Bannikova, G. E.

Blagova, E. V.

Dementiev, A. A.

Mikchailov, A. M.

Morgunova, E. Yu.

Shlyapnikov, S. V.

Vainshtien, B. K.

Varlamov, V. P.

Revistes Catalanes amb Accés Obert

CRYSTALLIZATION AND PRELIMINARY X-RAY
INVESTIGATION OF THE SERRATIA MARCESCENS
NUCLEASE
E.V. BLAGOVA'`, E. Yu. MORGUNOVA''-, G.E. BANNIKOVA'-",
A.A. DEMENTIEV''''' , A.M. MIKCHAILOV', S. V. SHLYAPNIKOV'
V.P. VARLAMOV", B.K. VAINSHTIEN"
O Institute of Crystallography of the USSR Academy of Sciences;
(`) "Bioengineriya " Centre at the Institute of Molecular Biology;
Institute of Molecular Biology of the USSR Academy of Sciences
The Serratia marccscens endonuclease (EC 3.1.4.9) splits DNA and
RNA with the formation of 5'-phosphorylated oligonucleotides. The en-
donuclease is the object of detailed investigations, being conducted in a num-
ber of scientific centers. At present, however, the results of various authors
on the characteristics of the enzyme and the study of its properties are large-
Iv contradictory. On the one hand, preparations of S. marccscens nuclease
differing in certain physico-chemical properties and structural parameters
have been isolated from various strains of bacteria [ 1-5]. On the other hand
(at least in the case of nucleases secreted by the strains W 225 and SM6), the-
re is weighty evidence for complete identity of the structures of the proteins
[6, 7].The ambiguity of the conclusions of individual publications evidently
may be associated with the absence of sufficient amounts of highly purified
protein. Inevitable consequence of this is the need to search for strains of
bacteria with increased expression of nuclease and, to develop effective met-
hods of isolation of preparative amounts of the enzyme.
We attempted to purify the enzyme expressed by S. marccscens bacte-
ria strain B10 Ml producing a considerable amount of nuclease activity
compared with the original material of so far investigated bacterial strains
(Bu 21 1, ATCC 9986 and 24) [8].
We report here the isolation of two isoforms of S. marccscens nuclease,
nucleases S.rn, and S.m„ in electrophoretically and structurally homoge-
neous states, the crystallization of the nuclease S.ml, the preparation of its
heavy atom derivatives and the results of the preliminary X-ray diffraction
analysis of the protein.
The crude preparations of S. marcescc'ns nuclease were obtained by
means of l1gand-exchange chromatography on the iminodiacetate-agarose
1 ButII.Soc. Cat . Cicn.J,SoI.XIII, Num.1,1992
:,^.t 1.1.151.:I(IC)I':11.1'11.
in Cu'' -torte (Table I), based upon an interaction of protein histidine resi-
dueswith chelaud n-ansition metal ions, and then by ion-exchange chronul-
u>graphy of the nuclease on phosphoccllulosc ^9]. After this two-steps pu-
rification procedure the enr.vme showed a specific activity of 5x10'' U/mg.
The nuclease was free from phosphar<ise, phosphomonoesterase and phosp-
hodiesterase acti^^ities. SI)S-gel electrophoresis indicated purit^^ of the pro-
teinaccording to the molecular weight (figure 1, N2). At the same time the-
re was some e^^idence for structural heterogeneit^• of the enz^^me. Non-de-
nanirating electrophoresis of the purified nuclrise sho^ti^ed two separau
protein bands containing active en^.vmes (figure I, N3). Further elecu-ofo-
cusing of the protein yielded two active peaks with isoelecu-ic points of 7.1
and 6J. Analysis of the N-terminus of the protein sample indicated the coe-
xistencc of two different forms of N-terminal amino acid residues, Glu and
Asp. F.^^cntually, we tried to crystallize the S. rrt^ircescerrs nuclease after the
abo^^c described purification procedure and obtained a lot of small cr^^srlls
in the protein drop (the sire Iess than 0.1 nu»), which were not suitable for
X-ra^^ diffraction analysis (figure ?).
We succeeded in separating t^ao electrophoreticall^^ different nucleases
b^^ ion-exchange chronritograph^° on DEAF-To^•opearl 6^OS (figure 3).
'These isozvmes, nucleases S.rn^ and S.m„ showed the same isoelectric
points and N-terminal amino acid residues as the initial enzyme compo-
nents and the same specific activities as unfi-actionatcd nuclease.
The structure analysis of nucleases S.m^ and .S. rn, showed a high degree
of similarity both to each other and to S. marcc^scrrrs nucleases from strains
W ZZ5 and SvI6 [ 10^, (figw-e -}). In conn-ast to the nuclease S.rn^, the nuclea-
se S.rn, contains a supplement n-ipeptide segment Asp-'I'hr-Leo in the N-
terminal portion of the molecule. The difference found in the N-terminal
portion of nuclease isoforms, containing a negati^z^l^^ charged dicarbosvlic
acid residue, can explain the corresponding differences in chromatographic
behaaiour of the isoforms and ^^alucs of their isoelecn-ic points.
Table 1. Purification scheme of Serratia marcescens nuclease.
Step
Volume
(n,l) A,^,=
Spec. acti^^.
(U/ml 10 ^)
Pacific.
Ic^^el
Yield
(°4,)
Culture fluid 10000 9.5C ?J I.C IOC
Cu''-II)A-agarose 18C 9.80 116.C ^1?.3 78
phosphoccllulosc 85 C.98 185.0 66.3 ?9
UI^:AI)l^:-'poaopearl
nuclraseS.m^ 45 0.18 -14.5 58.0 5
nucleaseS.rn, 50 0.95 ?56.G 96.0 33
^ Bu[ILti^^r.CacCicn.],b'ol.AI I I, ^lum.l,l 71?
CR YSTALLI%ATION AND PRLL I AfINAR Y X-RA 1. 385
1
41111111111011
!^ ". - z 3 y 5
Fig. 1. PAG electrophoresis under denaturation conditions ( 1-2) and without denaturants (3-
5): 1 - protein markers, 2, 3 - nuclease sample after phosphocellulose chromatography, 4 - nu-
clease S.m2, 5 - nuclease S.m,.
Fig. 2. Crystals of S.m nuclease after two-steps purification procedure.
[Butt I.So . (].u.Cien.],VoI.XII I,Num.1,1992
386 E.V. BLAG(VA FT AL.
-6
Activity, U/ml•10
,a
nuclease Sm 2
50
2
0
100 150 200 250
Volume, ml
Fig. 3. Chromatographic separation (1 - A,80, 2 - endonuclease activity) of the nucleases S.m1
and S. m2 on a column with DEAE - Toyopearl 650S.
SM6 MET -ARG-PHE- ASN-ASN -LYS-ASN-VAL-GLU-LEU-GLU--ALA-LEU-LEU-PHE-
SmI
Sm2
SM6 -ALA-ALA-GLN-ALA-SER-ALA-ASP-THR-LEU-GLU-SER-ILE-ASP-ASN-CYS-
S MI
S.m2 ASP-THR-LEU-GLU-SER-ILE-ASP-ASN-CYS-
SM6 -ALA -VAL-GLY-CYS-PRO-THR-GLY-GLY-SER- SER-ASN -VAL-SER-ILE-VAL-
S.mI -ALA -VAL-GLY-CYS-PRO-THR-GLY-GLY-SER- SER-ASN -VAL-SER-ILE-VAL-
S.m2 -ALA -VAL-GLY-CYS-PRO-THR-GLY-GLY-SER -SER-ASN -VAL-SER-ILE-VAL-
SM6 -ARG-HIS-ALA-TYR-THR-LEU-ASN-ASN-ASN-SER-THR-THR-LYS-PHE-ALA-
S.mI -ARG-HIS-ALA-TYR-THR-LEU-ASN-ASN-ASN-SER-THR-THR-LYS-PHE-ALA-
S.m2 -ARG-HIS-ALA-TYR-THR-LEU-ASN-ASN-ASN-SER-THR-THR-LYS-PHE-ALA-
Fig. 4. N-terminal region of amino acid sequences of nucleases S.mt, S.m, and SM6.
GLU-SER-ILE-ASP-ASN-CYS-
lButll.Soc.Cat . Cicn.1,Vol.XIIl, Nurn.1,1992
CRY7 ALLI/.-17 /ON AND PRELIMINARY X-RAY 387
A general prediction of the distribution of secondary structure ele-
ments of the proteins of this type applying a modified Chou- Fasman met-
hod [11 ] indicates that S. marcescens nucleases are a/(3-proteins, which con-
tain one large and three small regions identified as a-helices and six -
plea-ted structural conformations located between these a-helices (figure 5).
The isolation of two isoforms of the protein in a homogeneous state
made it possible to carry out a successful crystallization and to obtain high-
ly ordered crystals of S. marcescens nuclease suitable for X-ray analysis.
These crystals were grown at 4°C from 10 mM TRIS-HCI buffer, pH 8.3 in
"hanging" or "sitting" drops as well as by a microdialysis method. The pro-
tein concentration varied between 1 and 3.5%, and different precipitants
were applied.
Crystals of the best quality and maximum geometrical sizes were ob-
tained by the vapour diffusion technique in 10 pl drops with 1%, protein so-
lution and ammonium sulphate as a precipitant (1.2- 1.6M). Two crystal ha-
bits were found to be present in the same solution: prolongated (rod-like)
and rhombic prisms (figure 6,a,b), the rhombic crystals being usually obser-
ved in the presence of a small amount of dioxane (2-3%). Crystals of both
forms having the approximate dimensions of 0.7x0.3x0.2 mm (rod-like) and
0.4x0.4x0.2 mm (rhombic prisms) were suitable for X-ray analysis. Crystals
Fig. 5. Prediction of the secondary structure of the nuclease S.m strain W 225. NP denotes the
multiplicity of the u-helical (1) and 13-structure (2) conformational potentials of the hexa- and
pentapeptide segments of the protein.
40 80 120 160 200 240
Residue number
[But!1.Soc.Cat. Cien.],Vol. XI II,Nun.1,1992
i,AR / 1. /31 1GUI.1 /I I/.
I
v
l'ig. 6. Photographs of the nuclease S.mn, crystals: a. stick-like, b. rhombic, c. the third form of
crystals.
[BntII.ti
-
(:..t.Cien.],Vol.XI1I,Num.1,1992
CKFST10,ii. 770 A'; 1,ti'l91'Kh:Llall ;ti';1K)".A" 389
formed within two weeks and reached their maximum size within 1.5 - 2
months.
Another, third form of crystals was obtained in slightly different con-
ditions: the final pH of the solution in the protein drop was higher than in
the previous cases (figure 6,c).
Preliminary X-ray investigation was carried out on the rod-like crys-
tals. It was determined from X-ray precession photographs (Fig. 7), that the
crystals of S.m1 nuclease belong to an orthorhombic space group p2,2,2,
with unit cell dimensions a = 69.0, b = 106.7, c = 74.8 A; contain two mo-
lecules in an asymmetric unit and the value of packing density (V) is equal
to 2.3 A3/Da. The crystals diffract to at least 1.6 A resolution. The X-ray
diffraction experiments were carried out with radiation of a, = 1.54 A, mo-
nochromatized by a graphite monochromator. The intensities of control re-
flections decreased by 10% in 3 days.
We attempted to solve the phase problem in our case by the method of
isomorphous heavy-atom derivatives, as the structures of homologous ob-
jects have not been solved yet. More than 20 heavy-atom compounds were
tested in this work. The refinement of unit cell parameters and X-ray dif-
fraction data from the native crystals of nuclease S. m1 and crystals of its hea-
vy-atom derivatives were obtained on an automatic Syntex P2, diffractome-
Fig. 7. X-ray diffraction precession photograph of the nuclease S.m, crystals.
[ButII.Soc.Cat.Ci&n.],Vo1.XIII,NUm.1,1992
390 E.V. BLAGOVA ET AL.
ter. X-ray diffraction sets were measured by the ca-scan method. The angle
width of intensity peaks, as a rule, did not exceed 0.28°, that also indicated
a rather high quality of the crystals.
Among various heavy-atom compounds the Pt- and UO,- derivatives
were found to be the best ones (Tables 2, 3). Both derivatives were obtained
by soaking the native nuclease S.mi crystals in 0.5 mM K, Pt(SCN)4 solu-
tion for 5 days and in 1 mM UO, (Ac), solution for 120-130 hours, respec-
tively. The medium contrast for the Pt- derivative was 20%, for U02- deri-
vative - 16%.
Further crystallographic investigations of nuclease S.m, as well as the
crystallization of nuclease S.m, are in progress.
Table 2. Summary of data collection on the Syntex P2,.
Conditions, No. of No. of Diffraction R(I),,, R-fac,
Data set
mM tryst. unique refl. limit, /„ (YO
Native - 5 10197 2.87 4.8 4.6
K,Pt(SCN), 0.5 I 2002 5.2 3.7 20.4
UO,(AC), 1.0 1 3780 4.5 4.3 17.3
R-fac=<IIF,,l,I-IF,,II>/<IFpI>
Table 3. R-fac values distribution in the resolution areas.
K,Pt(SCN)4
d, A 74.5 -27.5 -16.9 -12.2 -9.5 -7.8 -6.6 -5.8 -5.1
R-fac, % 23.0 30.5 27.4 23.4 22.5 22.3 21.7 20.2
UO,(Ac)2
d, A 32.0 -18.1 -12.7 -9.7 -7.9 -6.6 -5.7 -5.0 -4.5
R-fac, % 28.3 16.8 12.0 9.8 8.8 8.6 8.2 7.8
ABSTRACT
Two isoforms of an extracellular endonuclease, nucleases S.m, and S.m, were pu-
rified from culture fluid of Serratia marcescens strain B10 Ml by the ligand-exchange
chromatography on Cu"-iminodiacetate-agarose and ion-exchange chromatography
[Budl.Soc.Cat.Cien.],Vol.XIII,Min. 1,1992
CRY.STALLIZATIOX AX'D PRELI,tIIXARY X-RAY 391
on phosphocellulose and DEAE-Toyopearl 650S. The pI-values for nucleases S.m, and
S.m, were found to be 7.1 and 6.7, respectively. The amino acid analysis and N-terminal
amino acid sequencing of the proteins showed a high degree of homology between the
enzymes. Three different forms of nuclease S.m1 crystals were obtained from ammo-
nium sulphate solution by a vapour diffusion technique. The crystals used for X-ray
analysis belong to the space group p2i2,2, with unit cell constants a = 69.0, b = 106.7,
c = 74.8 A, contain two molecules in an asymmetric unit, packing density V. = 2.313/
Da, and diffract to at least 1.61 resolution. The Pt- and U02-derivatives of the protein
were obtained. Preliminary X-ray investigation of nuclease S.m, crystals was carried
out.
[Butll.Soc.Cat. Cien.],Vol.XIII,Num.1,1992
392 F. V. BLAGOVA ET AL.
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[ButII.Soc.Cat. Cicn.],Vol.XI I I,Num.1,1992


Crystallization and preliminary X-ray investigation of the «Serratia Marcescens» nuclease

Crystallization and preliminary X-ray investigation of the «Serratia Marcescens» nuclease

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Revistes Catalanes amb Accés Obert