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 factor
Trang 1Solution structure and stability of the full-length excisionase
from bacteriophage HK022
Vladimir V Rogov1,2, Christian Lu¨cke3, Lucia Muresanu1, Hans Wienk1,*, Ioana Kleinhaus1, Karla Werner1, Frank Lo¨hr1, Primozˇ Pristovsˇek4and Heinz Ru¨terjans1
1
Institute of Biophysical Chemistry, J.W Goethe-University of Frankfurt, Germany;2Institute of Protein Research, Pushchino, Russia;
3
Max Planck Research Unit for Enzymology of Protein Folding, Halle, Germany;4National 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 Cys28fi 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 1H2O-2H2O 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 winged-helix 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 attPand 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: hruet@bpc.uni-frankfurt.de
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)
Trang 2X2 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 Cys28fi Ser
substitution)displays a tertiary fold characteristic for the
winged 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, Cys28fi 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 winged-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 Cys28fi 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 NH4Cl, 40 gÆL)1unlabelled glycerol and Trace Elements I solution [23] The cells were incubated in a fermenter, with intensive aeration, to slightly beyond the mid-log phase (A600¼ 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 15N-labelled protein)or containing neither nitrogen nor carbon sources (for the preparation of
13C,15N-labelled protein) After a short starvation period (20 min), the cells were supplemented with either 1.0 gÆL)1
of 15NH4Cl or 1.0 gÆL)1 of 15NH4Cl, 0.5 gÆL)1 of
13C-labelled glucose and 1.5 gÆL)1of13C-labelled glycerol Cell growth was continued for another 20 min before the addition of isopropyl thio-b-D-galactoside (1.0 mM 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 mM 2-mercapto-ethanol to protect the thiol group of the protein against oxidation The cleared cell lysate was subjected to Ni2+ chelating chromatography Protein elution was performed using a linear gradient of 50–400 mMimidazole
The collected protein was cleaved with thrombin, and a subsequent preparative gel filtration, through a
Trang 32.6· 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 mMsodium phosphate (pH 6.5),
100 mM NaCl, 0.2 mM EDTA (disodium salt), 0.03%
NaN3] and subsequently reduced in volume to a final Xis
concentration of 0.1–1.2 mM, 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)1Æcm)1(A0.1%1cm¼ 1.614)at 280 nm;
5% 2H2O as lock substance and 0.1 mM
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 mMdithiothreitol 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 mMbuffer species (sodium acetate for the pH range
4.5–5.5; sodium phosphate for the pH range 6.0–7.0)and
100–400 mM NaCl For all Xis_wt samples, 1 mM
dithio-threitol was added to the buffer prior to dialysis The pH
values were measured at 25C 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¢- and 3¢ 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 mM
K2HPO4and 100 mMNaCl, pH 6.0 The double-stranded
X1 DNA was concentrated and equilibrated with the
corresponding NMR buffer in an Amicon ultrafiltration
device (membrane MWCO¼ 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 programWSCAL, based on the
principles described by Filimonov et al [25] and Privalov &
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 (1H/13C/15N)probes with XYZ-gradient capa-bility Proton chemical shifts were referenced relative to internal 4,4-dimethyl-4-silapentane-1-sulfonate; 15N and
13C chemical shifts were referenced indirectly using the corresponding chemical shift ratios [31]
3D Triple-resonance [15N,1H]-TROSY-HNCO [32], (HCA)CO(CA)NH [33] and [15N,1H]-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 a15N-edited 3D TOCSY-HSQC [38] The resonances of aromatic ring protons were assigned using 2D clean [1H-1H]-TOCSY spectra [39,40] recorded with spin-lock times of 59.3 ms and 5.6 ms, and 2D [1H-1H]-NOESY spectra in1H2O and2H2O 3D Heteronuclear NMR spectra were collected for determining the interproton distances in HK022 Xis_C28S
A 15N-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 13C-Edited 3D NOESY-HSQC spectra (mixing time 70 ms)were recorded in two different versions; optimized for Ha/Hb NOE-correlations (3D NOESY-[13C,1H]-HSQC)and for methyl group NOE-correlations (3D NOESY-(CT)-[13C,1H]-HSQC)
The proton exchange experiments were carried out
at 600 MHz and temperatures of 288 K and 298 K A reference [1H,15N]-HSQC spectrum was recorded with fully protonated Xis_C28S The sample was then lyophilized and dissolved in the same volume of ice-cold2H2O A series of identical [1H,15N]-HSQC spectra were acquired every
15 min during the first 2 h, and thereafter every 30 min, until all amide protons were completely exchanged with2H (after 12 h)
[15N,1H]-TROSY and homonuclear 1D spectra were collected to establish the Xis–DNA interaction at 288 K A reference [15N,1H]-TROSY spectrum was recorded using 0.5 mL of a 0.4 mM Xis_C28S sample (20 mM sodium phosphate, 100 mMNaCl, 6% glycerol, pH 6.5) ; equivalent spectra were acquired after each titration step with 25 lL of 1.2 mMX1 (20 bp DNA duplex)until a protein/DNA ratio
of 1 : 3 was reached The reverse order of titration, when
25 lL of a 1 mMXis sample was added stepwise to 0.5 mL
of a 0.4 mM X1 solution, was monitored by 1D (SW¼ 32.2 p.p.m.)and [15N,1H]-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
Trang 4Restraint generation and structure calculation
The NOE-based distance restraints were extracted from 3D
13C- and 15N-edited NOESY-HSQC spectra and
homo-nuclear 2D NOESY spectra in1H2O and2H2O Automated
assignments of the NOEs, based only on chemical shifts,
were obtained using the programNMR2ST[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 c1/c2of six Val
residues and d1/d2of three Leu residues A cis-proline residue
entry was added to the standardDYANAresidue library for
correct calculation of Pro32, which has been identified as a cis
isomer No additions were made in the standardDYANA
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 found with the usage of
the standard Asn66 residue
For calculating the final structure ensemble, 868
NOE-derived distance restraints and 34 hydrogen bond restraints
(dHO£ 2.1 A˚ and dNO£ 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 bestDYANAconformers The minimized structures were
analyzed using PROCHECK-NMR 3.4 [48] The structure
images were prepared using theMOLMOLprogram [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 (Tm) 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 (Tm¼ 41.6 C)when recorded at pH 5.5, the thermodynamic analysis of this function indicated a significant denatured state population (FD)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)1ÆK)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)1ÆK)1)
DH
(kJÆmol)1)
DS
(kJÆmol)1ÆK)1)
DG
(kJÆmol)1) F N
a
Data for the Xis_wt thermal denaturation in the presence of 400 m NaCl.bData for the Xis_C28S thermal denaturation.
Trang 5pH 6.5 and 7.0 the protein stability was higher; the FDdid
not exceed 3%, even at 30C
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 mM In the latter, the Tmwas shifted by +5.7 (from
41.6 to 47.3C) 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 Cys28fi Ser amino acid substitution on
the protein stability was very small (Table 1) The difference
in Tmvalues (51.4C for Xis_wt vs 51.9 C for Xis_C28S)
was within the limits of experimental error
The high reversibility, the independence of Tmon 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 is shown in Fig 2B The specific (JÆg)1)presentation of DH revealed that the Xis denaturation enthalpy at 130C (DHspecific¼ 35 JÆg)1)was significantly lower than those values reported for small globular proteins (50 JÆg)1± 15% at 130C [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 DHspecific of 50 JÆg)1 at 130C 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 , pH 6.5) (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 DCpint (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).
Trang 6The calculated temperature dependences of FNand FDat
the NMR conditions used are presented for Xis_wt in
Fig 2C According to the analysis, FDstarts to increase
rapidly at temperatures only above 35C Thus, 30 C
(FD¼ 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
13C 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 [1H,15N]-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 3D15N-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 the15N-edited 3D NOESY-HSQC spectra
Fig 3 Assignment of the HN resonances in
HK022 Xis_C28S and Xis_wt spectra (A)
[1H,15N]-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.
Trang 7of 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 Caand Cbsignals
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 [15N,1
H]-TROSY-HNCACB spectrum
Figure 4B presents the sequential connectivities of
Xis_C28S residues 65–69, as observed in the 3D [15N,1
H]-TROSY-HNCACB (left panel) and 3D (HCA)CO(CA)NH
(right panel)spectra In the HNCACB experiment, the
resonance signals of13C directly coupled to15N are always
positive, whereas the relayed 13C resonance signals are
negative The sequential connectivity involving the Cb
resonance of IsoAsp66 is therefore positive in sign, whereas
the Casignal is negative (Fig 4B, left panel, G67 strip) In
contrast, the intraresidual connectivities involving the Ca
and Cb resonances of IsoAsp66 show the usual signs
(Fig 4B, left panel, N66 strip)
In the (HCA)CO(CA)NH experiment, the intraresidually
observed IsoAsp6613C(O)resonance [i.e the13C(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
Ccof 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 [15N,1H]-TROSY-HNCACB (left panel)and (HCA)CO (CA)NH (right panel) spectra of HK022 Xis_C28S at the1HN and15N 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.
Trang 8size 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 Caatom 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 random coil 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 wing 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.
Trang 9NOE 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)
A series of [15N,1H]-TROSY and 1D 1H 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 mM), 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 [15N,1H]-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
Maximal violation (A˚)0.36
Structural precision, RMSD (A˚)
Backbone atomsb(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˚, < 4.5 A˚ 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 Ha, white.
Trang 10a 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 Xis features only two regular
b-strands and two extended sequence segments [20] Second,
the wing 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 wing 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] At pH 5.0, 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 Cys28fi 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) [15N, 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.