ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
SL12. N-terminal, Histidine-containing Metal Binding Sites in Proteins:
Lessons from Model Studies
T. Gajda a , A. Jancsó a , A. Kolozsi a , A. Battistoni b , Z. Paksi a
a Dept. of Inorganic and Analytical Chemistry, University of Szeged, Hungary,
b Dept. of Biology, University of Rome Tor Vergata, Roma, Italy
e-mail: gajda@chem.u-szeged.hu
Recent biochemical studies pointed out the presence of relatively short, histidine-containing sequences with
strong metal binding ability, being independent from the other parts of the biomolecule, in a great number of
proteins and enzymes. According to the various biological studies, the metal ion coordination to these sites
determines or substantially contributes to the function of the given protein/enzyme, i.e. they have catalytic,
structural or some alternative functions, promoting the biological role of these biomolecules. The N-terminal
fragments of proteins often have no stable preorganized structures, therefore their metal binding and functional
properties can be assessed by studying oligopeptides with identical sequences. Here we report three examples
related to the above mentioned three different functions of N-terminal fragments.
During the last decade several nickel-containing SOD enzymes (Ni-SOD) have been isolated from aerobic soil
bacteria, with no apparent similarity to other SODs. Their crystal structure [1] indicates, that the metal ions are
bound to the N-terminal six amino acids in both the oxidized and reduced enzyme, thus the well conserved Nterminal
sequence 1 HCDXPC– (X=G or L) provides practically all critical interactions for metal ion binding and
catalysis. This offers a unique possibility for structural and functional modelling of these enzymes, since the
metal binding sites in metalloenzymes are generally well separated in the amino acid sequences, therefore the
structure of the active centres is almost impossible to mimic by small peptides. The solution chemical study of
the nickel(II)–HCDLPCG-NH2 (L 1 ) system indicated the formation of the square planar NiH–1L 1 species above
pH 6, with {NH2, N – , S – , S – } donor set, being identical with the active site of the reduced enzyme. This species
possesses high SOD-like activity, in contrast to the majority of nickel(II) complexes.
Cu, Zn-SOD enzymes of some Gram-negative bacteria are characterized by a His-rich N-terminal extension with
strong metal binding ability. The N-terminal sequence of the Cu, Zn-SOD from H. ducreyi has been suggested to
play a chaperoning role during the uptake of active-site copper, promoting the bacterial survival [2]. We have
undertaken equilibrium and solution structural study in the copper(II)- and zinc(II)- NH2-HGDHMHNHDTK-
OH (L 2 ) systems, in order to support this assumption and to investigate the possibility of similar function in zinc
uptake [3]. L 2 possesses extraordinary metal ion sequestering capacity in the neutral pH, provided only by side
chain donors, which approaches to the metal-trafficking proteins. The picomolar affinity for copper(II) supports
the proposed chaperoning role of the N-terminal His-rich region, while the nanomolar zinc(II) binding affinity
may suggest similar role in the zinc(II) uptake, too. Interestingly, the complex CuHL 2 has significant SOD-like
activity, suggesting multifunctional role of the N-terminal His-rich domain.
Endostatin, a fragment of collagen XVIII, is a special inhibitor of endothelial cell proliferation and migration,
and possesses marked anticancer properties without any side effects. Recently it was shown that the anticancer
activity of the N-terminal 25 amino acids and the protein itself are equivalent [4], which is of crucial importance
for future therapeutic usage. The presence of zinc(II), which is likely to have structural role, is necessary to exert
the anticancer effect in both cases, but details on the metal ion interaction of the N-terminal fragment is not
known. Here we report solution chemical investigation of the zinc(II) and copper(II) complexes of
HSHRDFQPVLHL–NH2 (L 3 ) peptide, which is identical with the first twelve amino acids of human endostatine,
and contains all possible metal binding sites of the N-terminal fragment. In presence of zinc(II) the ZnL complex
is formed in the neutral pH range with {NH2, 3Nim} coordination, creating huge macrochelates. Due to the
presence of an ATCUN motif, L 3 also binds copper(II) very efficiently. This finding may have biological
importance, since copper(II) is also deeply involved in angiogenesis.
The presentation will discuss some properties of the metallopeptides which uncovered some subtle details on the
functioning of the corresponding metalloproteins.
Acknowledgement: This work was supported by the Hungarian Scientific Research Found (NI61786, K63606).
References:
[1] J. Wuerges, J.-W. Lee, Y.-I. Yim, H.-S. Yim, K.D. Carugo, Proc. Natl. Acad. Sci. USA, 101, 8569 (2004).
[2] P. D'Angelo, F. Pacello, G. Mancini, O.Proux, J.L. Hazemann, A. Desideri, A. Battistoni, Biochemistry, 44,
13144 (2005).
[3] Z. Paksi, A. Jancsó, F. Pacello, N. Nagy, A. Battistoni, T. Gajda, J. Inorg. Biochem., in press (2008).
[4] R. M. Tjin Tham Sjin, R. Satchi-Fainaro, A. E. Birsner, V.M. S. Ramanujam, J. Folkman, K. Javaherian,
Cancer Res., 65, 3656-3663 (2005).
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