Allosteric regulation accompanied by oligomeric state changes of Trypanosoma brucei GMP reductase through cystathionine-β-synthase domain

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Allosteric regulation accompanied by
oligomeric state changes of Trypanosoma brucei
GMP reductase through cystathionine-β
-synthase domain
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Imamura Akira, Okada Tetsuya, Mase Hikaru,
Otani Takuya, Kobayashi Tomoka, Tamura
Manatsu, Kubata Bruno Kilunga, Inoue Katsuaki,
Rambo Robert P, Uchiyama Susumu, Ishii
Kentaro, Nishimura Shigenori, Inui Takashi
Nature Communications
11
Article number: 1837
1-10
2020-04-15
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http://hdl.handle.net/10466/00017469
doi: https://doi.org/10.1038/s41467-020-15611-3

ARTICLE
https://doi.org/10.1038/s41467-020-15611-3

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Allosteric regulation accompanied by oligomeric
state changes of Trypanosoma brucei GMP
reductase through cystathionine-β-synthase
domain
Akira Imamura1,7, Tetsuya Okada 1,7, Hikaru Mase1, Takuya Otani1, Tomoka Kobayashi 1, Manatsu Tamura1,
Bruno Kilunga Kubata2, Katsuaki Inoue 3, Robert P. Rambo 3, Susumu Uchiyama 4,5, Kentaro Ishii 5,6,
Shigenori Nishimura 1 & Takashi Inui 1 ✉

Guanosine 5′-monophosphate reductase (GMPR) is involved in the purine salvage pathway
and is conserved throughout evolution. Nonetheless, the GMPR of Trypanosoma brucei
(TbGMPR) includes a unique structure known as the cystathionine-β-synthase (CBS) domain,
though the role of this domain is not fully understood. Here, we show that guanine and
adenine nucleotides exert positive and negative effects, respectively, on TbGMPR activity by
binding allosterically to the CBS domain. The present structural analyses revealed that
TbGMPR forms an octamer that shows a transition between relaxed and twisted conformations in the absence and presence of guanine nucleotides, respectively, whereas the
TbGMPR octamer dissociates into two tetramers when ATP is available instead of guanine
nucleotides. These findings demonstrate that the CBS domain plays a key role in the allosteric
regulation of TbGMPR by facilitating the transition of its oligomeric state depending on ligand
nucleotide availability.

1 Department

of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai,
Osaka 599-8531, Japan. 2 AU/NEPAD Agency Regional Office for Eastern and Central Africa, Nairobi, Kenya. 3 Diamond Light Source, Harwell Science and
Innovation Campus, Didcot, Oxfordshire OX11 0DE, UK. 4 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka,
Suita, Osaka 565-0871, Japan. 5 Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama,
Myodaiji-cho, Okazaki 444-8787, Japan. 6Present address: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka,
Suita, Osaka 565-0871, Japan. 7These authors contributed equally: Akira Imamura, Tetsuya Okada. ✉email: inuit@bioinfo.osakafu-u.ac.jp
NATURE COMMUNICATIONS | (2020)11:1837 | https://doi.org/10.1038/s41467-020-15611-3 | www.nature.com/naturecommunications

1

ARTICLE

NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15611-3

T

he appropriate regulation of intracellular concentrations of
purine nucleotides is critical for the health of cells, and
most cells have the mechanism of either synthesizing de
novo or salvaging purine nucleotides to maintain their intracellular concentrations; nevertheless, this is not the case for most
parasitic protozoa. Trypanosoma brucei is one such parasite that
causes African trypanosomiasis by infecting to both human and
animals, and depends solely on salvaging the purines produced by
the host animals; interconversion between adenine and guanine
nucleotides is indispensable for this parasite1–4.
We recently have characterized the enzymes involved in purine
nucleotide salvage in T. brucei: guanosine 5ʹ-monophosphate
reductase (GMPR) and inosine 5ʹ-monophosphate dehydrogenase
(IMPDH)5,6. The former catalyzes the conversion of guanosine 5ʹmonophosphate (GMP) to inosine 5ʹ-monophosphate (IMP),
whereas the latter utilizes IMP to produce xanthosine 5ʹmonophosphate. Our previous studies have demonstrated that a
purine nucleotide analog, ribavirin 5ʹ-monophosphate, acts as an
inhibitor for both T. brucei GMPR (TbGMPR) and IMPDH
(TbIMPDH), but also shows an anti-trypanosomal effect in culture
when provided in the nucleoside form, ribavirin5,6. In general, both
GMPRs and IMPDHs have strong similarities in amino acid
sequence, and their catalytic domains share the common structure of
a (β/α)8 barrel, also referred to as a TIM barrel7; nevertheless, these
two enzymes are still distinctive each other by the presence or
absence of an additional domain8. This additional domain is known
as a cystathionine-β-synthase (CBS) or Bateman domain that consists of a tandem repeat of α-β-β-α folds, and is found in IMPDHs of
all organisms reported to date. The CBS domain of IMPDHs has
been shown to participate in the regulation of its activity and conformation in response to the concentrations of purine nucleotides8.
Despite of these previous findings, we and others have recently
revealed that GMPRs of trypanosomatids, including Trypanosoma
and Leishmania species, uniquely possesses a CBS domain that is
absent from the GMPRs of other species6,9, although the structure of
a GMPR harboring a CBS domain still remains undetermined.
These observations prompted us to investigate the structure and
reaction mechanism of TbGMPR.
In the present study, we investigated the biochemical and
structural effects of adenine and guanine nucleotides on TbGMPR.
We found that the binding of adenine and guanine nucleotides to
the CBS domain have opposing effect on the allosteric regulation
of TbGMPR activity. A combination of X-ray crystallography and
size-exclusion chromatography small-angle X-ray scattering (SECSAXS) analysis clearly revealed that TbGMPR can exist as a tetramer or an octamer depending on the nucleotide species that is
bound to the CBS domain. Our findings suggest that the change in
the oligomeric state of TbGMPR is responsible for allosteric regulation by nucleotide binding to the CBS domain.
Results
Opposite modulation of TbGMPR activity by purine nucleotides. Enzymes harboring a CBS domain usually change their
activities in the presence of purine nucleotides10–12; therefore, we
sought to examine whether purine nucleotides modify the activity
of TbGMPR. In the presence of GTP, the initial velocity of
TbGMPR was upregulated in a concentration-dependent manner
(Fig. 1a), and the EC50 value was estimated to be 4.8 µM. In
contrast, TbGMPR exhibited a decrease in its initial velocity in
the presence of ATP with an EC50 value of 160 µM (Fig. 1b). The
effect of each triphosphate nucleotide was maintained when
added as a magnesium complex, i.e. Mg-GTP or Mg-ATP
(Fig. 1a, b). These results indicate that TbGMPR is positively and
negatively regulated by GTP and ATP, respectively, with and
without magnesium ions. Kinetic analysis demonstrated that the
2

initial reaction velocity of TbGMPR without ligand nucleotides
showed a sigmoidal curve when plotted against the concentrations of GMP, and the plots were well-fitted to the Hill equation
(Fig. 1c, open circles). The kinetic parameters K0.5 and kcat values
for GMP were determined as 184 ± 3 µM and 16.7 ± 0.15 min−1,
respectively (mean ± s.d.; Table 1). The Hill constant (nHill) was
calculated to be 3.04 ± 0.12, meaning that GMP induced a positive
cooperativity effect on TbGMPR. A similar sigmoidal curve was
observed using recombinant TbGMPR prepared via an affinity
purification with glutathione-S-transferase (GST) tag and subsequent tag-removal (Supplementary Fig. 1). These results indicate that GMP induces a positive cooperative effect on TbGMPR,
both with and without a terminal tag.
The addition of GTP to the reaction mixture enhanced
TbGMPR activity by decreasing the K0.5 accompanied with the
increase of the kcat value in a concentration-dependent manner
(Table 1, Fig. 1c and Supplementary Fig. 2). The nHill value in the
presence of 1 mM GTP was 1.00 ± 0.34. In contrast, ATP showed
an inhibitory effect on TbGMPR activity by lowering the
substrate–enzyme affinity as indicated by the K0.5 value of 1200
± 12 µM, while the kcat and nHill values were relatively unchanged
compared to those obtained in the absence of ligands (Table 1,
Fig. 1c and Supplementary Fig. 2). The increase in the K0.5 value
was observed in an ATP concentration-dependent manner
(Supplementary Fig. 2a). The opposed effects of GTP and ATP
on TbGMPR activity were clearly observed when the values of
catalytic efficiency (kcat/K0.5) were plotted against each ligand
concentration. Although the kcat/K0.5 value of TbGMPR in the
absence of the ligands was determined as 0.913 × 105 M−1 min−1,
it was increased by the addition of 10 µM GTP, and finally
showed 4.36 × 105 M−1 min−1 with 1 mM GTP (Fig. 1d and
Table 1). In contrary, the kcat/K0.5 value was decreased by the
addition of ATP and reached to 0.111 × 105 M−1 min−1 with
1 mM ATP (Fig. 1d and Table 1). ...

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