag.
To generate dN-PFA, the arginase gene was amplified from
the C-terminal His-tagged construct PFA-C6H. NheI and
HindIII restriction sites were incorporated to delete the first 21
amino acids [primer 1, GAG CGC TAG CAA AAA CGT TTC
CAT TAT TGG TTC TCC; primer 2, CTC AAG CTT TTA
CAC TAT ATC GTA TCC (restriction sites underlined)].
Deletion of the corresponding N-terminal segment of hAII
facilitated its crystallization (31), so we reasoned that a compar-
able deletion would facilitate the crystallization of PFA. The
digested PCR product was ligated into PET28a (Novagen),
utilizing the N-terminal His tag and thrombin cleavage site,
and transformed into XL-1 Blue cells for DNA isolation and
sequencing. For the L2 loop deletion construct (dN-PFA-L2S),
an AgeI restriction site was incorporated into the dN(1-21)
construct L2 loop, resulting in the deletion of residues N84-
D157 [primer 1, CGG ACC GGT AAT ATA AGG AAT ATA
AA; primer 2, CGG ACC GGT TTT TTT TTC TTG TTT CAT
(restriction sites underlined)]. The digested product was purified
using a Qiagen extraction kit and transformed into XL-1 Blue
cells for DNA isolation and sequencing. Sequencing was per-
formed by the University of Pennsylvania DNA Sequencing
Facility, and the identified positive clone was then transformed
into Escherichia coli BL21-CodonPlus(DE3)-RIL cells for ex-
pression as previously described (21).
7
4-residue low-complexity region (LCR) inserted within loop L2
(
Figure 1). Amino acid sequence alignments of arginases from
P. falciparum, P. vivax, Plasmodium yoelii, P. knowlesi, and
Plasmodium berghei reveal that this insert is as large as 100
residues in P. vivax and as small as 15 residues in P. berghei and
shows no significant sequence conservation (22). Such LCRs are
found in more than 90% of all proteins in the P. falciparum
genome (23-26). A second ∼11-residue insert is also located in
loop L8 (Figure 1) (22). Despite these polypeptide insertions, PFA
still exists as an active trimer (21). The question remains as to what
effects, if any, these polypeptide insertions have on arginase
structure and function. Kinetic measurements on a PFA construct
bearing a C-terminal streptavidin affinity tag initially suggested that
malarial arginase is less catalytically efficient than the mammalian
3
-1 -1
arginases, with a kcat/KM value of 7.4 ꢀ 10 M
s
(21) [compared
5
-1 -1
with a k /K of 2.0 ꢀ 10 M
s
for rat arginase I (27) or 1.27 ꢀ
cat
M
5
-1 -1
10 M
s
for hAI (28)].
Here, we report the preparation of a PFA construct bearing an
N-terminal histidine tag instead of a C-terminal streptavidin
affinity tag to facilitate purification. Kinetic studies with this new
construct show that PFA actually exhibits catalytic efficiency
comparable to that of hAI. The 2.15 A resolution crystal
structure of PFA in a complex with the boronic acid inhibitor
˚

5
602 Biochemistry, Vol. 49, No. 26, 2010
Dowling et al.
Table 1: Kinetic Parameters for P. falciparum Arginase (PFA) Constructs
-1
-1 -1
construct
pH
K
M
(mM)
k
cat (s
)
k
cat/K
M
(M
s
)
K
i
(ABH) ( μM)
a
5
dN-PFA
8.5
3.3 ( 0.6
4 ( 1
25 ( 2
12 ( 1
13
440 ( 20
320 ( 40
77 ( 2
(1.3 ( 0.2) ꢀ 10
10 ( 1
12 ( 2
11 ( 2
4
8
.0
.4
(8 ( 2) ꢀ 10
3
3
7
(3.0 ( 0.3) ꢀ 10
(2.9 ( 0.4) ꢀ 10
b
PFA-C6H
PFA-Strep
8.0
8.0
8.0
8.0
8.0
9.0
76 ( 7
ND
c
3
b
96
7.4 ꢀ 10
ND
b,d
b
b
dN-PFA-L2S
4.0 ( 0.3
10 ( 1
20 ( 4
1.5
ND
ND
ND
3
dN-PFA-H381A
dN-PFA L8 chimera
28 ( 2
(2.7 ( 0.3) ꢀ 10
53 ( 2
e
b,f
b
b
ND
ND
ND
g
5
h
human arginase I
190
1.27 ꢀ 10
0.005
a
b
dN-PFA signifies the N-terminally truncated form of PFA based on sequence alignment with hAII. The N-terminal His tag is not cleaved. Not
determined. From ref 21.
ref 28. From ref 30; dissociation constant (K
c
d
V
e
f
V
g
max = 2.3 ( 0.1 μM/s. dN-PFA L8 chimera shows substrate inhibition with a K
i
of 130 ( 50 mM.
max = 2.6 ( 0.5 μM/s. From
h
d
) determined by isothermal titration calorimetry.
The H381A variant was generated using the QuikChange
mutagenesis kit (Stratagene) with the dN-PFA template
activity using a colorimetric method (32) with slight modifica-
tions. The reaction of urea with R-isonitroso propiophenone was
assessed at a wavelength of 550 nm using the Envision plate
reader (courtesy of the S. Diamond laboratory, Institute of
Medicine and Engineering, University of Pennsylvania). Product
formation was assessed by means of urea standard curves. No
background signal from substrate reacting with R-isonitroso
0
(
primer 1, 5 -GTT GAT AAA AAA GTT gcT GGA GAT
0
0
TCA TTG CC-3 ; primer 2, 5 -GGC AAT GAA TCT CCA gcA
ACT TTT TTA TCA AC-3 ). The PCR product was transformed
0
into XL1-Blue cells for DNA isolation and sequencing. The
construct for the L8 chimera was generated utilizing TA over-
hangs generated by Platinum Taq (Invitrogen) polymerase. The
dN-PFA plasmid without residues D374-T392 was elongated
propiophenone was observed when up to 400 mM
was used. At -arginine concentrations of >100 mM, a decrease
was observed for the dye developing reaction; therefore, standard
curves containing stoichiometric ratios of urea and -arginine were
used for reactions with >100 mM -arginine. The arginase reaction
was performed in 50 mM Tris (pH 8.0), 0.5 mM TCEP, 1 mM
MnCl , 0.05-1 μM protein, and 1-200 mM -arginine for 5-10
L-arginine
L
0
primer 1, 5 -AAA ACA GGC AAG TTG TGT TTA GAA CTT
(
0
0
ATC GCC-3 ; primer 2, 5 -CCC AAG TGA TGG ATT ATA
L
0
TTC TAC TAA ATC C-3 ). The insert was purchased from IDT
L
0
0
(
4
primer 3, 5 -AG ACA CCA GAA GAA GTA ACT-3 ; primer
0
0
, 5 -GTT ACT TCT TCT GGT GTC TT-3 ) and annealed
2
L
separately before being ligated with the PCR product. The
ligation product was transformed into XL-1 Blue cells for
DNA isolation and sequencing, and the identified positive clone
was then transformed into E. coli BL21-CodonPlus(DE3)-RIL
cells for expression as previously described (21).
min at 37 ꢀC. The assay mixture and protein were preincubated at
37 ꢀC for >1 min before the reaction was initiated. The assay
mixture (20 μL) was stopped with a sulfuric-phosphoric acid/R-
isonitroso propiophenone mixture (140 μL). Reaction points were
developed in a thermocycler (90 ꢀC for 1 h), followed by incubation
at room temperature (21 ꢀC for 15 min). The dN-PFA expressed in
minimal medium exhibited increased activity and was used for kcat
Protein Expression and Purification. Briefly, transformed
or streaked cells were grown on Luria-Bertani (LB) agar supple-
mented with 50 μg/mL kanamycin. The LB medium supplemented
with 50 μg/mL kanamycin was inoculated with a single colony and
grown for 8 h at 37 ꢀC and 250 rpm. This starter culture was then
transferred to 250 mL of LB medium supplemented with 50 μg/mL
kanamycin and 34 μg/mL chloramphenicol and grown for 12-
calculations as measured by linear rates at 200 mM
50 nM protein. Kinetic parameters were determined with Graph-
pad Prism (2008). Inhibition studies were performed with 25 mM
L-arginine with
L-
arginine at 37 ꢀC for 10 min to obtain significant absorbance signals
to plot IC data. The K value for ABH was calculated using the
50
i
16 h. The 250 mL cell growth was used to inoculate 5 L of either
Cheng-Prusoff equation (33). Results are listed in Table 1.
Isothermal Titration Calorimetry of Formation of the
dN-PFA-ABH Complex. Experiments were conducted on an
ITC200 isothermal calorimeter from MicroCal, Inc., in the
laboratory of W. DeGrado at the University of Pennsylvania.
Enzyme (50 μM) was dialyzed against 50 mM Tris (pH 8.0),
LB or minimal medium supplemented with 50 μg/mL kanamycin.
Cells were induced with 1 mM IPTG in the presence of 50 μg/mL
kanamycin, 10 μg/mL phenylmethanesulfonyl fluoride (PMSF),
and 1 μg/mL tosyl-arginine methyl ester (TAME) at 37 ꢀC.
Cells were harvested by centrifugation at 6000g and resus-
pended in 50 mM Tris (pH 8.0), 500 mM NaCl, and 3 mM
β-mercaptoethanol (BME) in the presence of protease inhibitors
200 mM NaCl, 1 mM MnCl , and 1 mM TCEP; 0.65 mM ABH
2
was dissolved in dialysis buffer. The sample cell (0.3 mL) was
overfilled, and the reference cell was filled with distilled water.
The inhibitor ABH was titrated into the sample cell with 20
sequential aliquots (2 μL each; an initial 0.2 μL injection was
made, but not used in data analysis). A control experiment
titrating the inhibitor into buffer was subtracted out, and data
analysis was performed using ORIGIN version 7.0.
(
10 μg/mL PMSF and 1 μg/mL TAME). Cells were lysed by
sonication and centrifuged at 30000g for 1 h. The supernatant
was loaded onto Ni-nitrilotriacetic acid resin and eluted with
increasing imidazole concentrations (10, 50, and 200 mM). Pulled
protein fractions were dialyzed into 50 mM Tris (pH 8.0),
2
00 mM NaCl, 1 mM MnCl , and 1 mM tris(2-carboxyethyl)-
2
phosphine (TCEP) and subsequently concentrated. Protein was
loaded onto a Superdex 26/60 size exclusion column preequili-
brated with dialysis buffer. The trimer protein peak was collected,
concentrated, and stored in aliquots at 4 ꢀC.
Kinetic Assays. dN-PFA, dN-PFA-L2S, dN-PFA L8 chi-
mera, dN-PFA-H381A, and PFA-C6H were assayed for catalytic
Crystallography. The inhibitor ABH was synthesized as
previously described (29). The construct dN-PFA was used
successfully in crystallization experiments. Briefly, a 2 μL sitting
drop of 5-10 mg/mL dN-PFA in 50 mM Tris (pH 8.0), 200 mM
NaCl, 1 mM MnCl , 1 mM TCEP, and 5 mM ABH was mixed
2
with a drop of 1.4 M sodium/potassium phosphate (pH 8.2) and

Article
Biochemistry, Vol. 49, No. 26, 2010 5603
and residues G382 and D383 within the loop L8 insert. All
refinement statistics are listed in Table 2. Buried surface area
calculations were performed using the PISA server (http://www.
ebi.ac.uk/msd-srv/prot_int/pistart.html) (38).
Table 2: Data Collection and Refinement Statistics
Data Statistics
˚
resolution limits (A)
37-2.15
Mice and Plasmodium Liver Infection. C57BL/6 mice were
housed in the Instituto de Medicina Molecular (IMM) facilities,
and experiments were performed under IMM Animal Care
Committee approval following National and EU guidelines.
The arg- (12) and wild-type P. berghei sporozoites were obtained
by dissection of Anopheles stephensi-infected mosquitoes bred at
the IMM insectarium. Mice were infected by intravenous inocu-
space group
R32 (hexagonal)
112.5, 112.5, 228.7
59546
˚
a, b, c (A)
total no. of reflections
no. of unique reflections
31095
completeness (%) (overall/outer shell)
100/100
redundancy (overall/outer shell)
8.4/8.3
a
R
merge (overall/outer shell)
0.104/0.420
19.4/5.4
I/σ(I ) (overall/outer shell)
4
lation of 3 ꢀ 10 sporozoites, and the parasite liver load was
Refinement
quantified 40 h postinfection by quantitative RT-PCR, as pre-
viously described (39). The relative amount of P. berghei ANKA
18S rRNA was calculated against the hypoxanthine guanine
b
R/Rfree
0.155/0.186
no. of atoms
c,d
protein atoms
2440
298
13
phosphoribosyltransferase (hprt) housekeeping gene. The PbA
c
0
water molecules
1
8S rRNA and hprt specific primer sequences were 5 -CGG CTT
c
ligand atoms
0
0
AAT TTG ACT CAA CAC G-3 and 5 -TTA GCA TGC CAG
AGT CTC GTT C-3 for PbA 18S rRNA and 5 -TGC TCGAGA
TGT GAT GAA GG-3 and 5 -TCC CCT GTT GAC TGG TCA
TT-3 for mouse hprt, respectively. External standardization was
c
metal ions
2
0
0
BME atoms
4
0
0
2
˚
average B factor (A )
0
main chain
30
34
43
21
33
38
side chain
performed using plasmids encoding the full-length P. berghei 18S
rRNA and hprt cDNA cloned into TOPO TA (Invitrogen).
water molecules
2
þ
Mn ions
inhibitor
BME
RESULTS AND DISCUSSION
Ramachandran plot (%)
most favorable
additional allowed
generously allowed
disallowed
Catalytic Activity of PFA Constructs. The catalytic effi-
90.0
10.0
0.0
ciency (kcat/K ) of dN-PFA increases with an increase in pH, and
M
kinetic parameters are comparable to those measured for human
arginase I (Table 1). Importantly, the dN-PFA construct is the
most catalytically active form of PFA reported to date. While the
KM values measured for the PFA constructs reported in Table 1
are in the same range as the KM values measured for mammalian
0.0
rmsd
˚
bond lengths (A)
0.007
1.1
bond angles (deg)
dihedral angles (deg)
17.1
P
P
arginases [KM = 1.4 mM for rat arginase I at pH 9.0 (40); KM =
a
R
merge
=
|I - ÆI æ|/ I, where I is the observed intensity and ÆI æ is the
b
1.5 mM for hAI at pH 9.0 (41), and KM = 0.55 mM for hAII at
pH 9 (42)], the KM value of 12 mM measured for the PFA-C6H
construct at pH 8.0 and the KM value of 13 mM measured for the
C-terminal streptavidin affinity tag construct reported by M u€ ller
and colleagues at pH 8.0 (PFA-Strep) (21) are notably higher.
Therefore, it appears that a C-terminal affinity tag on PFA is
detrimental for substrate binding and catalytic activity.
average intensity calculated for replicate data. Crystallographic R factor
R = ||F
P
P
(
o
| - |F
c
||/ |F
o
|) for reflections contained in the working set.
P
P
Free R factor (Rfree
test set excluded from refinement. |F
calculated structure factor amplitudes, respectively. Per asymmetric unit.
Residue C154 refined with an occupancy of 0.5.
=
||F
o
| - |F
c
||/ |F
o
|) for reflections contained in the
| and |F | are the observed and
o
c
c
d
equilibrated against a 500 μL reservoir of 40-50% 2-methyl-2,
The tightest binding inhibitor of rat and human arginases
4-pentanediol (MPD) at room temperature. Small crystals
appeared overnight and grew to typical dimensions of 50 μm ꢀ
known to date is the boronic acid analogue of
L-arginine,
5
0 μm ꢀ 50 μm. Crystals were harvested and cryoprotected in
ABH (29), which inhibits hAI with a K of 5 nM (30). Indeed,
d
30% Jeffamine ED-2001 and 0.1 M HEPES (pH 7.0) and then
ABH is an orally bioavailable drug lead that shows promising in
vivo activity for the treatment of cardiovascular diseases such as
erectile dysfunction and atherosclerosis in animal models (31, 43).
However, the inhibition of dN-PFA by ABH at physiological
flash-cooled in liquid nitrogen. Diffraction data were measured
on beamline 24-ID-E at the Advanced Photon Source (APS,
Argonne, IL). Crystal parameters and data collection statistics
are listed in Table 2.
pH is significantly weaker, with a K
competitive inhibition (Table 1). This affinity was confirmed by
isothermal titration calorimetry (pH 8.0) with a K of 11 ( 2 μM
of 11 ( 2 μM assuming
i
Data were indexed and merged using HKL2000 (34). Mole-
cular replacement calculations were performed with PHA-
SER (35) using the atomic coordinates of hAII less inhibitor
and solvent atoms [Protein Data Bank (PDB) entry 1PQ3] (31) as
a search probe for rotation and translation function calculations.
Iterative cycles of refinement and model building were performed
using PHENIX (36) and COOT (37), respectively, to improve
each structure as guided by Rfree. The N-terminal His tag is
completely disordered, and electron density is observable begin-
ning at residue K22 of the dN-PFA protein sequence. Residues
G72-N153 of the L2 loop insert are disordered and excluded
from the final model. Two cis-peptide linkages are found in the
refined structure: residues G190 and G191 immediately after β3
d
(Figure 2). The origin of weaker binding to dN-PFA is not
evident in the crystal structure of the enzyme-inhibitor complex,
since ABH appears to make identical interactions in the active
sites of hAI and dN-PFA (vide infra).
To assess the functions of the two amino acid sequence inserts
in PFA, two deletion constructs were generated. Approximately 70
residues (N84-D157) of the 74-residue LCR were deleted from
the L2 loop to generate the dN-PFA-L2S construct. A survey of
arginase crystal structures in the Protein Data Bank suggested
that this deletion construct would be able to fold properly.
Similarly, residues D374-T392 of loop L8 were replaced with

5
604 Biochemistry, Vol. 49, No. 26, 2010
Dowling et al.
the corresponding sequence from human arginase I, yielding the
dN-PFA-L8 chimera. While these constructs do not express well,
soluble protein expression is confirmed by Western blotting
analysis using an anti-His antibody (data not shown). Because
of the significant decrease in protein purity and the level of
expression, accurate protein concentrations were difficult to
estimate. However, a KM value of 4.0 ( 0.3 mM was determined
for the L2S construct with a Vmax of 2.3 ( 0.1 μM s- , and a KM
value of 20 ( 4 mM was determined for the L8 chimera with a
Vmax of 2.6 ( 0.5 μM s- . The KM value for the L2S construct is in
accord with that measured for the dN-PFA construct, indicating
that the LCR does not significantly impact substrate affinity. The
L8 chimera shows moderately decreased substrate affinity; more-
over, this construct shows substrate inhibition with a K of 130 (
i
50 mM (data not shown).
Parasitic LCR inserts are expected to exist primarily as
unstructured domains made up of hydrophilic and flexible amino
acids such as Asn, Lys, Glu, and Asp (26), and this is the case for
the 74-residue LCR in loop L2 of PFA. In PFA, the LCR insert
does not affect substrate binding, and the protein readily crystal-
lizes despite its presence (vide infra). While the possible functions
of LCR inserts are not fully understood, they are thought to be
external, nonglobular domains that do not necessarily affect
protein function. However, examples are known in which LCR
inserts affect enzyme activity and quaternary structure, including
dihydrofolate reductase-thymidylate synthase, subtilisin-like
protease-1, and S-adenosylmethionine decarboxylase/ornithine
decarboxylase of P. falciparum (44-46).
1
1
Crystal Structure. Since the dN-PFA construct was utilized
for the X-ray crystal structure determination, the acronym
“
PFA” refers specifically to this construct in the remainder of
this paper. PFA crystallizes as a loosely associated dimer of
trimers related by a crystallographic 2-fold axis. The observed
quaternary structure is identical to that of the hexameric argi-
nases from Thermus thermophilus and Bacillus caldovelox (47, 48),
except that there is less than 5% buried surface area between the
symmetry-related trimers of PFA. Dynamic light scattering
experiments indicate that PFA is a trimer in solution (data not
shown).
The PFA-ABH and hAI-ABH monomers superimpose with
˚
a rmsd of 0.93 A for 245 CR residues. Significant structural
differences are observed near the two sequence insertions in PFA
(Figure 3a). While the 11-residue insert in loop L8 is well-ordered,
the 74-residue LCR insert in loop L2 is largely disordered: only
flanking residues D70, N71, and C154-N158 are modeled into
visible electron density, and C154 is refined with half-occupancy.
The LCR is distant from the active site and is oriented away from
the trimer interface (Figure 3b). Thus, the LCR does not
significantly impact enzyme structure or function.
The structure of the binuclear manganese cluster in the
PFA-ABH complex is very similar to that observed in the rat
and human arginase I complexes (30, 49). Both metal ions exhibit
distorted octahedral coordination geometry (Figure 4a). The
F
IGURE 2: Isothermal titration calorimetry of the PFA-ABH com-
plex in 50 mM Tris (pH 8.0), 200 mM NaCl, 1 mM TCEP, and 1 mM
MnCl
at 25 ꢀC. Shown are the raw data obtained by titration of
2
5
0 μM arginase with 20 ꢀ 2 μL injections of 650 μM ABH. The area
under each peak is integrated and plotted vs [ABH]/[arginase]. The
solid line represents the best fit of the experimental data using
nonlinear least-squares fitting, indicating a stoichiometry n of
4
-1
1
.27 ( 0.03, an association constant (K
a
) of (8.8 ( 0.9) ꢀ 10 M
(and thus dissociation constant K
5.4 ( 0.2 kcal/mol.
d
= 11 ( 2 μM), and a ΔH of
-
F
IGURE 3: (a) Superposition of the hAI monomer [yellow, PDB entry 2AEB (30)] with the PFA monomer (green), both in complex with the
2
þ
inhibitor 2(S)-amino-6-boronohexanoic acid (ABH). Mn ions are shown as purple spheres, and the inhibitor ABH is colored as follows: C,
yellow (hAI) or black (PFA); O, red; N, blue; B, orange. Structural differences are observed for amino acid inserts in PFA: loop L2 (disordered
LCR, black dashed line) and loop L8 (red). (b) Trimeric PFA-ABH complex. Individual monomers are colored yellow, blue, and green; loops L2
and L8 are indicated as in panel a.

Article
Biochemistry, Vol. 49, No. 26, 2010 5605
F
IGURE 4: (a) Simulated annealing omit map of 2(S)-amino-6-boronohexanoic acid (ABH) contoured at 5σ. The binding mode of ABH is similar
B
to that observed for binding to mammalian arginases (30, 59). Intriguingly, H381 from an adjacent monomer (green) extends from the L8 loop
insertion into the active site of monomer A. Metal coordination interactions are indicated by solid black lines; selected hydrogen bond and van der
Waals interactions are indicated by dashed red and black lines, respectively. (b) Superposition of the PFA-ABH complex (colored as in panel a)
with the hAI-ABH complex (cyan). PFA and hAI residue labels are black and green, respectively.
electron density for the inhibitor ABH indicates that the boronic
acid moiety undergoes nucleophilic attack by the metal bridging
hydroxide ion to yield a tetrahedral boronate anion. This binding
mode mimics the tetrahedral intermediate and its flanking
hydrogen bond interaction with the central arginine residue.
However, in hAI, D204A makes a syn-oriented hydrogen bond
with R255 , whereas in PFA, E295 makes an anti-oriented
B
hydrogen bond with R346B.
transition states in the hydrolysis of
L
-arginine (50).
A second salt link network observed in mammalian arginases
and the arginase from T. thermophilus is not conserved within
PFA. This network consists of D204 , E262 , and R308 in hAI
The molecular recognition of the R-amino and R-carboxylate
groups of ABH is mediated by several direct and water-mediated
interactions with active site residues, most of which are also
observed in the hAI-ABH complex (30) (Figure 4b). Water-
mediated hydrogen bonds are particularly notable. Two water
molecules interact with the R-amino group of ABH. One water
molecule is within hydrogen bonding distance of E277 and the
backbone CdO group of G234, and the other is within hydrogen
bonding distance of the carboxylate side chains of D274 and D272
and the backbone CdO group of D272. This water molecule also
makes a van der Waals contact with H381 (vide infra). Three
water molecules are observed within hydrogen bonding distance
of the R-carboxylate moiety of ABH and mediate interactions
with S227, N231, H233, and H381 from an adjacent monomer.
Several hydrogen-bonded salt links in the bacterial and mam-
malian arginases are known to be important for stabilization of
the trimeric quaternary structure (27, 41, 51, 52), and some of
these salt links are conserved in PFA. Specifically, at the center of
the trimer interface, the hAI salt link network involving D204A,
E256 , and R255 is conserved as E295 , E347 , and R346 ,
A
B
B
and is linked to the conserved salt link network described above
through residue D204 , which accepts hydrogen bonds from
A
both R255B and R308B of an adjacent monomer (Figure 5).
Interestingly, arginase from B. caldovelox does not contain the
corresponding R308 residue, but a free L-arginine molecule is
reported to bind and make interactions similar to those of R308
in the mammalian arginases (47).
The 11-residue insertion in loop L8 influences the structure
and function of PFA. As previously noted in the structure
determination of rat arginase I (53), the C-terminal polypeptide
mediates more than 50% of the intermonomer contact surface
area. The enlarged L8 loop of PFA precedes helix H2 and the
C-terminal polypeptide and augments intermonomer contacts
(Figures 3b and 6). Interestingly, K340 from loop L7 protrudes
A
into a pocket in the adjacent monomer defined by residues from
helices G and H2 and loop L8 (Y345 , D377 , K378 , D383 ,
B
B
B
B
K393 , K396 , and E400 ). Of these residues, only Y345 is
B
B
B
conserved as Y254 in hAI, so it is clear that the complementary
surface area at the subunit interface has evolved to accommodate
the loop L8 insertion. Parenthetically, we note that electron
density for the side chain of K378 was not as well-defined as for
A
B
A
A
B
respectively, in PFA (subscripts A and B indicate adjacent
monomers) (Figure 5). The aspartate f glutamate substitution
results in a conformational change that nonetheless maintains the

5
606 Biochemistry, Vol. 49, No. 26, 2010
Dowling et al.
F
IGURE 6: Unique inter- and intramonomer interactions involving
loop L8 and helices G and H2 of PFA. PFA monomers are colored
green and yellow, and the L8 loop is colored red. The superimposed
hAI structure is colored gray. Red dashed lines represent hydrogen
F
IGURE 5: Arginase trimer containing several salt links at the trimer
˚
interface. The hAI subunit carbon atoms are colored red, blue, and
green; oxygen and nitrogen atoms are colored red and blue, respec-
tively. The PFA trimer subunits are colored similarly, except that
bonds and cyan dashed lines salt link interactions (∼3.5-4 A).
Oxygen and nitrogen atoms are colored red and blue, respectively.
Labels are black for PFA and red for hAI.
carbon atoms are colored gray. The R204
hAI are conserved in PFA as R346 -E347
second salt link cluster in hAI (R308 -E262
PFA by I410 , K353 , E295 , and F292
A
-E256
-E295 salt links. The
B
-D204 ) is replaced in
B B
-D204 saltlinks in
A
B
B
A
A
A
A
B
B
.
other residues in this pocket. However, the main chain atoms of
K378 were built into well-defined electron density, and the side
chain was modeled with a conformation such that the Nζ atom
was located near that of K340.
Another intermonomer interaction is observed for loop L8 of
PFA, in that H381A is within hydrogen bonding distance of
residue D272B in the active site; H381A also makes a solvent-
mediated interaction with the carboxylate group of ABHB
˚
FIGURE 7: P. berghei sporozoite infection shows a dependency on
(
Figures 3 and 4). Notably, H381A is 3.4 A from the Cβ atom
plasmodial arginase expression. Sporozoites from wild-type (WT)
and arg- (12) P. berghei strains were dissected from mosquito salivary
glands and used to infect C57BL/6 mice. Data for WT and arg-
P. bergheisporozoites showdecreased infectivity inthe arg- knockout
(p = 0.004; t = 40 h). Error bars represent the standard deviation
(n = 7).
of the inhibitor molecule ABH , interacting through van der
B
Waals interactions. A water molecule interacts with the R-amino
group of ABH , D272 , and D274 and is also located within
B
B
B
hydrogen bonding distance of H381 . Intermonomer interac-
A
tions with the bound inhibitor are unique to the active site of
PFA. Since H381 can make an intermonomer van der Waals
contact with a bound inhibitor, it can also make a similar
interaction with a bound substrate molecule. We probed the
influence of H381 on enzyme activity by preparing the H381A
variant, which exhibits modestly diminished kinetic parameters
in comparison with those of dN-PFA; the binding affinity of
ABH is also modestly compromised (Table 1). These results
suggest that H381 contributes to substrate binding and catalysis.
Parenthetically, we note that a new intramonomer hydrogen
bond network is observed in PFA involving R404 , E401 , and
Q349A (Figure 6). Although these residues are located near the
trimer interface, none make intermonomer interactions. How-
ever, the R404A mutation results in a partially active mono-
mer (22), suggesting that the R404-E401-Q349 hydrogen bond
network helps maintain the structure of the monomer near the
subunit interface to facilitate trimer assembly.
not compromise the in vivo viability in arginase knockout
parasites; i.e., proliferation in blood-stage infection is not
affected (12). This previous study used inoculations containing
6
1
0 parasitized red blood cells to infect BALB/c mice. Here, we
have compared the infectivity of arg- and wild-type P. berghei by
inoculation of mice using sporozoites dissected from mosquito
salivary glands. As opposed to direct infection with blood-stage
parasites, sporozoites must first infect liver cells to produce
merozoites that invade red blood cells and produce the manifes-
tations of clinical malaria. Using qRT-PCR of P. berghei 18S
rRNA to measure the degree of infectivity, we find a significant
reduction ( p < 0.01) in infectivity in the arg- strain 40 h postin-
fection (Figure 7), suggesting that liver-stage infection is com-
promised by parasite arginase deficiency. Wild-type Plasmodium
sporozoites and developing liver forms express arginase (arg),
A
A
which functionally degrades
L-arginine. We hypothesize that
Altered Infectivity of arg- Parasites. Previous work has
suggested a possible connection between the plasmodial arginase
and immune evasion by the parasite (12). However, disruption of
the parasitic arginase in the rodent malaria model P. berghei does
during infection with wild-type parasites,
L
-arginine levels are
lowered and as such the production of nitric oxide (NO) is likely
reduced. This may allow the parasite to evade an NO-dependent
immune response in the host. Indeed, the apparent reduction in

Article
Biochemistry, Vol. 49, No. 26, 2010 5607
liver-stage infection observed in arg- P. berghei parasites is consis-
tent with this hypothesis, although an increase in the production
of L-ornithine by the parasitic arginase may also be important for
parasite survival. The decreased liver-stage infectivity of arg-
parasites clearly warrants further investigation. Arginase inhi-
bitors, perhaps in synergistic combination with inhibitors of
parasite polyamine biosynthesis (54), may prove to be effective
against liver-stage infection and may possibly serve as prophy-
lactic agents in addition to their potential use as adjuvant
therapies.
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It is well documented that arginase activity can regulate NO
biosynthesis and thereby facilitate immune evasion (18, 55, 56).
The arginase can be that of the host, e.g., in certain cancer tumor
cells (57) or circulating myeloid-derived suppressor cells in cancer
patients (56), or it can be that of the invading pathogen, e.g., H. pylori
arginase (58). In this work, disruption of P. falciparum arginase
compromises the in vivo viability in arginase knockout sporo-
zoites, possibly by weakening the ability of the parasite to modify
the surrounding environment to its benefit and promote survival,
and possibly by exposing the less abundant and metabolically
active liver-stage parasite to NO-mediated killing. Interestingly,
exogenous arginase activity in L. major or H. pylori infection
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cell responses, and arginase inhibitors or supplemental L-arginine
attenuates infection and parasite growth (16-18).
Analysis of P. falciparum arginase may explain decreased
substrate and inhibitor potencies compared with that of human
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trimer interface due to the conserved 11-residue loop L8 inser-
tion, and H381 of this loop extends from one monomer into the
active site of an adjacent monomer in the trimer. The 74-residue
LCR domain is disordered in the crystal structure, and its
deletion does not affect substrate affinity. Thus, the three-
dimensional structure of PFA promises to guide the future design
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ACKNOWLEDGMENT
responses by arginase-induced L-arginine depletion in nonhealing
leishmaniasis. PLoS Negl. Trop. Dis. 3, e480.
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This work is based upon research conducted at the North-
eastern Collaborative Access Team beamlines of the Advanced
Photon Source, which are supported by Grant RR-15301 from
the National Center for Research Resources at the National
Institutes of Health. Use of the Advanced Photon Source is
supported by the U.S. Department of Energy, Office of Basic
Energy Sciences, under Contract DE-AC02-06CH11357. We
thank Dr. Scott Diamond for his assistance with kinetic colori-
metric measurements and Dr. William DeGrado and Emma
Magavern for assistance with the isothermal titration calorimetry
measurements.
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