Báo cáo khoa học: Solution structure and stability of the full-length excisionase from bacteriophage HK022 pot

Solution structure and stability of the full-length excisionase
from bacteriophage HK022
Vladimir V. Rogov
1,2
, Christian Lu¨cke
3
, Lucia Muresanu
1
, Hans Wienk
1,
*, Ioana Kleinhaus
1
, Karla Werner
1
,
Frank Lo¨hr
1
, Primoz
ˇ
Pristovs
ˇ
ek
4
and Heinz Ru¨ terjans
1
1
Institute of Biophysical Chemistry, J.W. Goethe-University of Frankfurt, Germany;
2
Institute of Protein Research, Pushchino, Russia;
3
Max Planck Research Unit for Enzymology of Protein Folding, Halle, Germany;

4
National Institute of Chemistry,
Ljubljana, Slovenia
Heteronuclear high-resolution NMR spectroscopy was
employed to determine the solution structure of the excisi-
onase protein (Xis) from the k-like bacteriophage HK022
and to study its sequence-specific DNA interaction. As wild-
type Xis was previously characterized as a generally unstable
protein, a biologically active HK022 Xis mutant with a single
amino acid substitution Cys28 fi Ser was used in this work.
This substitution has been shown to diminish the irreversi-
bility of Xis denaturation and subsequent degradation, but
does not affect the structural or thermodynamic properties
of the protein, as evidenced by NMR and differential scan-
ning calorimetry. The solution structure of HK022 Xis
forms a compact, highly ordered protein core with two well-
defined a-helices (residues 5–11 and 18–27) and five
b-strands (residues 2–4, 30–31, 35–36, 41–44 and 48–49).
These data correlate well with
1
H
2
O-
2
H
2
O exchange
experiments and imply a different organization of the
HK022 Xis secondary structure elements in comparison
with the previously determined structure of the bacterio-

phage k excisionase. Superposition of both Xis structures
indicates a better correspondence of the full-length HK022
Xis to the typical Ôwinged-helixÕ DNA-binding motif, as
found, for example, in the DNA-binding domain of the
Mu-phage repressor. Residues 51–72, which were not
resolved in the k Xis, do not show any regular structure in
HK022 Xis and thus appear to be completely disordered in
solution. The resonance assignments have shown, however,
that an unusual connectivity exists between residues Asn66
and Gly67 owing to asparagine-isoaspartyl isomerization.
Such an isomerization has been previously observed and
characterized only in eukaryotic proteins.
Keywords: excisionase; NMR spectroscopy; protein stabi-
lity; isoaspartyl linkage; cis-proline.
Knowledge about the molecular mechanisms of viral site-
specific integration/excision in prokaryotes and eukaryotes
can be widely employed in biotechnological and medical
applications, such as site-specific genomic targeting, drug
design, vector construction, etc. The structural determinants
of integrative recombination were studied extensively in
various viruses during the 1990s [1–4], and almost all
proteins participating in the bacteriophage k recombination
system have been structurally characterized [5–8]. However,
very little is known about the structural basis of excisionase
function, although its importance is generally recognized.
Two closely related bacteriophages – k and HK022 – use
common mechanisms for integration/excision of their
genomes during a life cycle. The phage-encoded integrase,
Int, recognizes attP (on the phage chromosome) and attB
(on the bacterial chromosome) core sites and performs site-

specific recombination with the help of the cell-encoded
integration host factor, IHF, resulting in integration of the
circular phage DNA into the cellular chromosome. The
attP and attB sites generate the prophage sites attR and
attL, which flank the inserted phage DNA. The reverse
reaction (called excisive recombination) leads to excision of
the prophage by recombination between attR and attL and
regeneration of the attP and attB sites. The excision requires
an additional enzyme – the excisionase (Xis). The cellular
protein factor for inversion stimulation, FIS, enhances the
excision, but cannot replace Xis [9,10]. Xis plays a key role
in reorienting the recombination directionality. It has been
shown that Xis from bacteriophage k binds cooperatively
to the two tandemly arranged specific DNA sites X1 and
X2, which are located in the long P-arms of attR [11]. The
Correspondence to H. Ru
¨
terjans, Institute of Biophysical Chemistry,
J.W. Goethe-University of Frankfurt, Marie-Curie Str. 9,
60439 Frankfurt, Germany.
Fax: + 49 69 798 29632, Tel.: + 49 69 798 29631,
E-mail:
Abbreviations: Cp,pr(T), experimental partial molar heat capacity
function; DSC, differential scanning calorimetry; DH, specific dena-
turation enthalpy; F
D
, population of protein in the denatured state;
F
N
, population of protein in the native state; FIS, factor for inversion

stimulation; RMSD, root mean square deviation; T
m
, denaturation
midpoint temperature; Xis, excisionase.
*Present address: Bijvoet Center for Biomolecular Research, Depart-
ment of NMR Spectroscopy, Utrecht University, the Netherlands.
Note: The chemical shift assignment of full-length HK022 Xis_C28S is
available in the BioMagResBank under the accession number BMRB-
5539; the atomic coordinates and the structure factors (PDB_ID
1PM6) have been deposited in the Protein Data Bank, Research
Collaboratory for Structural Bioinformatics, Rutgers University,
New Brunswick, NJ, USA.
(Received 22 July 2003, revised 14 October 2003,
accepted 21 October 2003)
Eur. J. Biochem. 270, 4846–4858 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03884.x
X2 site overlaps with the FIS binding site F, and in vivo the
k Xis bound to X1 cooperates either with FIS or with
a second k Xis molecule [12]. When k Xis occupies the X1
and X2 sites (or Xis and FIS occupy the X1 and F sites,
respectively) the DNA becomes significantly bent – up to
140° [13]. k Xis also enhances the binding of Int to its second
specific site, P2, whose affinity for Int is quite weak [11].
These two factors that are promoted by Xis, DNA bending
and Int binding to P2, have been proposed to change the
directionality of the recombination.
The Xis proteins from both k and HK022 phages are
identical 72-residue proteins, with the exception of Gly59
which is substituted by Ser in HK022 Xis [14]. Their binding
sites are very similar and the proteins can be interchanged
[14–16]. Structural studies of Xis were hampered by protein

instability and a high intracellular toxicity [17]. However,
site-directed mutagenesis, in combination with functional
studies, provided valuable information about the participa-
tion of different k Xis regions in DNA binding, and Xis–Int,
Xis–Xis and Xis–FIS interactions. Site-directed mutagenesis
revealed that the N-terminal part of k Xis (residues 1–53)
is involved in both the DNA specific recognition and the
interaction with FIS or a second Xis molecule, whereas
the C-terminal part (residues 54–72) is important for the
interaction with Int [18]. Alterations of the C-terminal part,
such as single amino acid substitutions or even its entire
deletion, did not completely inactivate the in vivo excisive
recombination activity of k Xis, although its efficiency was
substantially reduced [19]. For example, when k Xis is
mutated at positions 57, 60, 62, 63, 64 or 65, the k Xis–Int
interaction is prevented, probably as a result of an inability
to form the required structural motif. For this particular
region (residues 59–65) an a-helical structure has been
suggested by various prediction methods [19].
Recently, the 3D structure of the N-terminal fragment
(residues 1–55) of the excisionase from k phage was reported
[20]. This k Xis N-terminal fragment (with a Cys28 fi Ser
substitution) displays a tertiary fold characteristic for the
ÔwingedÕ helix family of DNA-binding proteins [20,21]. It
was found that at pH 5.0 the k Xis solution structure
consists of two antiparallel b-strands and two a-helices,
whereas residues 51–72 appeared not to show any regular
structure. Additionally, a model of sequence-specific DNA
interactions was proposed, based on the protein structure.
However, the reasons for Xis instability in vitro and in vivo,

as well as the role of the C-terminal tail, remained unclear.
In order to better understand the functional organization of
excisionases, a structural and thermodynamic study of the
excisionase from the k-like coliphage HK022 was carried
out in this work.
As wild-type k Xis was previously characterized as a
structurally unstable protein [17], in the present study the
3D structure of full-length HK022 Xis containing a single
amino acid substitution, Cys28 fi Ser (Xis_C28S)
(I. Kleinhaus, K. Werner, H. Ru
¨
terjans and V. V. Rogov,
unpublished results), was obtained by NMR spectroscopy.
The validity of this structure was demonstrated for a wide
range of external conditions by means of differential
scanning calorimetry (DSC) and NMR spectroscopy.
Comparison of the selected NOE patterns of HK022
Xis_C28S and wild-type HK022 Xis (Xis_wt) revealed
complete structural identity of the two proteins. The main
fold of the full-length HK022 Xis_C28S is very similar to
that reported for the k Xis N-terminal fragment, with a few
minor (but important) differences. Contrary to the structure
reported by Sam and co-authors [20], residues 2–4 of
HK022 Xis adopt a b-strand conformation, forming a
three-stranded antiparallel b-sheet together with residues
35–36 (b-strand 3) and residues 41–44 (b-strand 4). This
makes the full-length HK022 Xis structure more similar to
those of the typical ÔwingedÕ-helix proteins, e.g. the bac-
teriophage Mu repressor DNA-binding domain [21]. The
family of excisionases comprises 63 different enzymes that

are able to change or modulate the directionality of
recombination [22]. The structural and thermodynamic
study of HK022 Xis presented here provides a useful insight
into their structural organization and functionality.
Materials and methods
Preparation of protein and DNA samples
The previously constructed pPG14 plasmid [16], containing
the HK022 Xis gene linked with a His-tag and a thrombin
cleavage site at the protein N terminus, was used, in this
work, for the production of Xis_wt protein. The Cys28 fi
Ser single amino acid substitution was performed with
reference to the pPG14 construct, and the resulting plasmid
(pPG14_C28S;I.Kleinhaus,K.Werner,H.Ru
¨
terjans and
V. V. Rogov, unpublished results) was employed for the
production of Xis_C28S.
A previously designed protein isolation and purification
procedure (I. Kleinhaus, K. Werner, H. Ru
¨
terjans and
V. V. Rogov, unpublished results) was slightly modified in
order to achieve optimal stable isotope labelling. The
Escherichia coli BL21*(DE3)/pLysS strain was freshly
transformed with the plasmids pPG14 or pPG14_C28S
prior to protein overexpression in modified ECPM1-x
media (I. Kleinhaus, K. Werner, H. Ru
¨
terjans and V. V.
Rogov, unpublished results) [23], containing 1 gÆL

)1
unlabelled NH
4
Cl, 40 gÆL
)1
unlabelled glycerol and Trace
Elements I solution [23]. The cells were incubated in a
fermenter, with intensive aeration, to slightly beyond the
mid-log phase (A
600
¼ 1.3–1.6), after which the cells were
collected by centrifugation and resuspended in 4.0 L of
ECPM1-x media without any sources of nitrogen (for the
preparation of
15
N-labelled protein) or containing neither
nitrogen nor carbon sources (for the preparation of
13
C,
15
N-labelled protein). After a short starvation period
(20 min), the cells were supplemented with either 1.0 gÆL
)1
of
15
NH
4
Cl or 1.0 gÆL
)1
of

15
NH
4
Cl, 0.5 gÆL
)1
of
13
C-labelled glucose and 1.5 gÆL
)1
of
13
C-labelled glycerol.
Cell growth was continued for another 20 min before the
addition of isopropyl thio-b-
D
-galactoside (1.0 m
M
final
concentration) to induce expression of the Xis gene. After
2 h 45 min of induction, the cells were harvested by
centrifugation and lysed using a French press. All samples
of Xis_wt were supplemented with 10 m
M
2-mercapto-
ethanol to protect the thiol group of the protein against
oxidation. The cleared cell lysate was subjected to Ni
2+
chelating chromatography. Protein elution was performed
using a linear gradient of 50–400 m
M

imidazole.
The collected protein was cleaved with thrombin,
and a subsequent preparative gel filtration, through a
Ó FEBS 2003 Structure and stability of HK022 excisionase (Eur. J. Biochem. 270) 4847
2.6 · 60 cm Superdex 75 column, was employed as the
final step of protein purification. Homogeneity of the
protein was verified by SDS/PAGE and mass spectrometry
(MALDI-TOF). The fractions containing > 99% pure Xis
protein were used for the NMR sample preparation and
other applications.
For all NMR sample preparations, the selected Xis
fractions were exhaustively dialyzed against a 100-fold
excess of NMR buffer [50 m
M
sodium phosphate (pH 6.5),
100 m
M
NaCl, 0.2 m
M
EDTA (disodium salt), 0.03%
NaN
3
] and subsequently reduced in volume to a final Xis
concentration of 0.1–1.2 m
M
, depending on the experiment.
The protein concentration was derived from the optical
density of the samples using a calculated extinction coeffi-
cient of 13 940 mm
)1

Æcm
)1
(A
0.1%
1cm
¼ 1.614) at 280 nm;
5%
2
H
2
O as lock substance and 0.1 m
M
4,4-dimethyl-
4-silapentane-1-sulfonate as internal proton chemical shift
standard were added to the samples. All NMR samples of
Xis_wt also contained 1.0 m
M
dithiothreitol. Glycerol (6%)
was added to the NMR buffer for monitoring the Xis–
DNA interaction in order to reduce aggregation of the
complex. Usually, 300 lL samples were placed into 5 mm
NMR tubes (Shigemi, Allison Park, PA, USA) under argon
protection.
The Xis samples for DSC were dialysed against the
buffers, and then filtered and degassed prior to filling of the
calorimetric cell. The buffers used in this work consisted
of 25 m
M
buffer species (sodium acetate for the pH range
4.5–5.5; sodium phosphate for the pH range 6.0–7.0) and

100–400 m
M
NaCl. For all Xis_wt samples, 1 m
M
dithio-
threitol was added to the buffer prior to dialysis. The pH
values were measured at 25 °C without corrections for the
temperature dependence.
A synthetic 20 bp DNA duplex was used in this work for
the DNA binding experiment. It contained 15 bp of the
natural HK022 Xis binding site, X1 [14,24], with stabilizing
GCG and GC sequences at the 5¢-and3¢ termini,
respectively. Two single-stranded DNA oligonucleotides –
5¢-GCGATATGTTGCGTTTTGGC-3¢ and the comple-
mentary sequence (purchased from Carl Roth, Karlsruhe,
Germany) – were annealed, and the double-stranded X1
was purified by gel filtration in buffer containing 40 m
M
K
2
HPO
4
and 100 m
M
NaCl, pH 6.0. The double-stranded
X1 DNA was concentrated and equilibrated with the
corresponding NMR buffer in an Amicon ultrafiltration
device (membrane MWCO ¼ 0.5 kDa).
DSC
The DSC data were recorded using the SCAL-1 scanning

microcalorimeter (SCAL, Pushchino, Russia) at a pressure
of 2.0 atm. The optimal heating rate (60 KÆh
)1
) was
established experimentally. The data were sampled and
processed using the service program
WSCAL
,basedonthe
principles described by Filimonov et al.[25]andPrivalov&
Potekhin [26].
For calculating the experimental partial molar heat
capacity function, Cp,pr(T), the partial specific volumes
of Xis_wt and Xis_C28S were assumed to be 0.73 mLÆg
)1
[26]. The protein concentration, derived from the absorb-
ance of the samples, varied in the DSC samples from 1.0 to
2.9 mgÆmL
)1
. The molecular weights of Xis_wt and
Xis_C28S were calculated, from the amino acid sequence,
to be 8.635 kDa and 8.619 kDa, respectively.
The analyses of the Cp,pr(T) functions were performed
as described previously [25,27–30].
NMR spectroscopy
All NMR spectra for resonance assignments and structure
determination were collected at 303 K on Bruker DMX 500
and DMX 600 spectrometers, equipped with 5 mm triple-
resonance (
1
H/

13
C/
15
N) probes with XYZ-gradient capa-
bility. Proton chemical shifts were referenced relative to
internal 4,4-dimethyl-4-silapentane-1-sulfonate;
15
Nand
13
C chemical shifts were referenced indirectly using the
corresponding chemical shift ratios [31].
3D Triple-resonance [
15
N,
1
H]-TROSY-HNCO [32],
(HCA)CO(CA)NH [33] and [
15
N,
1
H]-TROSY-HNCACB
[34] spectra were collected for the sequential backbone
resonance assignments. Side-chain resonance assignments
were achieved using the following experiments: 3D
HBHA(CBCA)(CO)NH [35], 3D H(CC)(CO)NH-TOCSY
[36], 3D (H)C(C)(CO)NH-TOCSY [37] and a
15
N-edited 3D
TOCSY-HSQC [38]. The resonances of aromatic ring
protons were assigned using 2D clean [

1
H-
1
H]-TOCSY
spectra [39,40] recorded with spin-lock times of 59.3 ms and
5.6 ms, and 2D [
1
H-
1
H]-NOESY spectra in
1
H
2
Oand
2
H
2
O.
3D Heteronuclear NMR spectra were collected for
determining the interproton distances in HK022 Xis_C28S.
A
15
N-edited 3D NOESY-HSQC experiment [41],
employing water flip-back [42] and gradient sensitivity
enhancement [43], was acquired with a mixing time of
100 ms.
13
C-Edited 3D NOESY-HSQC spectra (mixing
time 70 ms) were recorded in two different versions;
optimized for H

a
/H
b
NOE-correlations (3D NOESY-
[
13
C,
1
H]-HSQC) and for methyl group NOE-correlations
(3D NOESY-(CT)-[
13
C,
1
H]-HSQC).
The proton exchange experiments were carried out
at 600 MHz and temperatures of 288 K and 298 K. A
reference [
1
H,
15
N]-HSQC spectrum was recorded with fully
protonated Xis_C28S. The sample was then lyophilized and
dissolved in the same volume of ice-cold
2
H
2
O. A series of
identical [
1
H,

15
N]-HSQC spectra were acquired every
15 min during the first 2 h, and thereafter every 30 min,
until all amide protons were completely exchanged with
2
H
(after 12 h).
[
15
N,
1
H]-TROSY and homonuclear 1D spectra were
collected to establish the Xis–DNA interaction at 288 K. A
reference [
15
N,
1
H]-TROSY spectrum was recorded using
0.5 mL of a 0.4 m
M
Xis_C28S sample (20 m
M
sodium
phosphate, 100 m
M
NaCl,6%glycerol,pH6.5);equivalent
spectra were acquired after each titration step with 25 lLof
1.2 m
M
X1 (20 bp DNA duplex) until a protein/DNA ratio

of 1 : 3 was reached. The reverse order of titration, when
25 lLofa1m
M
Xis sample was added stepwise to 0.5 mL
of a 0.4 m
M
X1 solution, was monitored by 1D
(SW ¼ 32.2 p.p.m.) and [
15
N,
1
H]-TROSY spectra up to a
protein/DNA ratio of 3 : 1.
The NMR spectra were processed and analyzed on
Silicon Graphics workstations using the
XWINNMR
2.6,
AURELIA
2.7.5 (Bruker BioSpin, Rheinstetten, Germany)
and
FELIX
97 (Accelrys, San Diego, CA, USA)
programs.
4848 V. V. Rogov et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Restraint generation and structure calculation
The NOE-based distance restraints were extracted from 3D
13
C- and
15
N-edited NOESY-HSQC spectra and homo-

nuclear 2D NOESY spectra in
1
H
2
Oand
2
H
2
O. Automated
assignments of the NOEs, based only on chemical shifts,
were obtained using the program
NMR
2
ST
[44].
The structures were calculated using a simulated annealing
protocol with torsion angle dynamics (
DYANA
1.5 [45]),
combined with an iterative structure refinement procedure
[46]. Using the program
GLOMSA
[47], 37 stereospecific
assignments were obtained for the prochiral methylene and
isopropyl groups of 29 residues, including c
1
/c
2
of six Val
residues and d

1
/d
2
of three Leu residues. A cis-proline residue
entry was added to the standard
DYANA
residue library for
correct calculation of Pro32, which has been identified as a cis
isomer. No additions were made in the standard
DYANA
residue library with respect to the IsoAsp66 residue, as the
C-terminal tail of HK022 Xis was shown to be disordered in
solution and no NOE violations were foundwith the usage of
the standard Asn66 residue.
For calculating the final structure ensemble, 868 NOE-
derived distance restraints and 34 hydrogen bond restraints
(d
HO
£ 2.1 A
˚
and d
NO
£ 3.1 A
˚
) were employed. Subse-
quent restrained energy minimization, carried out using the
DISCOVER
module of the
INSIGHT
97 software package

(Accelrys, San Diego, CA, USA), was performed with the
20 best
DYANA
conformers. The minimized structures were
analyzed using
PROCHECK
-
NMR
3.4 [48]. The structure
images were prepared using the
MOLMOL
program [49].
Results
Expression and purification of the Xis_wt
and Xis_C28S proteins
The isolation and purification of Xis was hampered by the
fact that the protein can adopt a non-native conformation,
characterized by a significant decrease in structural integrity
and functional activity. This conformation tends to form
high-order aggregates and seems to consist of disulfide-
bridged dimers, as evidenced by SDS/PAGE. Therefore, an
amino acid substitution was designed to replace the SH
group of Cys28 with the OH of Ser in order to decrease the
irreversibility of the Xis transition to the non-native state
(I. Kleinhaus, K. Werner, H. Ru
¨
terjans and V. V. Rogov,
unpublished results).
The replacement of Cys28 with Ser in Xis resulted in a
reasonable stabilization of the protein against aggregation

in various buffer systems and allowed optimization of the
isolation/purification scheme in order to reach a sufficient
protein yield. Interestingly, the optimized conditions
could also be successfully applied to the isolation, purifi-
cation and storage of Xis_wt. Comparison of the bio-
physical characteristics and biological activity of Xis_wt
and Xis_C28S revealed a high similarity in almost all
protein parameters (I. Kleinhaus, K. Werner, H. Ru
¨
terjans
and V. V. Rogov, unpublished results). The same amino
acid substitution was used recently to stabilize the
N-terminal fragment of k Xis; an equal ability of both
the wild-type and the mutant protein to bind specific DNA
and to initiate excisive recombination in vivo was also
demonstrated [20].
Thermodynamic characterization of HK022 Xis
The instability of the Xis protein has been a significant
obstacle for structural investigations. In this work, DSC was
used to study the denaturation of Xis in order to define the
stabilization energy (DG) of Xis_wt and Xis_C28S under
various experimental conditions and thus determine the
optimal conditions for NMR experiments. The thermo-
dynamic parameters of Xis denaturation under these
conditions are summarized in Table 1.
It was found that the thermal denaturation of Xis is
highly reversible and cannot be responsible for the previ-
ously observed irreversible inactivation of the protein. In
Fig. 1A, two repetitive scans of the same Xis_wt sample did
not show any significant difference, demonstrating the

ability of the protein to reconstitute the initial tertiary
structure after denaturation. This observation was also
supported by NMR data (not shown).
The influence of pH on the Xis_wt thermal denaturaion is
illustrated in Fig. 1B. A visible increase of the protein
stability was observed when the pH was raised from 4.7 to
7.0. At pH 4.7, the denaturation midpoint temperature (T
m
)
was so low, and consequently the denaturation enthalpy
(DH) so small, that the molar partial heat capacity function
[Cp,pr(T)] did not contain a substantial peak. Although the
Cp,pr(T) of the protein showed an intensive heat absorption
peak (T
m
¼ 41.6 °C) when recorded at pH 5.5, the
thermodynamic analysis of this function indicated a
significant denatured state population (F
D
) of Xis_wt
already at room temperature (Table 1). In contrast, at
Table 1. Thermodynamic parameters of the HK022 Xis_wt denaturation. The buffers used for data collection contained a 25 m
M
buffer species
(sodium acetate for pH 5.5; sodium phosphate for pH 6.5 and 7.0) and 100 m
M
NaCl. DCp calculated from the temperature dependence of DH is
equal to 3.1 kJÆmol
)1
ÆK

)1
. ° Indicates the value of the corresponding parameter at 25 °C. F
N
defines the protein native state population.
pH
T
m
(°C)
DH(T
m
)
(kJÆmol
)1
)
DCp(T
m
)
(kJÆmol
)1
ÆK
)1
)
DH°
(kJÆmol
)1
)
DS°
(kJÆmol
)1
ÆK

)1
)
DG°
(kJÆmol
)1
) F
N
°
5.5 41.6 138 2.8 82 0.26 6.9 0.91
5.5
a
47.3 153 2.8 82 0.25 8.2 0.96
6.5 51.4 165 3.1 76 0.22 9.8 0.98
6.5
b
51.9 165 3.0 74 0.21 9.9 0.98
7.0 54.0 174 3.0 74 0.21 11.0 0.99
a
Data for the Xis_wt thermal denaturation in the presence of 400 m
M
NaCl.
b
Data for the Xis_C28S thermal denaturation.
Ó FEBS 2003 Structure and stability of HK022 excisionase (Eur. J. Biochem. 270) 4849
pH 6.5 and 7.0 the protein stability was higher; the F
D
did
not exceed 3%, even at 30 °C.
Furthermore, it was found that the stability of Xis could
be significantly increased by raising the salt concentration.

Figure 1C shows two Cp,pr(T) functions obtained for
Xis_wt at pH 5.5 and NaCl concentrations of 100 and
400 m
M
. In the latter, the T
m
was shifted by +5.7° (from
41.6 to 47.3 °C). Thermodynamic analysis indicated that
this salt-induced stabilization is mostly entropic in nature as
the DH values were almost equal for low- and high-salt
conditions at the same temperature.
A detailed analysis of the Xis_wt and Xis_C28S thermal
denaturation was performed for Cp,pr(T) functions
obtained under the same conditions as the NMR experi-
ments (Fig. 2). Direct comparison of the thermodynamic
parameters of Xis_wt and Xis_C28S indicated that the
influence of the Cys28 fi Ser amino acid substitution on
the protein stability was very small (Table 1). The difference
in T
m
values (51.4 °C for Xis_wt vs. 51.9 °C for Xis_C28S)
was within the limits of experimental error.
The high reversibility, the independence of T
m
on protein
concentration, and the presence of only one heat absorption
peak suggest a simple monomolecular two-state scheme for
Xis denaturation. Indeed, a Cp,pr(T) function that was
simulated based on this assumption (Fig. 2A, dots) fits the
experimentally obtained curve reasonably well.

The temperature dependence of DH isshowninFig.2B.
The specific (JÆg
)1
) presentation of DH revealed that the Xis
denaturation enthalpy at 130 °C(DH
specific
¼ 35 JÆg
)1
) was
significantly lower than those values reported for small
globular proteins (50 JÆg
)1
± 15% at 130 °C [50]). This
difference suggests that the Xis molecule is not completely
structured. The structured region of the protein is appar-
ently limited to the segment encompassing residues 2–50.
After correction for the size of this cooperative unit
(apparent molecular mass of 6.070 kDa instead of 8.635
for full-length Xis), the DH
specific
of 50 JÆg
)1
at 130 °C
corresponds well with the expected value.
Fig. 1. DSC data of HK022 Xis_wt thermal denaturation under various
experimental conditions. (A) Reversibility of the HK022 Xis_wt ther-
mal denaturation. The first scan is shown by a dashed line, the second
scan by a solid line. (B) pH dependence of HK022 Xis_wt thermal
denaturation. The partial molar heat capacities of HK022 Xis_wt were
determined at pH 4.7, 5.5, 6.5 and 7.5 (each buffer contained 100 m

M
NaCl). (C) The partial molar heat capacities of HK022 Xis_wt at 100
and 400 m
M
NaCl (50 m
M
sodium acetate at pH 5.5 was used as a
buffer base).
Fig. 2. Thermodynamic analysis of HK022 Xis_wt denaturation under
the same conditions as the NMR experiments (50 m
M
sodium phosphate,
100 m
M
NaCl, 0.03% NaN
3
,pH6.5).(A) The experimentally deter-
mined partial molar heat capacity (solid line) and the best-fit partial
molar heat capacity (dots) of HK022 Xis_wt. The partial molar heat
capacities of the Xis_wt native (C
p
N
) and denatured (C
D
p
) states as well
as DCp
int
(dashed line) were calculated according to formulae 1–4
(supplementary material) using the experimental values presented in

Table 1. (B) Temperature dependence of the specific denaturation
enthalpy (DH) of full-length HK022 Xis_wt under the same experi-
mental conditions as the NMR experiments (dashed line). The thin
horizontal dotted lines present the area of the DH values expected for
small compact globular proteins [48]. The solid line indicates the
temperature dependence of a hypothetical DH calculated for only the
structured Xis part (residues 2–50). Experimentally observed DH val-
ues of HK022 Xis denaturation are indicated as circles. (C) The tem-
perature dependence of HK022 Xis_wt native (dotted line) and
denatured (solid line) populations under the same experimental con-
ditions as the NMR experiments. The populations were calculated
from DG values using the formula DG(T) ¼ DH(T) ) T*DS(T).
4850 V. V. Rogov et al. (Eur. J. Biochem. 270) Ó FEBS 2003
The calculated temperature dependences of F
N
and F
D
at
the NMR conditions used are presented for Xis_wt in
Fig. 2C. According to the analysis, F
D
starts to increase
rapidly at temperatures only above 35 °C. Thus, 30 °C
(F
D
¼ 0.03) was chosen as an optimal temperature for the
NMR experiments.
Although this thermodynamic study revealed an equal
stability of Xis_wt and Xis_C28S, the latter showed a
significantly lower tendency to aggregate at high concen-

trations and remained native for a longer time. Thus,
Xis_C28S was chosen for the structural study by means of
heteronuclear NMR spectroscopy.
Assignment of backbone and side-chain resonances
in HK022 Xis_C28S spectra
Nearly complete backbone and side-chain resonance assign-
ments were achieved for Xis_C28S (Fig. 3A), except for the
13
C resonances of the aromatic rings, the acidic carboxyl
groups and the amide resonances in the side-chains of Arg
and Lys. In contrast to the previously published k Xis
N-terminal fragment assignment [20], the backbone amide
proton resonances of Tyr2 and Thr4 have been identified for
HK022 Xis_C28S. Despite different experimental condi-
tions, the only other significant difference in the backbone
amide resonances of the two assignments is the position of
Glu45 HN.
The [
1
H,
15
N]-HSQC spectrum of Xis_wt corresponds
almost entirely to Xis_C28S (Fig. 3B). The most strongly
shifted HN resonances of HK022 Xis_wt (shown in red) were
assigned with the help of 3D
15
N-edited NOESY spectra.
Differences in chemical shift values occurred only in close
proximity to Cys28 (residues Arg26–Phe31) and did not
affect the amide resonances of residues sequentially more

than four amino acids distant from Cys28. Moreover,
comparison of the
15
N-edited 3D NOESY-HSQC spectra
Fig. 3. Assignment of the HN resonances in
HK022 Xis_C28S and Xis_wt spectra. (A)
[
1
H,
15
N]-HSQC spectrum with annotated HN
resonances of HK022 Xis_C28S. The position
of the Glu45 backbone HN, which shows a
significant difference from that observed for
the N-terminal fragment of k Xis20, is marked
by a square box. (B) Superposition of repre-
sentative sections of the HK022 Xis_C28S
(black contours) and Xis_wt (red contours)
[
1
H,
15
N]-HSQC spectra. The three inserts on
top show additional resonances with sub-
stantial chemical shift changes located outside
the plotted spectral region.
Ó FEBS 2003 Structure and stability of HK022 excisionase (Eur. J. Biochem. 270) 4851
of Xis_wt and Xis_C28S, revealed the almost complete
structural identity of these two proteins (data not shown).
In the course of the sequence-specific resonance assign-

ment, an unusual connectivity was observed between Asn66
and Gly67. This connectivity was identified as an isoaspartyl
linkage (Fig. 4A), which has been previously reported for
Asn–X pairs in several other proteins [51–53]. Recently, an
isoaspartyl linkage was described for the Asn306–Gly307
pair in malate synthase G, based on the juxtaposition of
expected and observed signs of the N306 C
a
and C
b
signals
in 3D and 4D heteronuclear NMR spectra [54]. In the
current study, the isoaspartyl linkage was determined based
on the relative sign of the signals in the 3D [
15
N,
1
H]-
TROSY-HNCACB spectrum.
Figure 4B presents the sequential connectivities of
Xis_C28S residues 65–69, as observed in the 3D [
15
N,
1
H]-
TROSY-HNCACB (left panel) and 3D (HCA)CO(CA)NH
(right panel) spectra. In the HNCACB experiment, the
resonance signals of
13
C directly coupled to

15
Narealways
positive, whereas the relayed
13
C resonance signals are
negative. The sequential connectivity involving the C
b
resonance of IsoAsp66 is therefore positive in sign, whereas
the C
a
signal is negative (Fig. 4B, left panel, G67 strip). In
contrast, the intraresidual connectivities involving the C
a
and C
b
resonances of IsoAsp66 show the usual signs
(Fig. 4B, left panel, N66 strip).
In the (HCA)CO(CA)NH experiment, the intraresidually
observed IsoAsp66
13
C(O) resonance [i.e. the
13
C(OO
–
) at
179.2 p.p.m.; Fig. 4B, right panel, N66 strip] is shifted
downfield owing to the negative charge; it does not
correspond to the sequentially observed carbonyl (i.e. the
C
c

of the IsoAsp66 residue at 176.2 p.p.m.; Fig. 4B, right
panel, G67 strip). Both spectra unambiguously demonstrate
the IsoAsp66–Gly67 linkage in Xis_C28S; resonances
corresponding to the usual Asn66–Gly67 residue pair were
not observed.
The solution-state structure of HK022 Xis
A compact, well-resolved structure has been calculated for
full-length HK022 Xis_C28S. The 20 final conformers,
superposed at the well-structured Tyr2–Val50 region of
Xis_C28S, are shown as a stereo-view representation in
Fig. 5A. The root mean square deviation (RMSD) value of
the backbone atoms in this region is 0.83 A
˚
; excluding
residues 12–17 (the flexible loop between the two a-helices),
these structures can be superposed with a backbone RMSD
of 0.71 A
˚
. The statistics of the final structure calculation are
summarized in Table 2.
The global structure of HK022 Xis consists of two small
antiparallel b-sheets and an L-shaped a-helical motif
(Fig. 5B). The a-helices 1 (residues Leu5–Arg11) and 2
(Leu18–Glu27) are separated by a loop L (residues Glu12–
Ser17) and are oriented nearly orthogonal to each other.
The first antiparallel b-sheet consists of three b-strands:
b-strand 1 (residues Tyr2–Thr4), b-strand 3 (residues
Val35–Lys36) and b-strand 4 (residues Tyr41–His44).
Residues Asp37–Glu40 form a reverse b-turn T between
b-strands 3 and 4. The b-strands 2 (residues Ile30–Phe31)

and 5 (residues Val48–Lys49) form the second b-sheet. The
Fig. 4. Identification of the IsoAsp66–Gly67 connectivity. (A) The deamidation of the Asn66 side-chain via a succinimide ring intermediate results in
an isoaspartyl linkage between Asn66 and Gly67. (B) Representative strips from the [
15
N,
1
H]-TROSY-HNCACB (left panel) and (HCA)CO
(CA)NH (right panel) spectra of HK022 Xis_C28S at the
1
HN and
15
N frequencies of residues Arg65–Lys69. The sequential connectivities are
indicated; positive and negative peaks are displayed in black and red contours, respectively. As Lys68 HN overlaps strongly with Val56 HN, the
resonances of Val56 are also indicated in the Lys68 planes.
4852 V. V. Rogov et al. (Eur. J. Biochem. 270) Ó FEBS 2003
size of this structure element is rather small and was not
recognized by any secondary structure prediction program.
On the other hand, the proton-exchange data and a detailed
structural analysis suggest that there is a strong hydrogen
bond interaction between the amide proton of Phe31 and the
carbonyl oxygen of Val48. The experimentally determined
Fig. 5. Solution state structure of HK022 Xis_C28S. (A) Stereoview of the 20 conformers representing the final HK022 Xis_C28S structure
ensemble, displayed as backbone C
a
atom traces from Met1 to Leu52. (B) Ribbon diagram of the average structure, calculated from the final 20
conformers of HK022 Xis_C28S. Residues Ser59–Ser72, which adopt a Ôrandom coilÕ conformation, are not shown. The two a-helices – a1 (residues
Leu5–Arg11) and a2 (residues Leu18–Glu27) – are shown in red and yellow; the loop between them is indicated by L. The five b-strands – b1
(residues Tyr2–Thr4), b2 (residues Ile30–Phe31), b3 (residues Val35–Lys36), b4 (residues Tyr41–His44) and b5 (residues Val48–Lys49) – are colored
in cyan. The triproline segment (residues Pro32–Pro34) is marked in blue. (C) Left panel: comparison of the ÔwingÕ b-sheet in the averaged structures
of the full-length HK022 Xis_C28S (PDB entry 1PM6, current work, cyan), k Xis N-terminal domain (PDB entry 1LX8, Sam et al. [20], magenta)

and DNA-binding domain of Mu repressor (PDB entry 1QPM, Ilangovan et al. [21], green). Right panel: comparison of the reverse b-turn T of
these three proteins. Backbone traces of all conformers are plotted (thin sticks of corresponding color) and the averaged backbone structures are
highlighted as thick sticks.
Ó FEBS 2003 Structure and stability of HK022 excisionase (Eur. J. Biochem. 270) 4853
NOE patterns within the b-sheets of the HK022 Xis_C28S
structure are presented in Fig. 6.
The triproline segment (residues Pro32–Pro34) acts as a
linker between b-strands 2 and 3. The first residue in this
segment, Pro32, adopts a cis conformation (as supported by
NOE data) whereas the other two residues, Pro33 and
Pro34, are trans prolines. The bulge structure (residues
45–47), which connects b-strands 4 and 5, is positioned just
opposite to the triproline segment (Fig. 5B).
The residues from Asp51 to Ser72 are not included in any
regular structure element in HK022 Xis, as indicated by the
lack of any medium- or long-range NOEs. Hence, the
C terminus is largely disordered and displays very high local
RMSD values.
Interaction of HK022 Xis with specific DNA (X1)
Aseriesof[
15
N,
1
H]-TROSY and 1D
1
H spectra was
recorded in order to identify the amino acids in the Xis
molecule that are directly involved in the protein–DNA
interaction. Unfortunately, strong association/aggregation
was observed under all conditions tested, both when the

DNA was titrated to the protein and vice versa. Even with
the usage of relatively dilute protein samples (0.1 m
M
),
association/aggregation was found to be prevalent. It
should be pointed out that aggregation had already started
at the very first titration steps (at a protein–DNA molar
ratio of 10 : 1) and affected nearly all Xis in solution. This
behavior suggests that Xis may change its structure when
bound to DNA, thus facilitating or inducing Xis–Xis and/or
Xis–FIS interactions.
Although the large size of the associates/aggregates did
not permit the identification of direct contacts between
protein and DNA, it was possible to define a part of Xis that
was not as strongly influenced by the protein–DNA
complex formation. The [
15
N,
1
H]-TROSY spectrum of
Xis_C28S in the presence of its site-specific DNA (a 20 bp
DNA duplex containing the X1 site) at a 1 : 3 molar ratio is
shown in Fig. 7. At this ratio, all Xis molecules are bound to
X1 DNA. Hence, only the HN resonances of very mobile
amino acids should be detected in this spectrum. Besides
Table 2. Structural statistics of the 20 energy-minimized conformers of
HK022 Xis_C28S. RMSD, root mean square deviation.
Restraint statistics
Total number of meaningful distance restraints 902 (34)
a

Intraresidual (i ¼ j) 141
Sequential (Œi – j Œ¼1) 256
Medium range (1<Œi – j Œ£ 4) 256 (20)
a
Long range (Œi – j Œ>4) 249 (14)
a
Restraint violations
0.20–0.30 A
˚
21
0.30–0.40 A
˚
3
Maximal violation (A
˚
) 0.36
Structural precision, RMSD (A
˚
)
Backbone atoms
b
(residues 2–50) 0.83 ± 0.14
All heavy atoms (residues 2–50) 1.86 ± 0.18
Backbone atoms
b
(residues 2–11; 18–50) 0.71 ± 0.13
All heavy atoms (residues 2–11; 18–50) 1.68 ± 0.19
Ramachandran plot analysis (%)
c
Residues in most favoured regions 83.9

Residues in additionally allowed regions 13.1
Residues in generously allowed regions 1.6
Residues in disallowed regions 1.5
a
The number of included H-bond restraints is indicated in par-
entheses.
b
N, C
a
,C¢.
c
Values for the structured part only (residues
2–50).
Fig. 6. Backbone NOE patterns showing the organization of the antiparallel b-sheet structure in HK022 Xis_C28S. Experimentally observed distances
of < 2.5 A
˚
,<4.5A
˚
and < 6.0 A
˚
are indicated by thick, thin and dotted double-ended arrows, respectively. The assumed hydrogen bond
positions, which were confirmed by exchange experiments, are shown as thick, green dashed lines. Backbone atoms are presented using the
following color code: C, grey; N, blue; HN, violet; O, red; and H
a
,white.
4854 V. V. Rogov et al. (Eur. J. Biochem. 270) Ó FEBS 2003
a few signals that could not be directly assigned, the
C-terminal amide resonances (starting with Leu52 HN)
were all observable at the same positions as in uncomplexed
Xis_C28S.

Discussion
Bacteriophages k and HK022 are closely related lambda-
like Enterobacteria viruses. They use common mechanisms
for integration/excision of their genomes during their life
cycle. Both phage excisionases show almost complete
sequence identity; only one out of 72 residues differs in
these two proteins.
Although the 3D structures of the two excisionases were
therefore expected to be very similar, some differences were
nevertheless clearly observed. First, HK022 Xis consists of
five b-strands, whereas k Xisfeaturesonlytworegular
b-strands and two extended sequence segments [20]. Second,
the ÔwingÕ b-sheet of HK022 Xis_C28S consists of three
b-strands and is therefore structurally more similar to other
proteins of this class, such as the DNA-binding domain of
the Mu repressor [21]. Third, the backbone atoms of the
region comprising the ÔwingÕ b-sheet (residues 2–4, 35–36
and 41–44 of HK022 Xis and residues 16–18, 47–48 and
58–61 of the Mu repressor) could be superposed with an
RMSD value of 0.49 A
˚
, whereas superposition with the
same region of k Xis led to an RMSD value of 1.30 A
˚
(Fig. 5C, left panel). In Fig. 5C (right panel), the reverse
b-turns of these three proteins are compared. Again, the
flexibility and direction of this structural element in full-
length HK022 Xis were more similar to those in the DNA-
binding domain of the Mu repressor [21].
This structural difference between k and HK022 Xis

may be the result of differences in the conditions of the
NMR investigations, i.e. pH 6.8 in the current study vs.
pH 5.0 in the work of Sam et al.[20].AtpH5.0,the
spectra of truncated k Xis did not reveal the Tyr2 and
Thr4 backbone amide proton resonances that are crucial
for the identification of the NOE contacts in the first
b-sheet. However, the spectra of full-length HK022
Xis_C28S and Xis_wt, acquired under the same
experimental conditions reported by Sam et al., still
displayed these resonances. This suggests that there may
be some basic differences in the structural organization
and thermodynamic stability between the full-length and
truncated Xis proteins.
Of particular interest is the segment of three consecutive
proline residues (Pro32–Pro33–Pro34) connecting b-strands
2 and 3. The NOE patterns and structure calculations of
Xis_C28S revealed a cis–trans–trans arrangement of these
prolines. Currently, only six protein structures are available
in the Brookhaven Data-Bank that have a triproline motif
with the first proline in a cis configuration. These include
cytochrome c oxidase (PDB entries 1AR1, 2OCC, and
1OCO), endo-b-N-acetylglucosaminidase F1 (2EBN), mye-
lin basic protein (1QCL), protocatechuate 4,5-dioxygenase
(1BOU and 1B4U), thiaminase I (2THI), and cytotoxic T
lymphocyte-associated antigen 4 (1DQT, 1I85, and 1I8L).
All of these proteins feature a cis–trans–trans triproline
motif, except for the latter which shows a cis–trans–cis
configuration. Interestingly, these triproline sequences are
always located on the protein surface – directly at the
interaction site, in those cases where protein–protein contacts

have been observed. This suggests a similar role for the
Pro32–Pro33–Pro34 segment on the Xis surface, upon
interaction with ligands such as FIS or another Xis molecule.
The amino acid substitution of Cys28 fi Ser did not
significantly change either the structure or stability of
HK022 Xis. The differences observed in the NMR spectra
of HK022 Xis_wt and Xis_C28S occurred almost exclu-
sively in the immediate vicinity of residue 28 (Fig. 3B),
indicating that the overall structure of Xis_wt is not
affected by the substitution. The DSC study revealed that
the stabilities of the two proteins are approximately equal
(Table 1). Thus, one can assume that the disulfide bridge
between two Xis molecules can be formed only after
perturbation of the native Xis structure, as observed during
Xis aggregation (I. Kleinhaus, K. Werner, H. Ru
¨
terjans
Fig. 7. Interaction of HK022 Xis_C28S with
specific DNA (X1). [
15
N,
1
H]-TROSY spec-
trum of an HK022 Xis_C28S mixture with X1
(molar ratio 1 : 3). The indicated resonances
did not shift in comparison to the spectrum of
uncomplexed HK022 Xis_C28S. All other
HN resonances of the protein cannot be
detected owing to significant line-broadening
after addition of the specific DNA.

Ó FEBS 2003 Structure and stability of HK022 excisionase (Eur. J. Biochem. 270) 4855
and V. V. Rogov, unpublished results) or the association/
aggregation upon interaction of Xis with specific DNA.
Spontaneous aspartate isomerization and deamidation of
asparaginyl residues can serve as an initial site of sponta-
neous nonenzymatic degradation of proteins or peptides
in vivo and in vitro [53]. Such a degradation plays an
important role in biological systems and serves as a
molecular clock [55]. In this work, an IsoAsp–Gly connec-
tivity was unambiguously identified for the prokaryotic
regulatory protein Xis, although it was previously observed
and described as an attribute of regulation only in
eukaryotic organisms. It is still not clear whether the
isomerization in Xis (and subsequent protein degradation)
also occurs in vivo; however, such a type of negative
regulation of both k and HK022 Xis may be physiological
relevant, as even a small amount of Xis in infected cells can
turn the phage life cycle to the lytic pathway. It has been
shown previously that the k Xis concentration in E. coli is
strongly regulated by cellular proteases [17]; this isomeriza-
tion could serve as a signal for protein self-degradation, thus
playing an additional regulatory role. In fact, spontaneous
degradation of Xis has been observed and found to cause
significant problems in the protein preparation
(I. Kleinhaus, K. Werner, H. Ru
¨
terjans and V. V. Rogov,
unpublished results).
On the other hand, the unusual IsoAsp66–Gly67 con-
nectivity in Xis does not affect the general fold or functional

properties of the free protein. It has been shown [56] that
this isomerization generally occurs in highly mobile protein
regions and is accelerated at neutral or basic pH. As a high
mobility of the HK022 Xis C terminus was reliably
demonstrated in the current work, a possible functional
meaning of this isomerization/degradation could be to
prevent the formation of the predicted C-terminal a-helix
(amino acids 59–64) [19] and to subsequently reduce the
ability of Xis to recruit Int to the P2 site.
The previous investigations of excisionase interaction
with specific DNA were usually performed with large
(> 100 bp) DNA duplexes containing X1, X2 or X1/X2
sequences [20,24,57]. Unfortunately, the tendency of Xis to
associate/aggregate when bound to short DNA duplexes (a
20 bp duplex containing the X1 site was used in the current
work) does not allow a direct structural investigation of the
protein–DNA complex. In our NMR experiments, no
changes in the positions of the protein amide and DNA
imide resonances were observed when one of the macro-
molecules was added in excess. [
15
N,
1
H]-TROSY spectra of
the protein bound to DNA only showed HN resonances
of C-terminal residues (Leu52–Ser71). These results sup-
port the previously suggested hypothesis that only the
N-terminal, structured part of Xis is involved in the
recognition of DNA [18,57]. On the other hand, the results
also indicate that the C-terminal a-helix, predicted in the

work of Wu and co-authors for Xis residues 59–64 [19], is
not induced by the Xis–DNA interaction.
Conclusions
We have determined the solution-state structure of the full-
length excisionase from k-like coliphage HK022, containing
the single amino acid substitution Cys28 fi Ser. This
structure was shown to be stable under a wide range of
experimental conditions and to be identical to the wild-type
HK022 Xis structure; the denaturation of HK022 Xis is
reversible and cannot be the origin of the previously
observed irreversible inactivation of Xis in vivo and in vitro.
The organization of the secondary structure elements in
HK022 Xis is slightly different compared with the closely
related bacteriophage k excisionase; the presence of an
additional antiparallel b-strand at the N-terminus (residues
2–4) makes the HK022 Xis structure more similar to the
Mu-repressor DNA-binding domain.
We have found that the triproline segment (residues
Pro32–Pro33–Pro34) adopts a cis–trans–trans conforma-
tion. This region could therefore play a key role in the
specific protein–protein interaction that occurs during
excisive recombination, similar to other proteins that
display a cis–trans–trans triproline motif at the surface.
According to the NMR data, the C-terminal part of Xis is
definitely not involved in the protein–DNA interaction, but
it might serve as a specific site for the Xis–Int interaction
that initiates excision. Moreover, the Asn66–IsoAsp iso-
merization at the C terminus could be involved in the
protein self-regulation in vivo.
Acknowledgements

We gratefully acknowledge support from the Frankfurt University
Center for Biomolecular Magnetic Resonance. We thank Prof. E. Yagil
for making the pPG14 plasmid available to us and Dr G. Ku
¨
llertz (Max
Planck Research Unit, Halle, Germany) for a database search of the
cisPro-Pro-Pro motif. P.P. thanks the Ministry of Education, Science
and Sport of Slovenia for financial support. V.R. thanks Dr S. Potekhin
for helpful discussion.
References
1. Gopaul, D.N. & Duyne, G.D. (1999) Structure and mechanism in
site-specific recombination. Curr. Opin. Struct. Biol. 9, 14–20.
2. Connolly, K.M., Wojciak, J.M. & Clubb, R.T. (1998) Site-specific
DNA binding using a variation of the double stranded RNA
binding motif. Nat. Struct. Biol. 5, 546–550.
3. Chen,Z.,Yan,Y.,Munshi,S.,Li,Y.,Zugay,M.,Xu,B.,Witmer,
M., Felock, P., Wolfe, A., Sardana, V., Emini, E.A., Hazuda, D.
& Kuo, L.C. (2000) X-ray structure of simian immunodeficiency
virus integrase containing the core and C-terminal domain
(residues 50–293) – an initial glance of the viral DNA binding
platform. J. Mol. Biol. 296, 521–533.
4. Lodi, P.J., Ernst, J.A., Kuszewski, J., Hickman, A.B., Engelman,
A., Craigie, R., Clore, G.M. & Gronenborn, A.M. (1995) Solution
structure of the DNA binding domain of HIV-1 integrase. Bio-
chemistry 34, 9826–9833.
5. Kwon, H.J., Tirumalai, R., Landy, A. & Ellenberger, T. (1997)
Flexibility in DNA recombination: structure of the lambda
integrase catalytic core. Science 276, 126–131.
6. Yuan, H.S., Finkel, S.E., Feng, J.A., Kaczor, G., Johnson, R.C. &
Dickerson, R.E. (1991) The molecular structure of wild-type and a

mutant Fis protein: relationship between mutational changes and
recombinational enhancer function or DNA binding. Proc. Natl
Acad. Sci. USA 88, 558–9562.
7. Kostrewa, D., Granzin, J., Koch, C., Choe, H.W., Raghunathan,
S., Wolf, W., Labahn, J., Kahmann, R. & Saenger, W. (1991)
Three-dimensional structure of the E. coli DNA-binding protein
FIS. Nature 349, 178–180.
8. Wojciak,J.M.,Sarkar,D.,Landy,A.&Clubb,R.T.(2002)Arm-
site binding by lambda-integrase: solution structure and functional
4856 V. V. Rogov et al. (Eur. J. Biochem. 270) Ó FEBS 2003
characterization of its amino-terminal domain. Proc. Natl Acad.
Sci. USA 99, 3434–3439.
9. Landy, A. (1989) Dynamic, structural, and regulatory aspects
of lambda site-specific recombination. Annu. Rev. Biochem. 58,
913–949.
10. Nash, H.A. (1996) Site-specific recombination: integration, exci-
sion, resolution, and inversion of defined DNA segments. In
Escherichia Coli and Salmonella: Cellular and Molecular Biology
(Neidhardt, F.C., Curtiss, R. III, Ingraham, J.L., Lin, E.C.C.,
Low, K.B., Magasanik, B., Reznikoff, W.S., Riley, M., Schaech-
ter, M. & Umbarger, H.E., eds), pp. 2363–2376. ASM Press,
Washington, DC.
11. Bushman, W., Yin, S., Thio, L.L. & Landy, A. (1984) Determi-
nants of directionality in lambda site-specific recombination. Cell
39, 699–706.
12. Numrych, T.E., Gumport, R.I. & Gardner, J.F. (1991) A genetic
analysis of Xis and FIS interactions with their binding sites in
bacteriophage lambda. J. Bacteriol. 173, 5954–5963.
13. Thompson,J.F.&Landy,A.(1988)Empirical estimation of protein-
induced DNA bending angles: applications to lambda site-specific

recombination complexes. Nucleic Acids Res. 16, 9687–9705.
14. Yagil, E., Dolev, S., Oberto, J., Kislev, N., Ramaiah, N. &
Weisberg, R.A. (1989) Determinants of site-specific recombination
in the lambdoid coliphage HK022. An evolutionary change in
specificity. J. Mol. Biol. 207, 695–717.
15. Weisberg, R.A., Gottesmann, M.E., Hendrix, R.W. & Little, J.W.
(1999) Family values in the age of genomics: comparative analyses
of temperate bacteriophage HK022. Annu. Rev. Genet. 33,
565–602.
16. Gottfried, P., Yagil, E. & Kolot, M. (2000) Core-binding
specificity of bacteriophage integrases. Mol. Gen. Genet. 263,
619–624.
17. Leffers, G.G. & Gottesman, S. (1998) Lambda Xis degradation
in vivo by Lon and FtsH. J. Bacteriol. 180, 1573–1577.
18. Numrych, T.E., Gumport, R.I. & Gardner, J.F. (1992) Char-
acterization of the bacteriophage lambda excisionase (Xis) protein:
the C-terminus is required for Xis-integrase cooperativity but not
for DNA binding. EMBO J. 11, 3797–3806.
19. Wu, Z., Gumport, R.I. & Gardner, J.F. (1998) Defining the
structural and functional roles of the carboxyl region of the bac-
teriophage lambda excisionase (Xis) protein. J. Mol. Biol. 281,
651–661.
20. Sam, M.D., Papagiannis, C.V., Connolly, K.M., Corselli, L.,
Iwahara, J., Lee, J., Phillips, M., Wojciak, J.M., Johnson, R.C. &
Clubb, R.T. (2002) Regulation of directionality in bacteriophage
lambda site-specific recombination: structure of the Xis protein.
J. Mol. Biol. 324, 791–805.
21. Ilangovan, U., Wojciak, J.M., Connolly, K.M. & Clubb, R.T.
(1999) NMR structure and functional studies of the Mu repressor
DNA-binding domain. Biochemistry 38, 8367–8376.

22. Lewis, J.A. & Hatfull, G.F. (2001) Control of directionality in
integrase-mediated recombination: examination of recombination
directionality factors (RDFs) including Xis and Cox proteins.
Nucleic Acids Res. 29, 2205–2216.
23. Bernard, A. & Payton, M. (1998) Fermentation and growth of
Escherichia coli for optimal protein production. In Current Pro-
tocols in Protein Science (Coligan, J.E., Dunn, B.M., Plough, H.L.,
Speicher, D.W. & Wingfield, P.T., eds), pp. 5.3.1–5.3.18. John
Wiley & Sons, New York.
24. Gottfried, P., Kolot, M. & Yagil, E. (2001) The effect of mutations
in the Xis-binding sites on site-specific recombination in coliphage
HK022. Mol. Genet. Genomics 266, 584–590.
25. Filimonov, V.V., Potekhin, S.A., Matveev, S.V. & Privalov, P.L.
(1982) Thermodynamic analysis of scanning microcalorimetry
data. 1. Algorithms for deconvolution of heat absorption curves.
Mol. Biol. (Moscow) 16, 551–562.
26. Privalov, P.L. & Potekhin, S.A. (1986) Scanning microcalorimetry
in studying temperature-induced changes in proteins. Methods
Enzymol. 131, 4–51.
27. Viguera, A.R., Martinez, J.C., Filimonov, V.V., Mateo, P.L. &
Serrano, L. (1994) Thermodynamic and kinetic analysis of the
SH3 domain of spectrin shows a two-state folding transition.
Biochemistry 33, 2142–2150.
28. Filimonov, V.V. & Rogov, V.V. (1996) Reversible association of
the equilibrium unfolding intermediate of lambda Cro repressor.
J. Mol. Biol. 255, 767–777.
29. Makhatadze, G.I. & Privalov, P.L. (1990) Heat capacity of pro-
teins. I. Partial molar heat capacity of individual amino acid
residues in aqueous solution: hydration effect. J. Mol. Biol. 213,
375–384.

30. Privalov,P.L. & Makhatadze, G.I.(1990)Heat capacity of proteins.
II. Partial molar heat capacity of the unfolded polypeptide chain
of proteins: protein unfolding effects. J. Mol. Biol. 213, 385–391.
31. Wishart, D.S., Bigam, C.G., Yao, J., Abildgaard, F., Dyson, H.J.,
Oldfield, E., Markley, J.L. & Sykes, B.D. (1995)
1
H,
13
Cand
15
N
chemical shift referencing in biomolecular NMR. J. Biomol. NMR
6, 135–140.
32. Salzmann, M., Pervushin, K., Wider, G., Senn, H. & Wu
¨
thrich, K.
(1998) TROSY in triple-resonance experiments: new perspectives
for sequential NMR assignment of large proteins. Proc. Natl
Acad. Sci. USA 95, 13585–13590.
33. Lo
¨
hr,F.&Ru
¨
terjans, H. (1995) A new triple-resonance experi-
ment for the sequential assignment of backbone resonances in
proteins. J. Biomol. NMR 6, 189–197.
34. Salzmann, M., Wider, G., Pervushin, K., Senn, H. & Wu
¨
thrich, K.
(1999) TROSY-type triple-resonance experiments for sequential

NMR assignments of large proteins. J. Am. Chem. Soc. 121,
844–848.
35. Grzesiek, S. & Bax, A. (1993) Amino acid type determination
in the sequential assignment procedure of uniformly
13
C/
15
N-
enriched proteins. J. Biomol. NMR 3, 185–204.
36. Montelione, G.T., Lyons, B.A., Emerson, S.D. & Tashiro, M.
(1992) An efficient triple resonance experiment using carbon-13
isotropic mixing for determining sequence-specific resonance
assignments of isotopically-enriched proteins. J. Am. Chem. Soc.
114, 10974–10975.
37. Grzesiek, S., Anglister, J. & Bax, A. (1993) Correlation of back-
bone amide and aliphatic side-chain resonances in
13
C/
15
N-
enriched proteins by isotropic mixing of
13
C magnetization.
J. Magn. Reson. 101B, 114–119.
38. Marion, D., Driscoll, P.C., Kay, L.E., Wingfield, P.T., Bax, A.,
Gronenborn, A.M. & Clore, G.M. (1989) Overcoming the overlap
problem in the assignment of
1
H NMR spectra of larger proteins
by use of three-dimensional heteronuclear

1
H-
15
N Hartmann-
Hahn-multiple quantum coherence and nuclear Overhauser-
multiple quantum coherence spectroscopy: application to inter-
leukin 1 b. Biochemistry 28, 6150–6156.
39. Davis, D.G. & Bax, A. (1985) Assignment of complex NMR
spectra via two-dimensional homonuclear Hartmann-Hahn spec-
troscopy. J. Am. Chem. Soc. 107, 2820–2821.
40. Griesinger, C., Otting, G., Wu
¨
thrich, K. & Ernst, R.R. (1988)
Clean TOCSY for
1
H spin system identification in macro-
molecules. J. Am. Chem. Soc. 110, 7870–7872.
41. Bax, A., Ikura, M., Kay, L.E., Torchia, D.A. & Tschudin, R.
(1990) Comparison of different modes of two-dimensional reverse-
correlation NMR for the study of proteins. J. Magn. Reson. 86,
304–318.
42. Grzesiek, S. & Bax, A. (1993) The importance of not saturating
H
2
O in protein NMR. Application to sensitivity enhancement and
NOE measurement. J. Am. Chem. Soc. 115, 12593–12594.
43. Schleucher, J., Sattler, M. & Griesinger, C. (1993) Coherence
selection by gradients without signal attenuation: application to
Ó FEBS 2003 Structure and stability of HK022 excisionase (Eur. J. Biochem. 270) 4857
three-dimensional HNCO experiments. Angew. Chem. Int. Ed.

Engl. 32, 1489–1491.
44. Pristovsek, P., Lu
¨
cke, C., Reincke, B., Ludwig, B. & Ru
¨
terjans, H.
(2000) Solution structure of the functional domain of Paracoccus
denitrificans cytochrome c552 in the reduced state. Eur. J. Bio-
chem. 267, 4205–4212.
45. Gu
¨
ntert, P., Mumenthaler, C. & Wu
¨
thrich, K. (1997) Torsion
angle dynamics for NMR structure calculation with the new
program DYANA. J. Mol. Biol. 273, 283–298.
46. Franzoni, L., Lu
¨
cke, C., Pe
´
rez, C., Cavazzini, D., Rademacher,
M.,Ludwig,C.,Spisni,A.,Rossi,G.L.&Ru
¨
terjans, H. (2002)
Structure and backbone dynamics of apo- and holo-cellular
retinol-binding protein in solution. J. Biol. Chem. 277, 21983–
21997.
47. Gu
¨
ntert, P., Braun, W. & Wu

¨
thrich, K. (1991) Efficient compu-
tation of three-dimensional protein structures in solution from
nuclear magnetic resonance data using the program DIANA and
the supporting programs CALIBA, HABAS and GLOMSA.
J. Mol. Biol. 217, 517–530.
48. Laskowski, R.A., Rullmann, J.A.C., MacArthur, M.W., Kaptein,
R. & Thornton, J.M. (1996) AQUA and PROCHECK-NMR:
Programs for checking the quality of protein structures solved by
NMR. J. Biomol. NMR 8, 477–486.
49. Koradi, R., Billeter, M. & Wu
¨
thrich, K. (1996)
MOLMOL
:apro-
gram for display and analysis of macromolecular structures.
J. Mol. Graph. 14, 51–55.
50. Makhatadze, G.I. & Privalov, P.L. (1995) Energetics of protein
structure. Adv. Protein Chem. 47, 307–425.
51. Robinson, N.E. & Robinson, A.B. (2001) Deamidation of human
proteins. Proc. Natl Acad. Sci. USA 98, 12409–12413.
52. Geiger, T. & Clarke, S. (1987) Deamidation, isomerization, and
racemization at asparaginyl and aspartyl residues in peptides.
Succinimide-linked reactions that contribute to protein degrada-
tion. J. Biol. Chem. 262, 785–794.
53. Simonovic, M. & Volz, K. (2002) Atomic resolution structure of
a succinimide intermediate in E. coli CheY. J. Mol. Biol. 322,
663–667.
54. Tugarinov, V., Muhandiram, R., Ayed, A. & Kay, L.E. (2002)
Four-dimensional NMR spectroscopy of a 723-residue protein:

chemical shift assignments and secondary structure of malate
synthase G. J. Am. Chem. Soc. 124, 10025–10035.
55. Robinson, N.E. & Robinson, A.B. (2001) Molecular clocks. Proc.
NatlAcad.Sci.USA98, 944–949.
56. Robinson, N.E. & Robinson, A.B. (2001) Prediction of protein
deamidation rates from primary and three-dimensional structure.
Proc.NatlAcad.Sci.USA98, 4367–4372.
57. Cho, E.H., Alcaraz, R., Gumport, R.I. & Gardner, J.F. (2000)
Characterization of bacteriophage lambda excisionase mutants
defective in DNA binding. J. Bacteriol. 182, 5807–5812.
Supplementary material
The following material is available from http://blackwell
publishing.com/products/journals/suppmat/EJB/EJB3884/
EJB3884sm.htm
Appendix S1. Formulae used for the partial molar heat
capacity function [Cp,pr(T)] analyses.
4858 V. V. Rogov et al. (Eur. J. Biochem. 270) Ó FEBS 2003