Further insight into the inhibitory action of a LIM/double zinc-finger motif of an agmatinase-like protein

Article abstract of DOI:10.1016/j.jinorgbio.2013.12.006

Agmatine is a precursor for polyamine biosynthesis also associated to neurotransmitter, anticonvulsant, antineurotoxic and antidepressant actions in the brain. It results from decarboxylation of l-arginine by arginine decarboxylase and it is hydrolyzed to urea and putrescine by agmatinase. Recently, we have described a new protein which also hydrolyzes agmatine although its sequence greatly differs from all known agmatinases. This agmatinase-like protein (ALP) contains a LIM-like double Zn-finger domain close to its carboxyl terminus, whose removal results in a truncated variant with a 10-fold increased k_cat, and a 3-fold decreased K_m value for agmatine. Our proposal was that the LIM-domain functions as an autoinhibitory, regulatory entity for ALP. Results in this report provide additional support for the postulated inhibitory effect. The purified isolated LIM domain was shown to be competitively inhibitory to a truncated variant ALP (lacking the LIM-domain), but not to the wild-type species. The C453A variant was shown to be a Zn^{2 +}-free enzyme with kinetic parameters similar to those of the truncated-ALP. A molecular dynamic simulation of a modeled LIM-domain 3D structure showed that, as a consequence of C453A mutation, the coordination of the zinc ion is broken and the structure of the zinc finger is melted. The inhibitory action of the LIM/double Zinc-finger motif was associated to a significant conformational change, as detected by tryptophan fluorescence studies, but was not related to changes in the association of the enzyme with the catalytically essential Mn^{2 +}.

Full text of DOI:10.1016/j.jinorgbio.2013.12.006

JIB-09436; No of Pages 4
Journal of Inorganic Biochemistry xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Further insight into the inhibitory action of a LIM/double zinc-finger motif of an
agmatinase-like protein
Jaime Cofre, Paola Montes, Alejandro Vallejos, José Benítez, David García, José Martínez-Oyanedel,
Nelson Carvajal, Elena Uribe ⁎
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 May 2013
Received in revised form 13 December 2013
Accepted 16 December 2013
Available online xxxx
Agmatine is a precursor for polyamine biosynthesis also associated to neurotransmitter, anticonvulsant,
antineurotoxic and antidepressant actions in the brain. It results from decarboxylation of L-arginine by arginine
decarboxylase and it is hydrolyzed to urea and putrescine by agmatinase. Recently, we have described a new pro-
tein which also hydrolyzes agmatine although its sequence greatly differs from all known agmatinases. This
agmatinase-like protein (ALP) contains a LIM-like double Zn-finger domain close to its carboxyl terminus,
m
whose removal results in a truncated variant with a 10-fold increased kcat, and a 3-fold decreased K value for
Keywords:
agmatine. Our proposal was that the LIM-domain functions as an autoinhibitory, regulatory entity for ALP. Results
in this report provide additional support for the postulated inhibitory effect. The purified isolated LIM domain
was shown to be competitively inhibitory to a truncated variant ALP (lacking the LIM-domain), but not to the
wild-type species. The C453A variant was shown to be a Zn2+-free enzyme with kinetic parameters similar to
those of the truncated-ALP. A molecular dynamic simulation of a modeled LIM-domain 3D structure showed
that, as a consequence of C453A mutation, the coordination of the zinc ion is broken and the structure of the
zinc finger is melted. The inhibitory action of the LIM/double Zinc-finger motif was associated to a significant con-
formational change, as detected by tryptophan fluorescence studies, but was not related to changes in the asso-
Agmatine
LIM-domain
Agmatinase-like protein
ciation of the enzyme with the catalytically essential Mn2+
.
©
2013 Elsevier Inc. All rights reserved.
1
. Introduction
respectively [7]. Moreover, ligands for binding of the metallic cofactor
Mn in bacterial agmatinase and other members of the arginase super-
family are fully conserved in human agmatinase.
2
+
Agmatine, produced by decarboxylation of L-arginine, binds to vari-
ous receptors and exhibits neurotransmitter actions in the brain [1]. At
physiological concentrations, agmatine is inhibitory to the neuronal ni-
tric oxide synthase [2] and participates in modulation of insulin release
from pancreatic cells and renal sodium excretion [3]. There is also evi-
dence for anticonvulsant, antinociceptive, anxiolytic and antidepressant
like actions associated to agmatine [4]. A fine control of agmatine levels
is evidently required, thus justifying the interest in enzymological as-
pects of agmatine synthesis and degradation in mammals.
Recently, we have described a new protein, immunohistochemically
detected in the hypothalamic region and hippocampal astrocytes and
neurons of the rat brain, which also hydrolyze agmatine although its se-
quence greatly differs from all known agmatinases and arginases [8,9].
The predicted amino acid sequence of the rat brain protein, designated
as agmatinase like protein (ALP) resulted to be remarkably different to
the sequences of all cloned agmatinases and the evolutionary related ar-
ginases, including the localization of residues which are strictly con-
served and critical for catalysis by these enzymes [8]. One particularly
interesting finding was a LIM-like domain closes the carboxyl terminus
of the ALP (Fig. 1), whose removal results in a truncated variant with a
Agmatine results from decarboxylation of L-arginine by arginine de-
carboxylase and it is hydrolyzed to urea and putrescine by agmatinase
[5]. The human agmatinase has been cloned but only poorly character-
ized, due to the considerably low activity exhibited by the obtained re-
combinant species. However, the functionality of the cloned human
gene was deduced from a functional complementation test with a
yeast strain which contains a disruption in the gene encoding ornithine
decarboxylase and thus requires exogenous polyamines for growth [6].
The amino acid sequence of the human agmatinase is about 30% and
b20% identical to bacterial agmatinases and mammalian arginases,
m
10-fold increased kcat, and a 3-fold decreased K value for agmatine
[10]. As expected for a LIM protein, zinc was detected only in the
wild-type ALP and amounted to 2 Zn2 /monomer [10].
+
The LIM domain is a zinc-coordinating domain, consisting of two
tandemly repeated zinc fingers, generally involved in cellular differenti-
ation and control of cellular growth, gene expression, interactions with
cytoskeleton, auto-inhibitory effects and possibly as biosensors that me-
diate communication between the cellular and nuclear compartments
[11,12]. The classic LIM consensus sequence includes a CX2CX16–23-
⁎
Corresponding author. Fax: +56 41 2239687.
E-mail address: auribe@udec.cl (E. Uribe).
HX2CX2CX2CX16–21CX2 (CH/D) sequence, associated with a highly
0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2013.12.006
Please cite this article as: J. Cofre, et al., Further insight into the inhibitory action of a LIM/double zinc-finger motif of an agmatinase-like protein, J.
Inorg. Biochem. (2013), http://dx.doi.org/10.1016/j.jinorgbio.2013.12.006

2
J. Cofre et al. / Journal of Inorganic Biochemistry xxx (2013) xxx–xxx
NaOH (pH 9.0) and 2 mM MnCl
the enzyme to the substrate, buffer and MnCl
2
. All the assays were initiated by adding
solution previously equil-
2
ibrated at 37 °C, and urea was determined by a colorimetric method
with α-isonitrosopropiophenone [15], measuring the absorbance at
540 nm.
Initial velocity and inhibition studies were performed in duplicate
and repeated three times. The inhibitory patterns were initially deter-
mined by double reciprocal plots and replots of intercepts versus inhib-
itor concentrations. Kinetic parameters were obtained by fitting
the experimental data to the appropriate Michaelis–Menten equation
i m
(v = VmaxS / K + S) by using nonlinear regression with Graph Pad
Prism version 5.0 for Windows (Graph Pad Software Inc., San Diego,
CA, USA). Protein concentration was determined by means of the stan-
dard Bio-Rad protein assay (Bio-Rad, CA, USA) with bovine serum albu-
min as standard.
2.4. Enzyme–metal interactions
Fig. 1. Proposed schematic representation of a LIM-domain of ALP. Postulated ligands to
the zinc ions are encircled and cysteine 453 is indicated [10].
The manganese and zinc content of ALP and mutant species was de-
termined by total reflection X-ray fluorescence analysis on a S2 Picofox
Bruker spectrometer equipped with an X-ray metal–ceramic tube and a
molybdenum target, working at 50 W of maximum power, at 50 kV and
variable sequence in the remainder of the domain that confers function-
al specificity [13]. Our proposal was that the LIM-domain functions as an
auto inhibitory, regulatory entity for ALP and that inhibition is reversed
by interaction of the domain with some yet undefined brain protein.
Similar results have been reported for the LIM-kinase [14].
1
mA and air-cooled. This technique is based in that when materials are
excited with high-energy, short wavelength radiation (e.g., X-rays),
they become ionized. If the energy of the radiation is sufficient to dis-
lodge a tightly-held inner electron, the atom becomes unstable and an
outer electron replaces the missing inner electron. When this happens,
energy is released due to the decreased binding energy of the inner elec-
tron orbital compared with an outer one. The emitted fluorescent radi-
ation is of lower energy than the primary incident X-rays. Because the
energy of the emitted photon is a characteristic of a transition between
specific electron orbitals in a particular element, the resulting fluores-
cent X-rays can be used to detect the abundances of elements in the
sample [16]. The stoichiometry calculations were based on a previously
determined subunit molecular mass of ALP 58 kDa [8].
We have now examined the interaction of ALP with the catalytically
2
+
2+
required Mn and the LIM-associated Zn , by using mutagenic and
kinetic approaches. The present report provides additional evidence
for the postulated effect of the LIM domain on the agmatinase activity
expressed by the protein ALP. The effect is shown to be strictly related
2
+
to the Zn ions, which are required for proper folding of the LIM do-
main, and not to the interaction of the enzyme with the catalytically es-
2
+
sential Mn
.
2
. Materials and methods
The affinity of Mn2 binding (ALP + Mn
+
2+
↔ ALP − Mn2+),was
evaluated by following the manganese reactivation of fully inactivated
2
.1. Materials
species, obtained by incubation with 50 mM EDTA in 50 mM Tris–HCl
(
pH 7.5) for 30 min at room temperature and then dialyzed for 6–8 h
Agmatine, glycine, Tris, SDS and all other reagents were of the
against ultrapure water. For reactivation, the enzymes were incubated
for 15 min at 37 °C with varying concentrations of Mn2 in 10 mM
Tris–HCl (pH 8.5), 50 m MKCl and 10 mM nitrilotriacetic acid as a
metal ion buffer. Then, agmatinase activities were determined in
highest quality commercially available (most from Sigma Aldrich
Chemical Co. Louis, MO, USA). Restriction enzymes, as well as enzymes
and reagents for PCR, are obtained from Invitrogen Co (Carlsbad, CA,
USA). The synthetic nucleotide primers were obtained from the Fermelo
Biotec Co. (Santiago, Chile).
+
5
0 mM Tris–HCl, pH 8.5. The studies were performed in duplicate and
repeated two times. Dissociation constants (K ) were estimated from
the hyperbolic dependence of agmatinase activity on free-Mn con-
centrations, with nonlinear regression using Graph Pad Prism 5.0. Free
Mn concentrations were calculated using a dissociation constant of
3
d
2
+
2
.2. Enzyme preparations
2
+
All species, including the wild-type, LIM-truncated species, C453A-
−8
.98 × 10
a
M and a pK value of 9.8 for nitrilotriacetic acid [17].
ALP variant and also the isolated LIM-domain, were directionally cloned
into the histidine-tagged pQE60 Escherichia coli expression vector, and
the histidine-tagged proteins were expressed in E. coli strain JM109, fol-
lowing induction with 0.05 mM isopropyl-β-D-thiogalactopyranoside.
All protein variants were purified by ion exchange chromatography on
a DEAE-cellulose column and affinity chromatography on NTA–Ni2+
columns. The purity of all preparations was ~90%.
2
.5. Fluorescence spectra
Fluorescence measurements were made at 25 °C on a Shimadzu RF-
301 spectrofluorimeter. The protein concentration was 40–50 μg/ml
5
and emission spectra were measured with the excitation wavelength
at 295 nm. The slit width for both excitation and emission was
1
buffer solution in the absence of protein. The buffer solution (pH 7.5)
contained 5 mM Tris–HCl and 250 mM KCl. In the fluorescence
quenching experiments, the acrylamide concentrations varied from 0
to 100 mM.
C453A-ALP variant was obtained by using the QuikChange® Site-
.5 nm, and spectra were corrected by subtracting the spectrum of the
Directed Mutagenesis Kit of Stratagene, with the plasmid H
6
pQE60-
2
9,4 containing the ALP cDNA as template. The presence of the desired
mutation and the absence of unwanted changes were confirmed by au-
tomated DNA sequence analysis.
2
.3. Enzyme and protein assays
2.6. Molecular modeling and molecular dynamic simulation
Routinely, agmatinase activities were determined by measuring the
formation of urea (product) from 80 mM agmatine in 50 mM glycine–
A model of the LIM-domain of ALP was generated with
MODELLER [18]. Even though its sequence identity is lowered to
Please cite this article as: J. Cofre, et al., Further insight into the inhibitory action of a LIM/double zinc-finger motif of an agmatinase-like protein, J.
Inorg. Biochem. (2013), http://dx.doi.org/10.1016/j.jinorgbio.2013.12.006

J. Cofre et al. / Journal of Inorganic Biochemistry xxx (2013) xxx–xxx
3
3
0%, the LIM 3 domain of Tes protein (pdb code 2XQN), was used as a
template. The PROCHECK program was used for stereo chemical
analysis of the model [19]. A 1 ns molecular dynamic simulation
for the LIM-domain, from the wild-type and mutant variants, was
performed using GROMACS with Gromos 9643b1 force field [20].
This allowed to relax the structure, improving the interactions in
the core of the protein model. It also allowed to eliminate side
chain steric clashes by changing the energetically incorrect confor-
mation of several amino acids and improving the hydrogen bond
network.
3
. Results and discussion
The present studies aimed to further support the postulated inhibi-
tory action of the LIM-domain on agmatine hydrolysis by ALP. One ini-
tial approach evaluates the isolated domain for effects on truncated,
LIM-free species. As expected, and as shown in Fig. 2, the isolated
Fig. 3. A. Structural model of the LIM-domain of ALP constructed using the MODELLER
2
+
2+
9v10 program. B. Atomic detail of Zn LIM-1 complex. C. Atomic detail of Zn –LIM-2
complex.
domain, cloned and expressed as indicated in the Materials and
methods section, was clearly inhibitory to the truncated species but
not to the wild-type species. The inhibition resulted to be competitive
with respect to the substrate agmatine, and the inhibition constant
(
K
i
) was calculated to be 0.2 μM.
We have previously shown the presence of 2 Zn2+/subunit of wild-
type species of ALP and the absence of the metal ion in the truncated
variant [10]. By using a site-directed mutagenesis approach, the atten-
tion was now addressed to ligands to the metal ion the LIM/double
zinc-finger motif of the ALP molecule. In the absence of crystallographic
information, mutagenic studies were guided by a bioinformatically gen-
erated molecular model for the LIM-domain (Fig. 3). In the present
study, Cys453one of the ligands predicted by the modeled structure,
was mutated to alanine and the metal contents and kinetic properties
of the resulting C453A variant were compared with those of the wild-
type and also with the previously described truncated variant lacking
the LIM-domain [10]. As shown in Table 1, like the truncated LIM-free
species, the C453A variant exhibited a significantly one order of magni-
tude higher kcat value than the wild-type species, without any change in
2
+
the interaction of the enzyme with the catalytically essential Mn . In
fact, using total reflection X-ray fluorescence, purified preparations of
the wild-type, truncated variant and C453A variants were found to
contain 1 Mn2 per subunit. Moreover, the interaction of all exam-
ined species with the activating metal ions was characterized by es-
+
sentially the same K
d
. However, Table 1 also shows that, in contrast
with the wild-type species, the C453A variant was found to devoid of
2
+
bound Zn
.
At first sight, the lack of bound Zn2 in the C453A variant appears as
unexpected for a molecular entity consisting of two tandemly repeated
zinc fingers and only one of the sites being altered by the mutation. One
explanation would be that occupation of the site containing Cys453 is
required for metal binding and proper folding of the second site. On
this basis, disruption of C453-containing site (LIM-1 in Fig. 3) is expect-
ed to eliminate the second LIM-2 site. A sequential folding pathway, like
that suggested by our present results, was also described for other LIM
proteins [21].
+
Table 1
Kinetic properties of ALP, ALP-truncated in LIM domain and C453A.
K
m
(mM)
k
cat (s−1)
Mn2+
Zn2+
K
d
Mn2+ (M)
content
content
Fig. 2. A. Effect of LIM-domain isolated on truncated-ALP activity at different concentra-
−8
ALP
Truncated-ALP
C453A
2.5
1.2
2.7
1
10
14.5
1.18
0.99
1.3
1.8
0
0.06
1.88 × 10
3.56 × 10
2.5 × 10
tions of agmatine. The activities were measured at 37 °C by 15 min with 2 mM MnCl
2
,
−8
−8
50 mM glycine, pH 9.5 and different concentrations of agmatine. B. Lineweaver–Burk
analysis of inhibitory effect of LIM-domain over truncated-ALP activity. The concentrations
of purified LIM-domain were 0–0.6–0.8–1 μM.
The experiment was repeated three times, standard deviations did not exceed 5%.
Please cite this article as: J. Cofre, et al., Further insight into the inhibitory action of a LIM/double zinc-finger motif of an agmatinase-like protein, J.
Inorg. Biochem. (2013), http://dx.doi.org/10.1016/j.jinorgbio.2013.12.006

4
J. Cofre et al. / Journal of Inorganic Biochemistry xxx (2013) xxx–xxx
folded LIM-domain, due to the absence of Zn2+ [22], resulted to be func-
tionally equivalent to deletion of the domain. Both lead to the release of
the auto inhibitory restraints and thereby activation of ALP, with a
marked (~10-fold) increase in kcat. This is schematically represented
in Fig. 4.
Changes induced by mutations in the Zn2+ binding residues, were
also analyzed by tryptophan fluorescence and acrylamide quenching
studies. ALP contains 5 tryptophan residues and none in the LIM do-
main; therefore, any change in fluorescent properties appears as an in-
dicator of structural changes transmitted to the rest of protein.
As shown in Fig. 5, the C453 → Ala mutation was accompanied by a sig-
nificant increase in fluorescence intensity and altered acrylamide
quenching profiles. The hyperbolic quenching curves are indicative of
the presence of tryptophan residues with different exposures to the sol-
vent in the case of mutants. Clearly, therefore, a significant conforma-
tional alteration is induced by the mutagenic replacement of C453.
Similar alterations in the fluorescence properties were previously re-
ported to be produced by deletion of the motif from the ALP molecule
Fig. 4. Schematic representation of the consequences of the C453A mutation, compared
with the removal of the LIM-domain from the ALP molecule.
In agreement with the experimentally determined metal-free state
of the C435A variant, a molecular dynamic simulation of the modeled
LIM-domain 3D structure showed the loss of zinc coordination in the
mutant. The result was an increased distance between the zinc ions
and the metal coordinating residues and the melting of the zinc finger
structure. Interestingly, and as predicted by our proposal, a nonproperly
[10].
In conclusion, the essentially identical consequences of removal of
2
+
the LIM domain or impediment of Zn binding by mutation of a ligand
in the domain, reinforce our conclusion that the Zn2 stabilized finger
structure is the auto inhibitory entity of ALP. Further studies are evi-
dently required for a better understanding of the regulatory conse-
quences of the LIM-domain mediated inhibition of the enzyme. One
interesting and plausible possible role would be in the control of
the physiological levels of the neurotransmitter/neuromodulatory
agmatine in the brain.
+
Acknowledgments
This research was supported by Grant Fondecyt 1120663.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2013.12.006.
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Please cite this article as: J. Cofre, et al., Further insight into the inhibitory action of a LIM/double zinc-finger motif of an agmatinase-like protein, J.
Inorg. Biochem. (2013), http://dx.doi.org/10.1016/j.jinorgbio.2013.12.006