Biomol NMR Assign
DOI 10.1007/s12104-013-9468-4
ARTICLE
1
H, 15N and 13C assignments of a putative peptidyl prolyl cis–trans
isomerase FKBP12 from Trypanosoma brucei
Rodolpho do Aido-Machado • Didier Salmon •
José R. Pires
Received: 9 November 2012 / Accepted: 18 January 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract TbFKBP12 is a putative peptidyl prolyl cis–
trans isomerase from Trypanosoma brucei, causative agent
of the African trypanosomiasis or sleeping sickness. It
interacts with the immunosuppressive drug rapamycin
inhibiting the formation of TORC2 complex leading to
parasite death by inhibiting cell proliferation through
cytokinesis blockade. Moreover, RNAi silencing of
TbFKBP12 revealed essential function in both procyclic
and bloodstream forms. Both facts make TbFKBP12 an
attractive target for ligand development and thus structural
data is desirable. In this work we report the NMR reso-
nance assignments for 1H, 15N and 13C nuclei in the
backbone and side chains of the TbFKBP12 as basis for
further studies of structure, backbone dynamics, interaction
mapping and drug screening.
Keywords FKBP12
NMR assignments

Trypanosoma brucei
Neglected diseases
Biological context
FK506 Binding Proteins (FKBPs) belong to the super
family of peptidyl prolyl cis–trans isomerases (PPIase).
They are conserved from bacteria to mammals and play a
crucial role in protein folding (some of them function as
chaperones). As the name suggests, they are able to bind
immunosuppressive drugs as the two macrolactones,
FK506 and rapamycin, which inhibits their PPIase activity.
R. do Aido-Machado
D. Salmon
J. R. Pires (&)
Instituto de Bioquı́mica Médica, CCS, Universidade Federal do
Rio de Janeiro, Av. Brigadeiro Trompowiski s/n, Rio de Janeiro,
RJ 21941-590, Brazil
e-mail: jrmpires@cnrmn.bioqmed.ufrj.br
In mammals, this immunosuppressive activity is unrelated
to their PPIase activity but is due to the formation of a
complex that binds either the phosphatidylinositol kinase
mTOR (rapamycin-FKBP) or the protein phosphatase
calcineurin (FK506-FKBP12) leading to the inhibition of
the downstream signaling cascades (Galat 2003).
The information obtained over the last 15 years reveals
that FKBP are involved in diverse biological processes
affecting the function and structure of target proteins, the
organism development and several signal transduction
pathways. Some of them might play a role in parasite viru-
lence (as TcMIP in Trypanosoma cruzi) and could therefore
be the target for anti-parasitic drugs (Moro et al. 1995).
In the parasite Trypanosoma brucei an archetypal
FKBP12 was identified (TbFKBP12) which is a cytoskel-
eton-associated protein localizing in the flagellar pocket
area of the bloodstream forms. Inhibition of TbFKBP12
expression by RNA interference revealed that TbFKBP12
is essential for parasite survival, affecting cytokinesis and
cytoskeleton architecture in bloodstream form and motility
in procyclic form (Brasseur et al. 2012). Interestingly,
whereas in mammals rapamycin was found to selectively
inhibit TOR complex 1 signaling, upon rapamycin binding
TbFKBP12 selectively binds TOR2 preventing TORC2
formation which inhibits polarized cell growth (Barquilla
et al. 2008). Both studies suggest TbFKBP12 as an
attractive target for development of ligands analogue to
rapamycin and thus structural data is necessary.
Therefore, we decided to perform NMR spectroscopy
studies on TbFKBP12 for generating chemical shift
assignments that will be useful for ligand screening pur-
poses as well as study of its structure and backbone
dynamics. In order to achieve these tasks, we have
expressed, isolated and assigned a 115-residue construct of
TbFKBP12.
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Methods and experiments program SPARKY (v.3.113) (T. D. Goddard and D. G.

Kneller, University of California, San Francisco).
Cloning, expression and purification of TbFKBP12

A full-length TbFKBP12 PCR fragment amplified from Extent of assignment and data deposition
wild-type T. brucei genomic DNA digested by BamHI/
EcoRI was cloned into BamHI/EcoRI digested pGEX-4T2 All non-proline, backbone amide 15N and 1H resonances
vector (Amersham Biosciences). The insert fused to GST at were assigned (except G1, S2, H3, M4, S92 and G97) as
the C-terminus was verified by sequencing (Genome were NH2 groups of asparagines and glutamines and
15
Express, France). TbFKBP12 was co-expressed as a GST Ne1/1He1 atoms of W67 side chains. The assigned 15N
fusion protein in E.Coli BL21 (DE3). Cells were grown at HSQC spectrum of TbFKBP12 is shown in Fig. 1. Almost
37 °C in LB medium containing 100 lg/mL of ampicillin. complete, around 90 % of 13Ca, 13CO, side chain aliphatic
Expression was induced by addition of 0.4 mM IPTG and and aromatic 13C and 1H resonances were assigned. Some
cells were grown for an additional 15 h at 17 °C before resonances deviated more than two standard deviations
centrifugation for 10 min at 6000 rpm at 4 °C, washed upfield from the expected values due to ring current effects
with buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM from nearby aromatic residues (e.g. G36 HA2 d =
EDTA, 1 mM PMSF) pH7.0, and lysed by sonication. This 1.98 ppm; V30 HG2 d = -0.17 ppm; V63 HG2 d =
lysate was then ultracentrifuged for 1 h at 18,000 rpm at 0.31 ppm; I99 HD1 d = 0.05 ppm). The deviations in Ha,
4 °C. GST-TbFKBP12 was then purified by affinity chro-
matography on a glutathione Sepharose column GSTrap
(GE Healthcare) and eluted with 10 mM reduced gluta-
thione in buffer (50 mM Tris–HCl, 150 mM NaCl) pH7.0.
The GST moiety was cleaved with thrombin (30 nM) and
then purified by size-exclusion chromatography on a
HiPrep 26/60 Sephacryl S-100 column (GE Healthcare) to
yield TbFKBP12 with an additional N-terminal glycine,
serine and histidine residues coming from the thrombin-
cleavable GST tag. Uniformly 15N and 13C, 15N-labeled
TbFKBP12 were grown in E. Coli BL21 (DE3) cells in M9
minimal medium containing 1.0 g/L of 15NH4Cl and 3.0
g/L [13C6] glucose, respectively, as the sole nitrogen and
carbon sources, and purified as described above.

NMR spectroscopy

All NMR experiments for 1H, 13C and 15N chemical-shift

assignments and structure determination (Kay 1995; Sattler
et al. 1999) were acquired at 298 K on TbFKBP12 dissolved
in 20 mM phosphate buffer (pH 7.0) 150 mM NaCl
and 100 mM 2-mercaptoethanol (except in the unlabeled
sample). The 2D-NOESY and total correlation spectroscopy
(TOCSY) spectra were acquired in 90 % H2O/10 %
D2O using a 0.8 mM unlabelled TbFKBP12 sample.
A 0.4 mM 15N-labelled sample in 90 % H2O (10 % D2O)
was used for 15N-HSQC, 3D 15N-edited NOESY and
TOCSY experiments. A 0.8 mM 13C, 15N labelled sample
was used to acquire 3D CBCA(CO)NNH, CBCANNH, Fig. 1 [15N, 1H]-HSQC spectrum of a 0.4 mM sample of TbFKBP12
HBHA(CO)NNH, HCCH COSY and TOCSY, 13C-edited in 20 mM phosphate buffer, 150 mM NaCl, 100 mM 2-mercap-
NOESY, and best-HNCO experiments. Spectra were toethanol at pH 7.0, 90 % H2O (v/v) 10 % D2O (v/v), at 298 K,
recorded at 800 MHz 1H frequency. The resonance assignments for
acquired in a Bruker AVIII800 spectrometer. Data were all backbone amides 15N–1H are shown, except for residues G1–M4,
processed using TopSpin (v.2.1) (Bruker BioSpin GmbH, S92 and G97. All side chain NH2 resonances of Asn and Gln residues
Germany). Assignment was carried out using the interactive could be assigned and are connected by horizontal lines

123
1 15 13
H, N and C assignments
Fig. 2 Secondary structure analysis based on TbFKBP12 assignment
data. Deviations of Ha chemical shifts of TbFKBP12 from corre-
sponding random coil values (upper panel) and Consensus chemical
shift index from Ha, Ca, Cb and CO data (lower panel). Secondary
Ca, Cb and CO chemical shifts from the corresponding
random coil chemical shifts were used to generate chemical
shift indexes (Wishart and Sykes 1994) and identify regular
secondary structure (Fig. 2), which was found to be similar
to the observed for other FKBP-type proteins deposited in
BMRB and pdb databanks, e.g. entries BMRB 15038,
39.4 % sequence identity to TbFKBP12 (Kang et al. 2007),
BMRB 16925, 51.5 % sequence identity to TbFKBP12
(Sapienza et al. 2011). Our data suggest that TbFKBP12
adopts a typical FBKP fold. The assignments were
deposited in the BioMagResBank (http://www.bmrb.wisc.
edu/) under the BMRB accession number 18810.
Acknowledgments Financial support was obtained from CNPq,
CAPES and FAPERJ (Brazilian funding agencies).
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