A dipicolinic acid tag for rigid lanthanide tagging of proteins and paramagnetic NMR spectroscopy

Article abstract of DOI:10.1021/ja803741f

A new lanthanide tag was designed for site-specific labeling of proteins with paramagnetic lanthanide ions. The tag, 4-mercaptomethyl-dipicolinic acid, binds lanthanide ions with nanomolar affinity, is readily attached to proteins via a disulfide bond, an

Full text of DOI:10.1021/ja803741f

Published on Web 07/19/2008
A Dipicolinic Acid Tag for Rigid Lanthanide Tagging of Proteins and
Paramagnetic NMR Spectroscopy
Xun-Cheng Su,† Bradley Man,‡ Sophie Beeren,‡ Haobo Liang,† Shane Simonsen,†
Christophe Schmitz,§ Thomas Huber,§ Barbara A. Messerle,‡ and Gottfried Otting*,†
Research School of Chemistry, Australian National UniVersity, Canberra ACT 0200, Australia, School of Chemistry,
UniVersity of New South Wales, Sydney NSW 2052, Australia, and School of Molecular and Microbial Sciences and
Australian Institute for Bioengineering and Nanotechnology, UniVersity of Queensland,
Brisbane QLD 4072, Australia
Received May 19, 2008; E-mail: gottfried.otting@anu.edu.au
Site-specific labeling of a protein with a lanthanide ion (Ln3+
)
provides access to a wealth of paramagnetic effects that contain
long-range structural information and can be measured by nuclear
magnetic resonance (NMR) spectroscopy.1 In particular, pseudo-
contact shifts (PCS) induced by lanthanides can be used to
determine the structure of protein-protein and protein-ligand
complexes rapidly from a minimum amount of NMR data2 and
the paramagnetically induced weak alignment of the protein in the
magnetic field leads to residual dipolar couplings (RDCs) which
contain detailed information about the structure and dynamics of
proteins.3 Correspondingly, many efforts have been directed toward
the development of tags for site-specific attachment of lanthanides
to proteins, including fusions with lanthanide-binding peptides4 and
chemical derivatization of cysteine residues with synthetic peptides
and lanthanide-chelating reagents.5–10
Figure 1. Superimposition of 15N HSQC spectra of uniformly 15N-labeled
ArgN derivatized with 1 at Cys68 in the presence of a 1:1 mixture of Lu3+
and Yb3+ (black) and in the presence of Lu3+ (red). The ratio of lanthanides
to protein was about 1.5:1. The spectra were recorded at 25 °C and pH 6.5
1
at a H NMR frequency of 800 MHz.
Several features must be considered in the design of suitable
lanthanide tags. (i) Rigidity of the attachment, as PCS and RDCs
are greatly reduced when the lanthanide tag reorientates with respect
to the protein.6,10 In addition, variation in the metal ion position
prevents interpretation of the PCS by a single magnetic susceptibil-
ity anisotropy (∆ꢀ) tensor. (ii) Enantiomeric purity, as metal ions
can act as chiral centers and different enantiomers of the metal-
chelate would lead to diastereomeric protein-tag constructs, there-
fore doubling the number of NMR peaks.5,7,11 (iii) Proximity of
the metal ion to the protein, as accurate determination of the ∆ꢀ
tensor requires adequate sampling of the space around the metal
ion.10 (iv) Ease of use. For example, lanthanide tags that are
anchored to the protein via two disulfide bonds immobilize the tag
well with respect to the protein. This requires, however, two cysteine
residues with thiol groups positioned at the correct distance to react
with a single tag molecule.8 Here we describe a simple lanthanide
tag and strategy for immobilization of a lanthanide close to the
protein using a single cysteine residue.
lanthanide ion by 1 and one or several protein carboxyl groups provides
an avenue for creating high-affinity lanthanide binding sites that
immobilize the lanthanide ion with respect to the protein.
For experimental verification, we derivatized the N-terminal
DNA-binding domain of the Escherichia coli Arginine repressor
(ArgN) with 1 to form ArgN-4MMDPA and recorded 15N HSQC
spectra in the presence of different lanthanide ions (Figure 1). For
each backbone amide, a single cross-peak was observed in the
presence of Lu3+ or any other lanthanide. Yb3+ induced PCS of
up to 2 ppm.
4-Mercaptomethyl-dipicolinic acid (4MMDPA, 1) coordinates metal
ions in a nonchiral fashion and can be readily attached to a cysteine
thiol group via a disulfide bridge using established dithionitrobenzoate
chemistry.9 Lanthanides bind dipicolinic acid (DPA) with nanomolar
affinity for the first DPA ligand and decreasing affinities for additional
DPA ligands up to the [Ln(DPA)3]3- complex.12 A protein derivatized
with one molecule of 1 thus leaves the free coordination sites on any
metal ion bound to 1 available for binding to additional ligands. In
the case of lanthanides, carboxyl groups of the protein are particularly
suitable ligands that can act as additional anchors for tethering the
lanthanide ion to the protein. Simultaneous coordination of the
Lanthanide binding was highly specific, that is, an up to 4-fold
excess of lanthanides did not change the protein NMR spectrum. The
lanthanide binding affinity of ArgN-4MMDPA was on par with that
of DPA, as titration of the ArgN-4MMDPA construct with a 1:1
complex of Lu3+ and DPA resulted in cross-peaks of metal-free and
Lu3+-bound ArgN-4MMDPA of similar intensity (Supporting Infor-
mation). In contrast, titration of ArgN-4MMDPA with [Yb(DPA)2]-
did not yield paramagnetically shifted cross-peaks, showing that ArgN-
4MMDPA cannot extract Yb3+ from the [Yb(DPA)2]- complex. The
ArgN-4MMDPA-Ln3+ complex seems incapable of binding an
additional DPA molecule with significant affinity, as 15N HSQC spectra
of ArgN-4MMDPA prepared with a 1:1 mixture of Lu3+ and Yb3+
yielded the same PCS as corresponding spectra with a 1:1 mixture of
[Lu(DPA)]+ and [Yb(DPA)]+ (Supporting Information).
† Australian National University.
‡ University of New South Wales.
§ University of Queensland.
9
10486 J. AM. CHEM. SOC. 2008, 130, 10486–10487
10.1021/ja803741f CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
the determination of the rhombic components prohibited comparison
(Supporting Information).
The magnitudes of the ∆ꢀ tensors observed for the ArgN-
4MMDPA-Ln3+ complexes are only about half as big as those
reported for proteins and peptides10,16 which may be a consequence
of lanthanide coordination with rapidly exchanging water molecules.
Nonetheless, the 4MMDPA tag offers many attractive advantages,
including its small size and thus reduced likelihood of interference
in studies of intermolecular interactions, its formation of stable
nonchiral 1:1 complexes with metal ions, the capability of im-
mobilizing lanthanides near the protein surface, and its straight-
forward chemical synthesis (Supporting Information).
Figure 2. Ribbon representation of the DNA-binding domain of the E.
coli arginine repressor:13 blue, N-terminus; red, C-terminus; magenta,
position of the lanthanide ion in the ArgN-DPA construct. The Cys68
(yellow) and Glu21 (red) side chains are shown as sticks.
Acknowledgment. Supported by the Australian Research
Council.
For maximal accuracy, PCS and RDCs were measured in samples
containing 1:1 mixtures of diamagnetic (Lu3+) and paramagnetic
(Tb3+, Tm3+, or Yb3+) lanthanides. The possibility of measuring
RDCs from aligned (paramagnetic) and unaligned (diamagnetic)
protein molecules present in the same sample is a unique advantage
over RDC measurements using alignment media. An EXSY
spectrum of a mixture of [Lu(DPA)]+ and [Yb(DPA)]+ complexes
showed that the metals exchange with a rate constant of about 0.1
s-1 at 25 °C, that is, the simultaneous presence of two different
metal ions does not result in noticeable exchange broadening.
The PCS measured for the ArgN-4MMDPA complexes with
Tb3+, Tm3+, and Yb3+ were used to determine the ∆ꢀ tensor
parameters of the respective lanthanides. The program Numbat14
afforded a simultaneous fit of all three tensors to a common
lanthanide position with respect to the protein. Performing the fit
for all 23 NMR conformers of ArgN, the metal position varied by
less than (1.2 Å, being on average within 4.6 Å of the sulfur atom
of Cys68 and within 2.6 Å of one of the carboxyl oxygens of Glu21.
The Ln3+-oxygen distance agrees with Ln3+-oxygen distances in
the literature,15 indicating that Glu21 coordinates the lanthanide
ions. The carboxyl group of Glu21 is the only carboxyl group in
the vicinity of Cys68.
Supporting Information Available: Protocols for the synthesis of
4MMDPA and ligation to proteins; 15N-HSQC spectra of ArgN-
4MMDPA with Lu3+, Tb3+, and Tm3+; plot of amide chemical shift
differences ArgN/ArgN-4MMDPA-Lu3+ versus amino acid sequence;
amide chemical shifts of ArgN-4MMDPA with Lu3+, Tb3+, Tm3+
,
and Yb3+; RDC data; Sanson-Flamsteed plots of the ∆ꢀ and alignment
tensors; correlation plots of experimental and calculated PCS and RDCs.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Pintacuda, G.; John, M.; Su, X.-C.; Otting, G. Acc. Chem. Res. 2007, 40,
206–212.
(2) (a) Pintacuda, G.; Park, A. Y.; Keniry, M. A.; Dixon, N. E.; Otting, G.
J. Am. Chem. Soc. 2006, 128, 3696–3702. (b) John, M.; Pintacuda, G.;
Park, A. Y.; Dixon, N. E.; Otting, G. J. Am. Chem. Soc. 2006, 128, 4997–
5009.
(3) (a) Bax, A.; Grishaev, A. Curr. Opin. Struct. Biol. 2005, 15, 563–570. (b)
Tolman, J. R.; Ruan, K. Chem. ReV. 2006, 106, 1720–1736. (c) Lakomek,
N. A.; Carlomagno, T.; Becker, S.; Griesinger, G. J. Biomol. NMR 2006,
34, 101–115. (d) Bouvignies, G.; Markwick, P. R. L.; Blackledge, M.
Chemphyschem 2007, 8, 1901–1909.
(4) (a) Ma, C.; Opella, S. J. J. Magn. Reson. 2000, 146, 381–384. (b) Wo¨hnert,
J.; Franz, K. J.; Nitz, M.; Imperiali, B.; Schwalbe, H. J. Am. Chem. Soc.
2003, 125, 13338–13339. (c) Martin, L. J.; Ha¨hnke, M. J.; Nitz, M.;
Wo¨hnert, J.; Silvaggi, N. R.; Allen, K. N.; Schwalbe, H.; Imperiali, B.
J. Am. Chem. Soc. 2007, 129, 7106–7113.
(5) Ikegami, T.; Verdier, L.; Sakhaii, P.; Grimme, S.; Pescatore, B.; Saxena,
K.; Fiebig, K. M.; Griesinger, C. J. Biomol. NMR 2004, 29, 339–349.
(6) Rodriguez-Castan˜eda, F.; Haberz, P.; Leonov, A.; Griesinger, C. Magn.
Reson. Chem. 2006, 44, S10–S16.
(7) Prudeˆncio, M.; Rohovec, J.; Peters, J. A.; Tocheva, E.; Boulanger, M. J.;
Murphy, M. E. P.; Hupkes, H. J.; Kosters, W.; Impagliazzo, A.; Ubbink,
M. Chem.sEur. J. 2004, 10, 3252–3260.
To assess the rigidity of lanthanide binding, we compared the
∆ꢀ tensors determined from PCS with the alignment tensors
determined from the RDCs of the backbone amides of the regular
secondary structure elements of the ArgN-4MMDPA complexes
with Tb3+, Tm3+, and Yb3+. In the case of a molecule aligned
with the magnetic field by virtue of its paramagnetism, the
alignment tensor should be directly proportional to the ∆ꢀ tensor
(see also footnotes of Table S3):
(8) (a) Keizers, P. H. J.; Desreux, J. F.; Overhand, M.; Ubbink, M. J. Am.
Chem. Soc. 2007, 129, 9292–9293. (b) Vlasie, M. D.; Comuzzi, C.; van
den Nieuwendijk, A. M. C. H.; Prudeˆncio, M.; Overhand, M.; Ubbink, M.
Chem.sEur. J. 2007, 13, 1715–1723.
B02
(9) Su, X.-C.; Huber, T.; Dixon, N. E.; Otting, G. Chembiochem 2006, 7, 1599–
1604.
Aax,rh
)
∆ꢀax,rh
(1)
15µ0kT
(10) Su, X.-C.; McAndrew, K.; Huber, T.; Otting, G. J. Am. Chem. Soc. 2008,
130, 1681–1687.
(11) Pintacuda, G.; Moshref, A.; Leonchiks, A.; Sharipo, A.; Otting, G. J. Biomol.
NMR 2004, 29, 351–361.
where B0 is the magnetic field strength, µ0 is the induction constant,
k is the Boltzmann constant, and T is the temperature. Flexibly
attached lanthanide ions result in smaller ∆ꢀ and alignment tensors
as well as in discrepancies between the orientations of the tensor
axes.10 In the case of the three paramagnetic ArgN-4MMDPA-
Ln3+ complexes, the orientations of the principal axes of alignment
and ∆ꢀ tensors were in close agreement as expected for rigid
lanthanide coordination. In addition, the axial components of the
tensors closely obeyed equation 1, while the large uncertainties in
(12) Grenthe, I. J. Am. Chem. Soc. 1960, 83, 360–364.
(13) Sunnerhagen, M.; Nilges, M.; Otting, G.; Carey, J. Nat. Struct. Biol. 1997,
4, 819–826.
(14) Schmitz, C.; Stanton-Cook, M. J.; Su, X.-C.; Otting, G.; Huber, T. J. Biomol.
NMR 2008, 41, 179–189.
(15) Parker, D.; Dickins, R. S.; Puschmann, H.; Crossland, C.; Howard, J. A. K.
Chem. ReV. 2002, 102, 1977–2010.
(16) Bertini, I.; Janik, M. B. L.; Lee, Y.-M.; Luchinat, C.; Rosato, A. J. Am.
Chem. Soc. 2001, 123, 4181–4188.
JA803741F
9
J. AM. CHEM. SOC. VOL. 130, NO. 32, 2008 10487