Fungal human pathogens such as Cryptococcus neoformans are becoming an increasingly prevalent cause of human morbidity and mortality owing to the increasing numbers of susceptible individuals. The few antimycotics available to combat these pathogens usually target fungal-specific cell-wall or membrane-related components; however, the number of these targets is limited. In the search for new targets and lead compounds, C. neoformans has been found to be susceptible to mycophenolic acid through its target inosine monophosphate dehydrogenase (IMPDH); in contrast, a rare subtype of the related C. gattii is naturally resistant. Here, the expression, purification, crystallization and preliminary crystallographic analysis of IMPDH complexed with IMP and NAD+ is reported for both of these Cryptococcus species. The crystals of IMPDH from both sources had the symmetry of the tetragonal space group I422 and diffracted to a resolution of 2.5 A for C. neoformans and 2.6 A for C. gattii

Allison

Anna Stamp

Bostjan Kobe

Carl A. Morrow

Chen

Collaborative Computational Project, Number 4

Elion

Escobar-Henriques

Eugene Valkov

Guillen Schlippe

Hedstrom

Hyle

Jackson

James A. Fraser

Kabsch

Kaur

Kohler

McMillan

Park

Prosise

Riera

Shaw

Sintchak

Umejiego

Zhang

Morrow, CA

Stamp, A

Valkov, E

Kobe, B

Fraser, JA

University of Queensland eSpace

crystallization communications
1104 doi:10.1107/S1744309110031659 Acta Cryst. (2010). F66, 1104–1107
Acta Crystallographica Section F
Structural Biology
and Crystallization
Communications
ISSN 1744-3091
Crystallization and preliminary X-ray analysis of
mycophenolic acid-resistant and mycophenolic
acid-sensitive forms of IMP dehydrogenase from the
human fungal pathogen Cryptococcus
Carl A. Morrow,a‡ Anna
Stamp,a‡ Eugene Valkov,a,b
Bostjan Kobea,b and James A.
Frasera*
aCentre for Infectious Disease Research, School
of Chemistry and Molecular Biosciences, The
University of Queensland, Brisbane, QLD 4072,
Australia, and bInstitute for Molecular
Bioscience, The University of Queensland,
Brisbane, QLD 4072, Australia
‡ These authors contributed equally to this
work.
Correspondence e-mail: jafraser@uq.edu.au
Received 29 June 2010
Accepted 6 August 2010
Fungal human pathogens such as Cryptococcus neoformans are becoming an
increasingly prevalent cause of human morbidity and mortality owing to the
increasing numbers of susceptible individuals. The few antimycotics available to
combat these pathogens usually target fungal-specific cell-wall or membrane-
related components; however, the number of these targets is limited. In the
search for new targets and lead compounds, C. neoformans has been found to
be susceptible to mycophenolic acid through its target inosine monophosphate
dehydrogenase (IMPDH); in contrast, a rare subtype of the related C. gattii
is naturally resistant. Here, the expression, purification, crystallization and
preliminary crystallographic analysis of IMPDH complexed with IMP and
NAD+ is reported for both of these Cryptococcus species. The crystals of
IMPDH from both sources had the symmetry of the tetragonal space group I422
and diffracted to a resolution of 2.5 A˚ for C. neoformans and 2.6 A˚ for C. gattii.
1. Introduction
Fungal infections of humans are highly refractive to pharmacological
intervention because of the similarities in our shared eukaryotic
physiology. Emerging drug resistance is further exacerbating this
problem, as is the growing cohort of immunocompromised indivi-
duals susceptible to opportunistic infections. One such opportunist is
Cryptococcus neoformans, a basidiomycete yeast that predominantly
infects immunocompromised patients and causes fungal meningo-
encephalitis (Casadevall & Perfect, 1998). Recent figures from the
US Centers for Disease Control and Prevention estimate an annual
global incidence of cryptococcal meningitis of almost 958 000 cases,
resulting in more than 624 000 deaths per year. The vast majority of
cases involve AIDS patients in sub-Saharan Africa and mirror the
prevalence of HIV in the region (Park et al., 2009). The development
of more effective, readily available and cheaper antifungals is
therefore a matter of urgency.
Rational drug design was pioneered in the purine metabolic
pathway, which has continued to serve as a fertile source of drug
targets and therapeutic agents for over fifty years (Elion, 1989). One
of the key well studied enzymes from this pathway is inosine
monophosphate dehydrogenase (IMPDH), which is the rate-limiting
enzyme and catalyses the first committed step of de novo GTP
biosynthesis: the conversion of inosine monophosphate (IMP) to
xanthosine monophosphate (XMP). The mechanism involves two
reactions: initially, both IMP and the cofactor NAD+ bind and IMP is
oxidized, forming the covalent intermediate E–XMP*, while NAD+ is
reduced to NADH. The E–XMP* complex undergoes a conforma-
tional change and is subsequently hydrolysed to form XMP (Jackson
et al., 1977; Kohler et al., 2005; Guillen Schlippe & Hedstrom, 2005;
Sintchak et al., 1996). XMP is then converted to guanosine mono-
phosphate (GMP) via GMP synthase.
As a rate-limiting central metabolic enzyme, IMPDH is highly
expressed in proliferating cells and has become a therapeutic target
for antimicrobial, antiproliferative and immunosuppressive treat-
ments (Kaur et al., 2005; Allison & Eugui, 2000; Chen & Pankiewicz,
2007). One such therapeutic agent is mycophenolic acid (MPA), an
# 2010 International Union of Crystallography
All rights reserved
uncompetitive inhibitor of IMPDH that acts as an NAD+ analogue
and traps the covalently bound E–XMP* enzyme intermediate.
IMPDH displays a variability in MPA sensitivity across different
species; mammalian enzymes are largely sensitive and in humans
MPA acts as a potent immunosuppressant via inhibition of B and T
lymphocyte proliferation and is commonly prescribed following renal
transplantation. Microorganisms, including bacteria, protozoans and
fungi, range from extremely sensitive to highly resistant (Hedstrom,
2009; Zhang et al., 1999). Treatment with MPA dramatically reduces
the intracellular GTP pool and evokes a range of phenotypes,
including abnormal cell morphology, polarity and cell-cycle pro-
gression, as well as inhibition of translation and G-protein signalling,
in the yeast Saccharomyces cerevisiae (Escobar-Henriques et al., 2001;
Sagot et al., 2005).
MPA inhibits the growth of C. neoformans, yet we have found that
a rare subtype of the closely related C. gattii is naturally resistant.
Kinetic and structural studies have consistently revealed significant
differences between microbial IMP dehydrogenases and their human
counterparts. Subtle changes in the enzyme conformation, structure
of the active site and structure of the cofactor-binding site can lead to
substantial changes in IMPDH kinetic parameters and drug resis-
tance, suggesting that highly species-specific inhibitors that do not
affect a human host could be developed as novel therapeutics
(McMillan et al., 2000; Prosise et al., 2002; Umejiego et al., 2004;
Kohler et al., 2005; Riera et al., 2008).
The available structures of eukaryotic IMPDHs include human
type I and type II IMPDH and those from Cricetulus griseus
(Chinese hamster) and a number of protozoan parasites including
Tritrichomonas foetus and Cryptosporidium parvum. The Crypto-
coccus enzymes (both 544 residues) share 70% identity with human
type I (563 residues) and type II (514 residues) IMPDH, but only
44% identity with the well characterized T. foetus enzyme (503
residues). Cryptococcus IMPDH is slightly longer than most
comparable enzymes owing to a unique insertion in part of the active
site. No IMPDH structures from the fungal kingdom are available,
despite the extensive genetic and biochemical characterization of the
enzymes from the model yeast Saccharomyces cerevisiae and the
human pathogen Candida albicans (Shaw et al., 2001; Hyle et al., 2003;
Kohler et al., 2005).
To investigate the mechanism of resistance and the potential of
Cryptococcus IMPDH as an effective therapeutic target, here we
describe the crystallization of an MPA-sensitive IMPDH from the
clinically prevalent C. neoformans and of a rare naturally occurring
MPA-resistant IMPDH from C. gattii.
2. Materials and methods
2.1. Cloning
Total RNAwas isolated from C. neoformans var. grubii strain H99
and C. gattii strain MMRL2651 using TRIzol (Invitrogen, USA).
Intron-free cDNA was subsequently synthesized using the Bioline
cDNA Synthesis Kit (Bioline, UK) and the IMPDH-encoding IMD1
gene from both species was PCR-amplified using primers designed
to introduce novel restriction sites. The resulting amplicons were
digested and cloned in-frame into the pQE-30 expression vector
(Qiagen, Netherlands) to introduce an N-terminal 6His tag
(MRGSHHHHHHGS).
The C. neoformans strain H99 IMPDH nucleotide sequence (locus
designation CNAG_00441) is available from the Broad Institute un-
published genome (http://www.broadinstitute.org/annotation/genome/
cryptococcus_neoformans/MultiHome.html), while the C. gattii strain
MMRL2651 IMPDH sequence is available in GenBank (FJ418781).
2.2. Expression and purification
IMD1 plasmids were cotransformed with the lac repressor vector
pREP4 into Escherichia coli strain BL21 (DE3) pLysS (Novagen,
Japan). The transformed cells were grown in LB medium at 310 K
with 100 mg ml1 ampicillin and 35 mg ml1 kanamycin to an OD600 of
1.0 and induced with 1 mM IPTG. The cells were subsequently
grown for 5 h at 293 K before harvesting. The pellets were resus-
pended in lysis buffer (50 mM HEPES pH 8.0, 1M KCl, 30 mM
imidazole, 1 mM benzamidine–HCl and 1 mM PMSF) prior to
disruption by sonication. The expressed protein was purified via Ni-
immobilized metal-affinity chromatography using 5 ml HisTrap
columns (GE Healthcare, USA). The supernatant was loaded onto
the columns and eluted in a linear gradient of 30–500 mM imidazole.
Fractions were analysed via SDS–PAGE; the peak fractions were
concentrated and buffer-exchanged into gel-filtration buffer (50 mM
Tris–HCl pH 8.0, 150 mM KCl, 2 mM EDTA, 2 mM DTT). Both
proteins were loaded onto Superdex 200 size-exclusion columns and
eluted at a rate of 2.5 ml min1 using an A¨KTA FPLC system (GE
Healthcare). The peak fractions were again identified via SDS–
PAGE, pooled and concentrated to 5 mg ml1 before buffer-
exchange into crystallization buffer (50 mM Tris–HCl pH 8.0, 50 mM
KCl, 2 mM EDTA, 1 mM DTT).
2.3. Crystallization
The optimal protein concentration for crystallization was 5 mg ml1
as determined using the Hampton PCT screen (Hampton Research,
USA). For the initial screening, only the IMPDH inhibitor MPA
(dissolved in dimethylsulfoxide) was added to a concentration of
100 mM prior to crystallization. Crystallization screening for both
proteins was performed using hanging-drop vapour diffusion at 277 K
and commercial sparse-matrix screens (Qiagen; Hampton Research;
Emerald Biosciences, USA). The plates were set up using a Mosquito
Nanodrop crystallization robot (TTP LabTech, UK) by combining
200 nl protein solution and 200 nl reservoir solution and inverting the
drops over 100 ml reservoir solution. The drops were monitored and
imaged using the Rock Imager system (Formulatrix, USA). An initial
crystal cluster was observed for C. gattii IMPDH with reservoir
solution containing 1.8M lithium sulfate, 6.8%(v/v) 2-methyl-2,4-
pentanediol and 0.09M imidazole–HCl pH 6.5. The crystals were
further optimized using factorial grid screening around this condition
using sitting-drop vapour diffusion, varying the protein and salt
concentration and the pH. In addition, we used an Additive Screen
kit (Hampton Research). 1 ml protein solution was mixed with 1 ml
reservoir solution and equilibrated against 100 ml reservoir solution.
Small crystals obtained in 1.6–1.9M lithium sulfate pH 6.4–6.6 were
further optimized by using larger drop sizes and microseeding.
For the final round of crystallization, the IMPDH proteins from
C. neoformans and C. gattii were mixed with a tenfold molar excess
of the substrate IMP and the cofactor NAD+. Large crystals were
obtained using wells containing 1.9M lithium sulfate and 0.09M
imidazole–HCl pH 6.5 plus either 3.0–12.0%(v/v) 2-methyl-2,4-
pentanediol, 3.0–12.0% pentaerythritol ethoxylate (15/04 EO/OH) or
no additive.
2.4. Data collection and processing
Crystals were mounted in nylon loops and immersed in the cryo-
protectant Paratone-N (Hampton Research) before flash-cooling in
crystallization communications
Acta Cryst. (2010). F66, 1104–1107 Morrow et al.  IMP dehydrogenase 1105
liquid nitrogen. Diffraction data were collected from cryocooled
single crystals on the MX2 undulator beamline of the Australian
Synchrotron (Clayton, Australia). Reflections were indexed and
integrated using the program XDS (Kabsch, 2010) and then scaled
using SCALA as implemented within the CCP4 suite (Collaborative
Computational Project, Number 4, 1994).
3. Results and discussion
Both C. neoformans and C. gattii IMPDH were expressed hetero-
logously in soluble form in E. coli after 5 h induction at 293 K. Both
proteins appeared as a single band of approximately 58 kDa on
reducing SDS–PAGE gels, matching the estimated size of His-tagged
IMPDH monomers, and their purity was estimated to be >99%.
Following initial screening, three further rounds of optimization
led to large diffraction-quality crystals in 1.9M lithium sulfate and
0.09M imidazole–HCl pH 6.5 plus either 3–12% 2-methyl-2,4-
pentanediol, 3–12% pentaerythritol ethoxylate (15/04 EO/OH) or no
further additive. Crystals of both proteins (Fig. 1) appeared after 3 d
at 277 K.
The crystals of IMPDH from the two species visually had different
morphologies, with the largest IMPDH crystals growing to maximum
dimensions of 100  150  200 mm. However, the crystals of
C. neoformans and C. gattii IMPDH both had the body-centred
tetragonal symmetry of space group I422. The unit-cell parameters
for theC. neoformans andC. gattii IMPDH crystals were a = b = 148.8,
c = 106.4 A˚ and a = b = 148.4, c= 106.2 A˚, respectively. The resolution
of the best diffracting crystals was 2.5 A˚ for C. neoformans IMPDH
and 2.6 A˚ for C. gattii IMPDH. Data-collection and refinement
statistics are summarized in Table 1.
Current work is focused on refining the structure of IMPDH for
both species and obtaining data sets for alternate ligand-bound forms,
including inhibitor-boundMPA-resistant andMPA-sensitive IMPDH.
The authors would like to acknowledge the UQ ROCX facility
(The University of Queensland) and the Australian Synchrotron. This
work was supported by grants from the National Health and Medical
Research Council (NHMRC) and the Australian Research Council
(ARC). CAM is supported by an ANZ Trustees PhD Scholarship in
Medical Research. JAF is the recipient of an NHMRC Career
Development Award. BK is an ARC Federation Fellow and an
NHMRC Honorary Research Fellow.
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1106 Morrow et al.  IMP dehydrogenase Acta Cryst. (2010). F66, 1104–1107
Figure 1
Crystals of cryptococcal IMP dehydrogenases in complex with IMP and NAD+,
space group I422. (a) C. neoformans IMP dehydrogenase, (b) C. gattii IMP
dehydrogenase.
Table 1
Data-collection and refinement statistics.
Values in parentheses are for the outer shell.
C. neoformans IMPDH C. gattii IMPDH
No. of crystals 1 1
Beamline Australian Synchrotron
MX2
Australian Synchrotron
MX2
X-ray wavelength (A˚) 0.95 0.95
Detector ADSC Quantum 315r ADSC Quantum 315r
Crystal-to-detector distance (mm) 350 320
Rotation range per image () 1 1
Total rotation range () 180 180
Exposure time per image (s) 2 2
Data-collection temperature (K) 100 100
Space group I422 I422
Unit-cell parameters (A˚, ) a = b = 148.8, c = 106.4,
 =  =  = 90
a = b = 148.8, c = 106.2,
 =  =  = 90
Resolution range (A˚) 20–2.5 (2.5–2.42) 20–2.6 (2.74–2.6)
Unique reflections 20908 18484
Total observations 305209 491039
hI/(I)i 23.6 (4.9) 22.5 (6.1)
Rmerge† (%) 11.7 (78.1) 13.5 (79.6)
Rmeas = Rr.i.m.‡ (%) 12.1 (80.9) 13.8 (81.3)
Wilson B factor (A˚2) 46.6 52.7
Completeness (%) 99.8 (100) 99.8 (100)
Mosaicity () 0.40 0.41
Multiplicity 14.6 26.6
Monomer molecular weight (kDa) 57.9 57.9
† Rmerge =
P
hkl
P
i jIiðhklÞ  hIðhklÞij=
P
hkl
P
i IiðhklÞ, where Ii(hkl) is the intensity of
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mean intensity of that reflection. Calculated for I > 3(I). ‡ Rmeas = Rr.i.m. =P
hkl ½N=ðN  1Þ1=2
P
i jIiðhklÞ  hIðhklÞij=
P
hkl
P
i IiðhklÞ, where Ii(hkl) is the intensity
of the ith individual measurement of reflection hkl and hI(hkl)i is the weighted average
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crystallization communications
Acta Cryst. (2010). F66, 1104–1107 Morrow et al.  IMP dehydrogenase 1107


Crystallization and preliminary X-ray analysis of mycophenolic acid-resistant and mycophenolic acid-sensitive forms of IMP dehydrogenase from the human fungal pathogen Cryptococcus

Fungal human pathogens such asCryptococcus neoformansare becoming an increasingly prevalent cause of human morbidity and mortality owing to the increasing numbers of susceptible individuals. The few antimycotics available to combat these pathogens usually target fungal-specific cell-wall or membrane-related components; however, the number of these targets is limited. In the search for new targets and lead compounds,C. neoformanshas been found to be susceptible to mycophenolic acid through its target inosine monophosphate dehydrogenase (IMPDH); in contrast, a rare subtype of the relatedC. gattiiis naturally resistant. Here, the expression, purification, crystallization and preliminary crystallographic analysis of IMPDH complexed with IMP and NAD+is reported for both of theseCryptococcusspecies. The crystals of IMPDH from both sources had the symmetry of the tetragonal space groupI422 and diffracted to a resolution of 2.5 Å forC. neoformansand 2.6 Å forC. gattii.</jats:p

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Acta Crystallographica Section F Structural Biology and Crystallization Communications

Crystallization and preliminary X-ray analysis of mycophenolic acid-resistant and mycophenolic acid-sensitive forms of IMP dehydrogenase from the human fungal pathogen<i>Cryptococcus</i>

http://espace.library.uq.edu.au/view/UQ:217184/UQ217184_OA.pdf

Crystallization and preliminary X-ray analysis of mycophenolic acid-resistant and mycophenolic acid-sensitive forms of IMP dehydrogenase from the human fungal pathogen Cryptococcus

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