Pyrimidine-specific ribonucleoside hydrolase from the
archaeon Sulfolobus solfataricus – biochemical
characterization and homology modeling
Marina Porcelli
1,2
, Luigi Concilio
1
, Iolanda Peluso
1
, Anna Marabotti
3
, Angelo Facchiano
3
and
Giovanna Cacciapuoti
1
1 Dipartimento di Biochimica e Biofisica ‘F. Cedrangolo’, Seconda Universita
`
di Napoli, Italy
2 Consorzio Interuniversitario Biostrutture e Biosistemi ‘INBB’, Rome, Italy
3 Istituto di Scienze dell’Alimentazione del CNR, Avellino, Italy
Nucleoside hydrolases (NHs; EC 3.2.2.–) catalyze the
irreversible hydrolysis of the N-glycosidic bond of
b-ribonucleosides, forming ribose and the free purine
or pyrimidine base [1–3]. All characterized members
are metalloproteins with a unique central b-sheet
topology and a cluster of aspartate residues
(DXDXXXDD motif) at the N-terminus of the
enzyme [2–5].
In nature, a widespread distribution of NHs in dif-
ferent protozoa [6–11], bacteria [12–14], yeasts [15–17],

whose 3D structures have been solved indicates that the amino acid resi-
dues involved in the calcium- and ribose-binding sites are preserved.
SsCU-NH is highly thermophilic with an optimum temperature of 100 °C
and is characterized by extreme thermodynamic stability (T
m
= 106 °C)
and kinetic stability (100% residual activity after 1 h incubation at 90 °C).
Limited proteolysis indicated that the only proteolytic cleavage site is local-
ized in the C-terminal region and that the C-terminal peptide is necessary
for the integrity of the active site. The structure of the enzyme determined
by homology modeling provides insight into the proteolytic analyses as well
as into mechanisms of thermal stability. This is the first nucleoside hydro-
lase from Archaea.
Abbreviations
Cf, Crithidia fasciculata; CU-NH, pyrimidine-specific ribonucleoside hydrolases; Ec, Escherichia coli; IAG-NH, purine-specific inosine-
adenosine-guanosine nucleoside hydrolases; IG-NH, 6-oxo-purine-specific inosine-guanosine nucleoside hydrolases; IPTG, isopropyl thio-b-
D-galactoside; IU-NH, purine-nonspecific inosine-uridine nucleoside hydrolases; Lm, Leishmania major; MTA, 5¢-methylthioadenosine; MTAP,
5¢-methylthioadenosine phosphorylase; MTAPII, 5¢-methylthioadenosine phosphorylase II; MTI, methylthioinosine; NH, nucleoside hydrolase;
NP, nucleoside phosphorylase; PNP, purine nucleoside phosphorylase; PVDF, poly(vinylidene fluoride); Ss, Sulfolobus solfataricus;
Tv, Trypanosoma vivax.
1900 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS
protozoa [6–11]. In these organisms, the nucleoside sal-
vage pathway is vital because, in contrast to most
other living organisms, they lack a de novo biosynthetic
pathway for purines [6–11,22]. All protozoa therefore
utilize salvage enzymes such as NHs and phospho-
ribosyltransferases to form nucleotides [22,23]. Because
neither NH activity, nor the encoding genes have ever
been detected in mammals, the parasitic NHs have
been studied extensively in recent years as attractive

To elucidate the mechanisms by which hyperthermo-
philic enzymes acquire their unusual thermostability
and to increase our knowledge on the structure of
NHs, we carried out the expression, purification and
physicochemical characterization of a NH from S. sol-
fataricus, (SsCU-NH), aiming to elucidate the struc-
ture ⁄ function ⁄ stability relationships in this enzyme and
to explore its biotechnological applications. A detailed
kinetic investigation was also performed to define
the substrate specificity of SsCU-NH and to study
the functional role played by this enzyme in
the purine ⁄ pyrimidine nucleoside metabolism. Finally,
the 3D structure of the enzyme was constructed by
homology modeling using the crystal structure of
Escherichia coli pyrimidine-specific NHs Yeik [29] and
Ybek [30] as templates. The structure provided insight
into the active site architecture of SsCU-NH as well as
into the features of the protein that may contribute to
its thermostability. This is the first example of a NH
reported in Archaea.
Results and Discussion
Analysis of SsCU-NH gene and primary structure
comparison
The analysis of the complete sequenced genome of
S. solfataricus revealed an ORF (SSO0505) encoding a
311 amino acid protein homologous to a NH, which is
annotated as iunH-1. The putative molecular mass of
the protein predicted from the gene was 35.21 kDa
and the estimated isoelectric point was 5.17.
The coding region starts with an ATG triplet at the

cant sequence identity with pyrimidine-specific NHs
from E. coli YeiK (34%) and YbeK (30%).
Figure 1 shows the multiple sequence alignment
of SsCU-NH with homologous enzymes whose 3D
M. Porcelli et al. CU-NH from S. solfataricus
FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS 1901
structures have been solved, such as the purine-non-
specific NHs from Crithidia fasciculata (CfIU-NH)
[26] and Leishmania major (LmIU-NH) [9], the
pyrimidine-specific NHs from E. coli such as YeiK
(Ec-YeiK) [29] and YbeK (Ec-YbeK) [30], and with
the purine-specific NH from Trypanosoma vivax
(TvIAG-NH) [11].
The analysis of the sequence alignment shows that
the amino acid residues involved in the calcium-bind-
ing site and in the ribose binding site of these enzymes
are well conserved in SsCU-NH. Figure 1 also com-
pares the nucleoside base specificity in the active sites
of the TvIAG-NH and Ec-YeiK. In this regard, it
should be noted that TvIAG-NH binds the purine ring
Fig. 1. Multiple sequence alignment of SsCU-NH, CfIU-NH, LmIU-NH, Ec-YeiK, Ec-YbeK and TvIAG-NH. The calcium ( ) ribose (d) and base
(+) binding sites of Ec-YeiK are indicated above the alignment. The residues at the active site of TvIAG-NH are indicated below the sequence
with the same symbols. Identical and conserved residues are highlighted in dark and pale gray respectively. DXDXXXDD motif is shown in
white lettering on a black background. Numbers on the right are the coordinates of each protein.
CU-NH from S. solfataricus M. Porcelli et al.
1902 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS
with N12, D40, W83 and W260, whereas the base-
binding pocket of Ec-YeiK is composed of N80, I81,
H82, F159, F165, T223, Q227, Y231 and H239. From
the comparison, it appears that SsCU-NH maintains

found to be of 170 nmol of uridine cleavedÆmin
)1
Æmg
)1
at 80 °C, confirming that the SsCU-NH gene had been
cloned and expressed.
Direct evidence that this putative NH is present
in S. solfataricus comes from experimental results
obtained measuring the nucleoside hydrolase activity
of the crude extract after extensive dialysis against
10 mm Tris ⁄ HCl (pH 7.4) to make the cell homogenate
phosphate-free and to assure that the degradation of
nucleoside substrate cannot be ascribed to NP activity.
The results obtained indicate that NH activity of
S. solfataricus cells is approximately 10 nmol of uri-
dine cleavedÆmin
)1
Æmg
)1
at 80 °C.
Recombinant SsCU-NH was easily purified to
homogeneity by a fast and efficient two-step procedure
that utilizes a heat treatment and affinity chromatogra-
phy on 5¢-methylthioinosine (MTI)-sepharose. Approx-
imately 2 mg of the recombinant enzyme with a 20%
yield was obtained from 1 L of culture (data not
shown). No processing occurred at the amino terminus
of the enzyme in the E. coli system, as demonstrated
by sequence determination of the first ten amino acids
of SsCU-NH.

is required in maintaining the active
site structure.
Substrate specificity and kinetic characterization
With the aim of gaining insight on the physiological
role of SsCU-NH, we carried out a detailed kinetic
characterization of this enzyme. The enzymatic charac-
terization defines SsCU-NH as a pyrimidine-specific
NH. This enzyme was completely inactive towards
adenosine and guanosine. SsCU-NH, in analogy with
Ec-Yeik enzyme, is specific for uridine and cytidine
and is unable to hydrolyze the deoxyribonucleosides
such as thymidine and deoxycytidine. This evidence
confirms a common characteristic for all NHs that
bind the 2¢-hydroxyl of the ribose ring with specific
hydrogen bonds by the conserved Asp residues in the
active site. In addition, SsCU-NH is not active with
nucleoside 5¢-phosphates as substrate and the catalytic
efficiency towards inosine is at least 100-fold below
that for uridine.
Initial velocity studies carried out with increasing
concentrations of pyrimidine nucleosides gave typical
Michaelis–Menten kinetics. The recombinant enzyme
shows Michaelis constants for uridine and cytidine of
the same order of magnitude, within the experimental
errors, with K
m
values of 310 lm and 970 lm respec-
M. Porcelli et al. CU-NH from S. solfataricus
FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS 1903
tively. Moreover, as shown in Table 1, the relative effi-

characterized by the hexameric quaternary structure
distinctive of bacterial PNP, exhibits catalytic proper-
ties reminiscent with human MTAP, recognizing only
6-aminopurine nucleoside as substrates and showing
an extremely high affinity for 5¢-methylthioadenosine
(MTA).
Homology-based database searches in the complete
genomic sequence of S. solfataricus revealed the pres-
ence of an additional putative NP gene (SSO1519).
To accomplish detailed structural and functional
studies on this enzyme and to verify its substrate
specificity, we carried out the expression of the pro-
tein in E. coli. The catalytic activity of recombinant
enzyme was assayed utilizing purine and pyrimidine
ribonucleosides or deoxyribonucleosides as substrate
of the phosphorolytic reaction. By contrast to our
expectations, no NP activity was detectable with all
nucleosides tested, even when modifying the assay
conditions in different ways. Therefore, we think that
the annotation of this gene as putative NP is not
correct. On the basis of the obtained results, SsCU-
NH is the only known enzyme physiologically
involved in the pyrimidine nucleoside catabolism in
this archaeon.
Thermal properties and limited proteolysis
The temperature dependence of the activity of SsCU-
NH in the range 40–130 °C is shown in Fig. 2. The
enzyme is highly thermoactive; its activity increased
sharply up to the optimal temperature of 100 °C and a
50% activity was still observed at 110 °C. This behav-

⁄ K
m app
(s
)1
ÆM
)1
)
Uridine 310 ± 20 7.1 ± 0.2 (22.9 ± 0.8) · 10
3
Cytidine 970 ± 50 39.4 ± 1.2 (40.6 ± 0.8) · 10
3
Fig. 2. The effect of temperature on SsCU-NH activity. The activity
observed at 100 °C is expressed as 100%. The assay was per-
formed as indicated in the Experimental procedures. Arrhenius plot
is reported in the inset; T, temperature (°K).
CU-NH from S. solfataricus M. Porcelli et al.
1904 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS
To explore the correlation between the resistance to
proteolysis and the conformational protein stability
and to obtain information about the flexible regions of
SsCU-NH exposed to the solvent and susceptible to
proteolytic attack, we subjected the enzyme to limited
proteolysis. The application of limited proteolysis can
often provide useful information about conformational
changes resulting in protection of the cleavage sites or
uncovering new sites [44–46]. SsCU-NH was com-
pletely resistant to trypsin, whereas proteinase K, sub-
tilisin and thermolysin were able to cleave the enzyme.
Therefore, proteolytic degradation of SsCU-NH was
investigated by measuring the residual activity after

SsCU-NH. The optimal alignment between SsCU-NH
and its structural templates was obtained by extracting
the sequences of the target and the templates from a
global alignment with 30 sequences belonging to the
NH family. The type and position of the predicted sec-
ondary structures, with few exceptions, are superim-
posed on those present in the templates, supporting
the correctness of the final alignment that was used to
create the structure of the monomeric SsCU-NH (data
not shown).
Among the ten models obtained using the two ver-
sions of the program modeller, we chose the best one
both in terms of stereochemical parameters (91.1%
of the amino acids in the most favored regions of
the Ramachandran plot) and ProsaII z-score
(z-score = ) 10.30, analogous to that of the template,
which is equal to )10.84). Experimental evidence con-
firms that SsCU-NH is a tetramer. Therefore, we
assembled its oligomeric form using the 3D structure
of YeiK enzyme as template.
The superposition of the tetrameric model with its
template YeiK shows an RMSD of 0.53 A
˚
, indicating
that no major differences are present between target
and template in terms of global architecture (Fig. 4A).
Each subunit of SsCU-NH is made of a central b-sheet
composed of seven parallel and one antiparallel
Fig. 3. Thermostability of SsCU-NH. (A) Residual SsCU-NH activity after 10 min of incubation at the temperatures shown. Apparent T
m

binding and adaptation of this portion of SsCU-NH
to the active site of the protease could be facilitated by
the concerted motion of these two segments. Neverthe-
less, because we were unable to isolate the proteolytic
fragment of 10.6 kDa, which was completely digested
by the proteases, we cannot exclude the possibility that
a first proteolytic cleavage could occur in loop 275–
288, which is a flexible and exposed loop protruding
towards the exterior of each monomer and, subse-
quently, the digestion was prolonged until segment
228–238.
Residues involved in Ca
2+
-coordination and in
substrate binding are shown in Fig. 5A. Residues D9,
D14, I121 and D238 participate in Ca
2+
coordina-
tion. These residues, with the exception of I121,
which coordinates the ion with the oxygen of its main
chain, are strictly conserved in the NH family
(Fig. 1), and are almost perfectly superimposed on
the structures of SsCU-NH and of the templates
(Fig. 5B). Residues D13, N37, N156, E162 and N164,
and again D238, are able to form hydrogen bonds
with the oxygen moieties of the sugar. Furthermore,
these residues are strictly conserved in the NH family
as well as H79, which is near the oxygen O1¢ and is
considered to be one of the catalytic residues of the
protein [29,30]. Other neighboring residues of ribose

H6H5
H4
H3
S7
S8
S10
S11
S9
S6
S5
S4
S1
S2
S3
N-ter
Fig. 4. 3D structure of SsCU-NH. (A) Tetrameric assembly of SsCU-NH (cyan) compared to the template YeiK (yellow). The Ca ions in the
active site of YeiK are represented as orange spheres. Capital letters indicate the monomers. (B) Structure of the monomer. Helices are rep-
resented as red cylinders and b-strands as yellow arrows. Secondary structures are labelled with a progressive number, from N- to C-end.
The arrow indicates the putative site of cleavage by proteases (see text).
CU-NH from S. solfataricus M. Porcelli et al.
1906 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS
O2 atoms in the hydrolysis of uridine [29,30]. How-
ever, mutagenic studies showed that H239A mutant of
YeiK has an increased K
m
but an unchanged k
cat
with
respect to the wild-type enzyme, therefore suggesting a
role for H239 in substrate binding but not in direct

H79
D14
D238
D13
H236
I121
P237
N164
E162
N156
Q229
Ca
Ribose
F163
V78
N37
D9
H79
D14
D238
D13
H236
I121
P237
N164
E162
N156
Q229
Ca
Ribose

18 25
Helix stability contributions (kcalÆmol
)1
)
c
22.3 31.5
Volume of cavities in monomer (A
˚
3
)
d
Buried 56 483
Surface 1220 1014
Total 1276 1497
Volume of cavities in tetramer (A
˚
3
)
d
Buried 867 2985
Surface 5672 3489
Total 6539 6474
Ile + Leu residues at monomers interface
A ⁄ B and C ⁄ D 1 Ile + 5 Leu 1 Ile
A ⁄ C and B ⁄ D 5 Ile + 1 Leu 2 Leu
a
Calculated using the program DSSP.
b
Calculated according to Fac-
chiano et al. [48].

terms of presence of cavities in the interior of the pro-
tein. Using a probe of 0.5 A
˚
, we were able to calculate
the volume of buried and surface cavities for SsCU-
NH and the template, both in the monomer and in the
tetramer, and we found that the volume of buried cavi-
ties found in SsCU-NH is significantly lower than that
of YeiK (Table 2). This could be due to the higher
number of bulky residues (especially Trp, Tyr, Ile),
which are also generally shielded or partially shielded
from the solvent and therefore create a high compact
core in the structure of SsCU-NH. However, this result
should be interpreted with caution because it has been
reported that the estimation of packing density and
cavity volumes in homology models is intrinsically
noisy and may be inaccurate for the possible incorrect
modeling of nonconserved side chains between tem-
plate and target. This effect is dependent also on tem-
plate-target sequence identity [47].
A previous study [48] analyzed different factors that
concur together to stabilize helices in proteins. In the
present study, we applied the same analysis to our
model and to the template. The results obtained are
summarized in Table 2. Among the helix stabilizing
factors evaluated, the most significant one is the lower
content of b-branched residues in the helices of the
thermophilic protein (18% versus 25%). Indeed,
b-branched residues are known to destabilize helices.
Moreover, the evaluation of energetic contribution to

CU-NH from S. solfataricus M. Porcelli et al.
1908 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS
Japan). Pfu DNA polymerase was purchased from Strata-
gene (La Jolla, CA, USA). Thermolysin was obtained from
Boehringer (Mannheim, Germany). Trypsin, proteinase K,
subtilisin, nucleosides, purine and pyrimidine bases,
O-bromoacetyl-N-hydroxysuccinimide and standard pro-
teins used in molecular mass studies were obtained from
Sigma (St Louis, MO, USA). IPTG was from Applichem
(Darmstadt, Germany). Sephacryl S-200 and AH-Sepharose
4B were obtained from Amersham Pharmacia Biotech (Pis-
cataway, NJ, USA); poly(vinylidene fluoride) (PVDF)
membranes (0.45 mm pore size) were obtained from Milli-
pore (Bedford, MA, USA). All reagents were of the purest
commercial grade.
Enzyme assay
Nucleoside hydrolase activity was determined following the
formation of purine ⁄ pyrimidine base from the correspond-
ing nucleoside by HPLC using a Beckman system Gold
apparatus (Beckman Coulter Inc., Fullerton, CA, USA).
Unless otherwise stated, the standard incubation mixture
contained: 10 mmol Tris ⁄ HCl buffer (pH 7.4), 200 nmol of
the nucleoside and the enzyme in a final volume of 200 lL.
The incubation was performed in sealed glass vials for
5 min at 80 °C, except where indicated otherwise. The vials
were rapidly cooled in ice, and the reaction was stopped by
the addition of 100 lL of 10% trichloroacetic acid. Control
experiments in the absence of the enzyme were performed
to correct for nucleoside hydrolysis. When the assays were
carried out at temperatures above 80 °C, the reaction mix-

parameters were determined from Lineweaver–Burk plots
of initial velocity data. K
m
and V
max
values were obtained
from linear regression analysis of data fitted to the Michael-
is–Menten equation. Values given are the mean ± SE from
at least three experiments. The k
cat
value was calculated by
dividing V
max
by the total enzyme concentration. Calcula-
tions of k
cat
were based on an enzyme molecular mass of
140 kDa.
Analytical methods for protein
Protein concentration was determined by the Bradford
method [49] using BSA as standard. Protein eluting from
the columns during purification was monitored at A
280
.
The concentration of purified SsCU-NH was estimated
spectrophotometrically using e
280
= 57870 m
)1
Æcm

acids, was approximately 95%.
Stability and thermostability studies
The stability of SsCU-NH activity was examined at the
indicated temperatures. Immediately after the addition of
the compound, (time-zero control) and at different time
intervals, aliquots were removed from each sample and
analyzed for activity in the standard assay. Activity val-
ues are expressed as a percentage of the zero-time control
M. Porcelli et al. CU-NH from S. solfataricus
FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS 1909
(100%). Enzyme thermostability was tested by incubating
the protein in sealed glass vials at temperatures in the
range 90–110 °C in an oil bath. Samples (2 lg) were
taken at time intervals and residual activity was deter-
mined by the standard assay at 80 °C. Activity values are
expressed as a percentage of the zero-time control
(100%).
Cell culture and homogenate preparation
Sulfolobus solfataricus was isolated from an acidic hot
spring in Agnano (Naples, Italy) and grown at 87 °Cas
previously reported [51]. For the homogenate preparation,
10 g of S. solfataricus cell paste was disrupted by sonication
in 10 mm Tris ⁄ HCl (pH 7.4). After centrifugation for 1 h
at 25 000 g, the supernatant was extensively dialyzed
against the same buffer.
Cloning and expression of the SsCU-NH-encoding
gene
The putative SsCU-NH gene SSO0505 (GenBankÔ acces-
sion number AE006681.1) from S. solfataricus was cloned
into the pET-22b(+) expression vector via two engineered

600
of 3.0), IPTG was added
at 1 mm final concentration and the induction was pro-
longed for 16 h. Cells (10 g) were harvested by centrifu-
gation and lysed as described by Sambrook et al. [52].
The cell debris was removed by centrifugation at 20 000 g
for 60 min at 4 °C and the supernatant was used as a
cell extract.
Preparation of MTI-Sepharose
The preparation of MTI-Sepharose was performed as
described by Kim et al. [53] by treating AH-Sepharose 4B
(5 g) with 1 mmol of O-bromoacetyl-N-hydroxysuccinimide
and then coupling the resin with 10 mg of MTI. MTI was
prepared by enzymatic deamination of MTA utilizing non-
specific adenosine deaminase (adenosine aminohydrolase;
EC 3.5.4.4) purified from Aspergillus oryzae. MTA
(30 lmol) was incubated with adenosine deaminase at
37 °C for 16 h in Tris ⁄ HCl 0.1 m (pH 7.4) as previously
described [54] and the reaction was stopped by the addition
of 10% tricarboxylic acid. The formation of MTI was
checked by reverse-phase HPLC on a 4.6 · 250 mm Ultra-
sphere ODS (5 l m particle size) column (Beckman) using
an Agilent 1100 series cromatograph (Agilent Technologies,
Palo Alto, CA, USA). The column, was equilibrated and
eluted with a 20 : 80 (v ⁄ v) mixture of 95% methanol
and 0.1% trifluoroacetic acid in H
2
O at a flow rate of
1mLÆmin
)1

ride (final concentration 250 lm) and the samples were
assayed for SsCU-NH activity. Proteolytic inactivation of
SsCU-NH with thermolysin was carried out in the same
CU-NH from S. solfataricus M. Porcelli et al.
1910 FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS
substrate ⁄ protease final mass ratio in a solution of 20 mm
Tris ⁄ HCl (pH 7.4), containing 10 mm CaCl
2
at 60 °C.
At the desired time intervals, aliquots were removed,
the reaction was stopped by the addition of 5 mm final
concentration EDTA and samples were assayed for activ-
ity.
The degradation of the intact protein over 2 h of incuba-
tion was monitored by SDS ⁄ PAGE of the digested material
followed by staining with Coomassie blue R-250. For the
amino acid sequence analysis, samples of the digested
recombinant SsNH from SDS ⁄ PAGE analysis were electro-
phoretically blotted onto a PVDF membrane utilizing a
Bio-Rad Mini trans-blot transfer cell apparatus. Trans-
ferred proteins were stained with Coomassie blue (0.1% in
50% methanol) for 5 min, destained in 50% methanol and
10% acetic acid solution for 30 min at room temperature,
and allowed to air dry. Stained protein bands were excised
from the blot and their NH
2
-terminal sequences were
determined.
Multiple sequence alignment
Protein similarity searches were performed using the data

USA), and version 8.2 for Windows (available at: http://
salilab.org/modeller/about_modeller.html) was used to
model the monomeric structure of SsCU-NH. Five models
were created using each version of the program, setting
the highest level of optimization. The quality of the mod-
els and their stereochemical properties were analyzed using
the programs procheck [65] and prosaii [66], and the
best monomer, created by modeller, version 6.1, was
chosen for the creation of the tetrameric form of SsCU-
NH enzyme. The monomeric chains were superimposed
on the structure of YeiK, to maintain the same relative
orientation of the subunits, with the aid of insightii tools.
A mild optimization of the tetrameric structure was then
applied to reduce steric clashes, using 500 steps of the
Steepest Descent method, with a final gradient of
0.01 kcalÆmol
)1
. All atoms were allowed to relax with no
constraints. This procedure represents the best compromise
between the need for relieving steric clashes and the risk
of distorting the geometry of the protein with deep and
extensive minimization. This concept was previously
applied to other models of oligomeric proteins and com-
plexes [67].
The control of the final quality of all models was per-
formed again with procheck [65]. After the assembly and
the subsequent mild energy minimization applied for
reduction of steric clashes [67], the quality of the model in
terms of stereochemical parameters was slightly worse
(86.1% of residues in most favored regions of the Rama-

).
Consistent valence force-field developed for insightii was
adopted to assign potentials and charges both to the pro-
tein and the ligand. Hydrogen bonds were calculated with
the tool Measure ⁄ H-bonds provided in insightii.
M. Porcelli et al. CU-NH from S. solfataricus
FEBS Journal 275 (2008) 1900–1914 ª 2008 The Authors Journal compilation ª 2008 FEBS 1911
Acknowledgements
The authors thank Dr Susan Costantini, Laboratory
of Bioinformatics, ISA, Avellino, for providing a tool
for the calculation of the percentage of secondary
structures from a dssp file. This research was sup-
ported by a grant from ‘Regione Campania’ L.R.
n.5 ⁄ 2002 and was partially supported by the CNR-Bio-
informatics project.
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