The 2.0 A˚ X-ray structure shows that 90-MtCM is an all a-helical homodimer Protein Data Bank ID: 2QBV with the topology of Escheri-chia coliCM EcCM, and that both protomers contribute t
Trang 1the intracellular chorismate mutase (Rv0948c) from
chorismate mutase (y2828) from Yersinia pestis
Sook-Kyung Kim*, Sathyavelu K Reddy, Bryant C Nelson, Howard Robinson, Prasad T Reddy and Jane E Ladner
Biochemical Science Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg,
MD, USA
Keywords
chorismate mutase; Mycobacterium
tuberculosis; pathogenesis; shikimate
pathway; Yersinia pestis
Correspondence
P T Reddy, Biochemical Science Division,
Bldg 227, Rm B244, Chemical Science and
Technology Laboratory, National Institute of
Standards and Technology, Gaithersburg,
MD 20899, USA
Fax: +1 301 975 8505
Tel: +1 301 975 4871
E-mail: prasad.reddy@nist.gov
J E Ladner, Biochemical Science Division,
Bldg 227, Rm B244, Chemical Science and
Technology Laboratory, National Institute of
Standards and Technology, Gaithersburg,
MD 20899, USA
Fax: +1 240 314 6225
Tel: +1 240 314 6206
E-mail: jane.ladner@nist.gov
Present addresses
*Division of Metrology for Quality Life,
Korea Research Institute of Standards and
Science, Daejeon, Republic of Korea
Division of Molecular Biology, Department
of Zoology, Sri Venkateswara University,
Tirupati, Andhra Pradesh, India
Biology Department, Brookhaven National
Laboratory, Upton, NY, USA
(Received 9 May 2008, revised 25 July
2008, accepted 30 July 2008)
doi:10.1111/j.1742-4658.2008.06621.x
The Rv0948c gene from Mycobacterium tuberculosis H37Rv encodes a 90 amino acid protein as the natural gene product with chorismate mutase (CM) activity The protein, 90-MtCM, exhibits Michaelis–Menten kinetics with a kcat of 5.5 ± 0.2 s)1 and a Km of 1500 ± 100 lm at 37C and
pH 7.5 The 2.0 A˚ X-ray structure shows that 90-MtCM is an all a-helical homodimer (Protein Data Bank ID: 2QBV) with the topology of Escheri-chia coliCM (EcCM), and that both protomers contribute to each catalytic site Superimposition onto the structure of EcCM and the sequence align-ment shows that the C-terminus helix 3 is shortened The absence of two residues in the active site of 90-MtCM corresponding to Ser84 and Gln88
of EcCM appears to be one reason for the low kcat Hence, 90-MtCM belongs to a subfamily of a-helical AroQ CMs termed AroQd. The CM gene (y2828) from Yersinia pestis encodes a 186 amino acid protein with an N-terminal signal peptide that directs the protein to the periplasm The mature protein, *YpCM, exhibits Michaelis–Menten kinetics with a kcatof
70 ± 5 s)1and Kmof 500 ± 50 lm at 37C and pH 7.5 The 2.1 A˚ X-ray structure shows that *YpCM is an all a-helical protein, and functions as a homodimer, and that each protomer has an independent catalytic unit (Protein Data Bank ID: 2GBB) *YpCM belongs to the AroQc class of CMs, and is similar to the secreted CM (Rv1885c, *MtCM) from M tuber-culosis
Abbreviations
*MtCM, secreted chorismate mutase from Mycobacterium tuberculosis; *YpCM, secreted chorismate mutase from Yersinia pestis; CM, chorismate mutase; EcCM, chorismate mutase domain of chorismate mutase–prephenate dehydratase from Escherichia coli; IPTG, isopropyl-thio-b- D -galactoside; MtCM, intracellular chorismate mutase from Mycobacterium tuberculosis; PfCM, chorismate mutase from Pyrococcus furiosus; ScCM, allosteric chorismate mutase from Saccharomyces cerevisiae; TSA, transition state analog; TtCM, chorismate mutase from Thermus thermophilus.
Trang 2Chorismate mutase (CM) (EC 5.4.99.5), a shikimate
pathway enzyme [1], catalyzes the pericyclic
rearrange-ment of chorismate to prephenate [2] Subsequent to
this conversion, prephenate dehydrogenase and
prephenate dehydratase catalyze the biosynthesis of
tyrosine and phenylalanine, respectively As this
bio-synthetic pathway is absent in mammals but is
essen-tial for the survival of bacteria and fungi, CM is often
targeted for the development of inhibitors for
micro-bial pathogens This work was aimed at the
character-ization of CM from Mycobacterium tuberculosis
H37Rv, a dreaded pathogen that claims two million
human lives annually [3]
Annotation of the genome of M tuberculosis H37Rv
revealed two genes for CM [4] The Rv1885c gene
encodes a secreted CM (*MtCM) and the Rv0948c
gene encodes an intracellular CM (MtCM) Sasso et al
[5], Prakash et al [6] and Kim et al [7] have
character-ized *MtCM Kim et al [7] have shown that *MtCM
has in fact an extracellular destination in M
tuber-culosis Prakash et al [6] conducted a brief study of
the recombinant MtCM Our work is aimed at the
fur-ther characterization of MtCM The true primary
sequence of MtCM is complicated by virtue of a
number of presumptive in-frame initiator methionines
preceded by a reasonable ribosome-binding sequence
The annotation Rv0948c for MtCM in a laboratory
strain of M tuberculosis H37Rv would encode a 105
amino acid protein (105-MtCM) [4], whereas the
anno-tation MT0975 for MtCM in the CDC1551 strain
would encode a 217 amino acid protein (217-MtCM)
[8] Furthermore, alignment of 105-MtCM with the
genetically engineered Escherichia coli CM (EcCM) [9]
shows that the 105 amino acid protein has extra amino
acids beyond the N-terminus of EcCM Hence, we
cloned 90-MtCM beginning with Met16 in Rv0948c
We determined the 3D structure of the 90-MtCM and
kinetic parameters of all three proteins The 90-MtCM
is an AroQ class CM and the protein functions as a
dimer In this article, we also report on the cloning of
the gene encoding the secreted CM from Yersinia
pestis (*YpCM, y2828), purification of the protein,
investigation of the properties of the enzyme, and the
crystal structure analysis of the protein
Results and Discussion
Annotation of CMs in M tuberculosis H37Rv
The difference in annotation of MtCM arose from an
in-frame initiator ATG codon in MT0975 (217-MtCM)
and in Rv0948c (105-MtCM) Furthermore, the
N-ter-minus of 105-MtCM has 22 more residues than the
CM domain of E coli prephenate dehydratase (Fig 1) There is an in-frame methionine at position 16 of 105-MtCM and a purine-rich sequence analogous to the Shine–Dalgarno sequence about 10 nucleotides upstream of Met16 We reasoned that this Met16 could be the real initiator and consequently would pro-duce a 90 amino acid protein (90-MtCM) We charac-terized all three versions of the putative intracellular
CM, i.e 217-MtCM, 105-MtCM, and 90-MtCM In a recent publication, Schneider et al [10] observed simi-lar ambiguity about the translation start site(s) in the gene MSMEG5513 for an intracellular CM, a homolog
of Rv0948c, from the annotation of the Mycobacte-rium smegmatis genome They determined the transla-tion start site by translation fusion with the b-galactosidase gene, and showed that the first methio-nine in MSMEG5513 is the ‘real initiator’ Schneider
et al [10] did not determine the translation start site for Rv0948c
Production and purification of MtCM The 105-MtCM was overproduced as a fusion protein with the subtilisin prodomain (Fig 2, lane 2) The fusion protein was completely soluble (Fig 2, lane 3) Cleavage of 105-MtCM from the prodomain was trig-gered by fluoride-induced subtilisin activity (Fig 2, lane 5) We observed three major protein products at this stage of purification: intact fusion protein (per-haps not very tightly bound), 105-MtCM, and an unidentified lower molecular mass protein Hence, 105-MtCM was further purified by molecular sieve chromatography to near homogeneity (Fig 2, lane 6) The molecular mass of 105-MtCM determined
by MALDI-TOF MS was 11 771 Da (theoretical mass = 11 771 Da), and established that the protein is intact Similarly, 90-MtCM was overproduced as a fusion protein with the subtilisin prodomain, and the protein was purified to homogeneity (Fig 3, lane 5)
As can be seen in lane 5 of Fig 3, 90-MtCM migrated
as a 6 kDa protein Hence, we determined the molecular mass of 90-MtCM by MALDI-TOF MS as
10 090 ± 1 Da, which is identical to the theoretical mass of 10 090 Da The yield of 105-MtCM and 90-MtCM was 1 mg per 1 L of culture Activity mea-surements for CM using these two proteins showed that both proteins catalyze the conversion of choris-mate to prephenate (see kinetic measurements for kcat) The 217-MtCM, purified from the subtilisin column, had 1⁄ 50th of the CM activity of 90-MtCM and 105-MtCM at the same stage of purification Hence, we did not further purify or characterize 217-MtCM We conclude that the annotation of the MT0975 gene was
Trang 3misled by an upstream in-frame initiator methionine
preceded by the Shine–Dalgarno sequence
Purification of *YpCM from the periplasmic fluid
of E coli
*YpCM production was induced with 10 lm
isopro-pyl-thio-b-d-galactoside (IPTG) Periplasmic proteins
were isolated as described for *MtCM [7] The peri-plasmic fluid was concentrated to 500 lL in a Milli-pore centrifugal tube with a 5000 Da molecular mass cutoff *YpCM was purified on a 210 mL Biosep SEC-3000 HPLC column (Phenomenex, Torrance, CA, USA), equilibrated and eluted with 50 mm Tris⁄ HCl (pH 7.5), 1 mm EDTA, and 100 mm NaCl
Fig 1 Alignment of 90-MtCM with AroQ a CMs The alignment begins with amino acid 13 in 90-MtCM (the numbering begins with amino acid 1 in the 90 amino acid protein) Amino acids 1–12 were not seen in the electron density map; their sequence is shown above the align-ment In the structural alignment of MtCM, EcCM, PfCM, and TtCM by MATRAS [12], the top line indicates the average secondary structure (AVE_SECSTR); H, helical; T hydrogen-bonded turn; C, coil; S, bend Capital letters indicate agreement for all structures The active site resi-dues in EcCM are highlighted, and are shadowed when similar in the other sequences C-terminal resiresi-dues that were not visible in the struc-tures are shown as lower-case letters for MtCM, PfCM, and TtCM At the top of the figure, the 15 N-terminal residues of the 105-MtCM construct are shown.
32.5 Subtilisin prodomain:105 aa MtCM fusion protein 25.0
16.5
105 aa MtCM monomer 6.5
Fig 2 SDS ⁄ PAGE (16%) of the production and purification of
105-MtCM Lane 1: uninduced cell-free extract of E coli BL21(DE3)
harboring the pG58–105-MtCM clone (25 lg of protein) Lane 2:
induced cell-free extract of E coli BL21(DE3) harboring the pG58–
105-MtCM clone (25 lg of protein) Lane 3: 48 000 g supernatant
of induced cells (same volume as used in lane 2) Lane 4: flow
through from subtilisin column (same volume as used in lane 2).
Lane 5: 10 lg of protein(s) eluted after equilibration with 100 m M
sodium fluoride Lane 6: 5 lg of purified 105-MtCM from a
Sepha-dex G-75 column Lane 7: molecular mass markers.
32.5
25.0
16.5
Subtilisin prodomain: 90 aa MtCM fusion protein 6.5
90 aa MtCM monomer
Fig 3 SDS ⁄ PAGE (16%) of the production and purification of 90-MtCM Lane 1: induced cell-free extract of E coli BL21(DE3) harboring the pG58–90-MtCM clone (25 lg of protein) Lane 2:
48 000 g supernatant of induced cells (same volume as used in lane 1) Lane 3: flow through from subtilisin column (same volume
as used in lane 1) Lane 4: 10 lg of protein(s) eluted after equilibra-tion with 100 m M sodium fluoride Lane 5: 13 lg of purified 90-MtCM from a Sephadex G-75 column Lane 6: molecular mass markers.
Trang 4Kinetic measurements
Assays for CM were performed with 90-MtCM and
105-MtCM at chorismate concentrations of 100 lm to
4 mm Both MtCMs catalyzed the conversion of
chorismate to prephenate with a kcat of 5.5 ± 0.2 s)1
and a Km of 1500 ± 100 lm at 37C and pH 7.5
(Table 1) The kcat for MtCM is about 14-fold lower
than that reported for EcCM (72 s)1) [11] The Km of
1500 lm chorismate for MtCMs is five times higher
than that observed for EcCM One obvious reason for
the low kcat and high Km exhibited by MtCM is the
absence of two of the substrate-binding residues found
in the C-terminus of EcCM (Fig 1) In contrast,
*YpCM, in which all the catalytic site residues are
pre-served, exhibits a high kcat of 70 ± 5 s)1, similar to
that for EcCM
Crystal structure of 90-MtCM The crystal structure of 90-MtCM shows clearly that the molecule is an all a-helical homodimer (Protein Data
Table 1 Comparison of the catalytic properties of MtCM, EcCM,
and *YpCM MtCM proteins and *YpCM were purified as
described in Experimental procedures One microgram of MtCM or
200 ng of *YpCM in 10 lL was used in each assay of 0.3 mL of
buffer The buffer consisted of 50 m M Tris ⁄ HCl (pH 7.5), 0.5 m M
EDTA, 0.1 mg BSAÆmL)1, and 10 m M b-mercaptoethanol
Choris-mate concentrations were varied from 0.25 to 4 m M Assays were
performed at 37 C for 5 min [34], and the reaction was stopped
with 0.3 mL of 1 M HCl After further incubation for 10 min at
37 C to convert prephenate to phenylpyruvate, 0.6 mL of 2.5 M
NaOH was added The absorbance of the phenylpyruvate
chromo-phore was read at 320 nm Blanks without the enzyme were
main-tained for each of the chorismate concentrations to account for the
nonenzymatic conversion of chorismate to prephenate Results are
the average of three experiments The kinetic data for EcCM were
from the literature [11], measured at 30 C.
A
B
C
Fig 4 Crystal structure of 90-MtCM (A) The homodimer is shown
as a stereo cartoon on the top with one polypeptide chain in blue
and the other in green (B) The superimposition of a single chain of
TtCM, PfCM and EcCM onto 90-MtCM is shown, with the TSA
from EcCM marking the active site The approximate positions of
the N-termini and C-termini are labeled in the same color as the
polypeptide chain The three helices are labeled H1, H2, and H3.
(C) Helix 3 from each of the four structures is shown The helices
were taken from the superimposed structures and then separated
by translating each horizontally in the plane of the page The figures
were drawn with PYMOL (http://www.pymol.org).
Trang 5Bank ID: 2QBV; Fig 4) The polypeptide chain has one
long 36 residue helix (helix 1), an eight residue loop, an
11 residue helix (helix 2), a two residue loop, and a 15
residue helix (helix 3) The buried surface area of the
dimer is 3810 A˚2 This crystal form has one protomer in
the asymmetric unit; the complete molecule is generated
by a crystallographic two-fold The data and refinement
statistics are shown in Table 2 The Ramachandran plot
has 96.9% of the residues in the most favored region
and 3.1% in the additional allowed region Five residues
were modeled with alternative conformations No
inter-pretable density was observed for the first 12 residues or
for the last five residues When the model is numbered
according to the 90 residue protein, residues 13–85 are
seen in the electron density map Using matras [12] to
compare the structure with a representative library of
structures, three structures stood out as very similar;
these are Protein Data Bank IDs 2D8D, 1YBZ and
1ECM 2D8D and 1YBZ are annotated as CMs from
Thermus thermophilus (TtCM) and Pyrococcus furiosus
(PfCM), respectively, from Structural Genomics
pro-jects on these organisms 1ECM [13] is a genetically
engineered 109 amino acid CM domain from E coli
The dimer of 90-MtCM is shown in Fig 4A, and the
superimposition of the four structures for a single
poly-peptide chain is shown in Fig 4B It is apparent from
the structural alignment that helix 3 of 90-MtCM is a
shorter version of helix 3 of EcCM, TtCM and PfCM lacking two of the binding site residues (Fig 4C) corre-sponding to Ser84 and Gln88 of EcCM as discussed below
During the preparation of this article, we were made aware of another deposited Protein Data Bank file for 90-MtCM, 2VKL (M Okvist, K Roderer, S Sasso,
P Kast, and U Krengel, unpublished data) In the structure of 90-MtCM in 2VKL, there is a malate ion from the buffer in the active site of the enzyme Malate mimics endo-oxabicyclic dicarboxylic acid, the transition state analog (TSA), in much the same fashion as citrate that we observed in our *YpCM structure (Protein Data Bank ID: 2GBB) We crystallized 90-MtCM in the pres-ence of citrate but did not see citrate in the active site However, we studied the effect of citrate on 90-MtCM activity, and found citrate to have some kind of inhibi-tory effect from preliminary results (data not shown) The inhibition is not a salt effect, because sodium chlo-ride and sodium acetate had no effect on the activity The rmsd on C-alphas between the two 90-MtCM structures is 0.69 A˚ One difference between the struc-tures is that five residues at the C-terminal end are dis-ordered in our structure (2QBV) and only one residue
is disordered in 2VKL In fact, although the space group is the same for both structures, the c-axis is
10 A˚ shorter in 2VKL This difference is due to crystal packing, which allows the C-terminal residues of neighboring molecules (not in the same dimer) to inter-act and the tail of one protomer to almost reach the active site of the neighbor
Active site of 90-MtCM The structure of EcCM includes the TSA, which clearly identifies the active site From structural and sequence homology with EcCM, the active site residues
of 90-MtCM can be identified, and are shown in Fig 5 The striking difference between EcCM and 90-MtCM is that the EcCM residues Ser84 and Gln88 are absent in 90-MtCM (Fig 1) Structurally, Ser84 of EcCM lines up with Gly84 of 90-MtCM The final five residues, GRLGH, of 90-MtCM are not seen in the electron density map However, none of these residues
is a candidate for performing the role of Gln88 in EcCM Of the other two structures, PfCM has the conserved Ser70 and Gln74, and TtCM has Ser81 and Glu85 There are two Protein Data Bank files for TtCM; in the file 2D8D, Glu85 is only seen in one of the two chains in the structure, which has a dimer in the asymmetric unit, and in the file 2D8E, which has one chain in the asymmetric unit, all of the C-terminal residues are seen Another difference is the loop
orien-Table 2 Diffraction data and refinement statistics showing
over-all ⁄ high-resolution shell (2.18–2.10 A˚) values where appropriate.
Diffraction data
Cell dimensions (a, b, c) (A ˚ ) 59.9, 59.9,
47.5
89.0, 144.1, 116.6
No of measured intensities 91 040 566 550
No of unique reflections 6192 ⁄ 815 43 510 ⁄ 4106
Mean redundancy 14.7 ⁄ 14.8 13.0 ⁄ 8.0
R merge 0.056 ⁄ 0.323 0.113 ⁄ 0.469
0% Completeness 100.0 ⁄ 100.0 99.4 ⁄ 95.3
Refinement
Resolution limits (A ˚ ) 20.0–2.0 20.0–2.1
R-factor (95% of the data) 0.219 0.207
Rfree(5% of the data) 0.298 0.258
Bond length rmsd (A ˚ ) 0.021 0.019
Average B (main
chain ⁄ side chain) (A˚ 2
)
42.2 ⁄ 44.0 34.3 ⁄ 36.5 Average B for water (A˚2 ) 41.5 34.5
Trang 6tation between the first long helix and the second helix.
Figure 4B shows that for the EcCM, TtCM and PfCM
structures, this loop aligns very well, but that the
90-MtCM structure is significantly altered; however,
examination of the surface (not shown) demonstrates
that even with this change, the active site remains bur-ied We superimposed the EcCM structure with the TSA onto 90-MtCM to see the TSA in the active site
of 90-MtCM (Figs 5 and 6) As the residues corre-sponding to Ser84 and Gln88 of EcCM are absent in 90-MtCM, chorismate is unlikely to be as well stabi-lized in its active site This at least partly explains the low kcatfor 90-MtCM (Table 1)
In an attempt to substantiate the notion that the lower kcat and higher Km are due to the missing sub-strate-binding residues, we engineered a modified version of helix 3 in 90-MtCM We replaced the C-ter-minal seven amino acids GRGRLGH in 90-MtCM with amino acids SVLTQQALL or SVLTEQALL, cor-responding to the C-terminus of EcCM, thus providing Ser84 and Gln88⁄ Glu88 in corresponding positions in 90-MtCM Production of glutamine and glutamic acid
H 2 N
H 2 N
NH 2 NH
Arg18′
(Arg127)
(Lys54) (Asp63)
(Gln66)
(Arg43)
Arg58
Arg35
(Ala99)
(Gln 103) (Glu67)
Residues in EcCM missing in 90-MtCM
Arg46 Val55
Glu59
NH
O
HN
OH
HN
N H
H N
H
H H O O
O
-+
+ +
O O
HO O
O O
O
NH 2
NH 2
H 2 N
H 2 N
H 2 N
Fig 6 The active site of 90-MtCM: a diagrammatic view of the active site of 90-MtCM is shown, with the superimposition of the TSA from the EcCM structure The corresponding residue numbers for *YpCM are shown in parentheses.
A
B
C
Fig 5 Stereo view of the active sites of EcCM, 90-MtCM, and
*YpCM: a stereo view of the active site of EcCM is shown in (A), and the corresponding view of 90-MtCM is shown in (B) The active site residues are shown in stick form, and the rest of the structure is in cartoon form In EcCM, one polypeptide chain is gray and the other is rose In 90-MtCM, one polypeptide chain is blue and the other is green The TSA from the EcCM structure is shown with yellow carbon atoms in the 90-MtCM structure for orientation The active site residues are labeled, and the N-terminus of the chain that contributes one residue (R11¢ in EcCM and R18¢ in 90-MtCM) to the active site is labeled In 90-MtCM, the observed C-terminus of the structure visible in the electron density is indi-cated for the second chain The citrate from the crystal structure of
*YpCM is shown in the active site with yellow carbon atoms in (C) All of the active site residues belong to the same chain The figure was drawn with PYMOL (http://www.pymol.org).
Trang 7variants of the 90-MtCM clones resulted in inclusion
bodies of the overproduced protein(s) under various
conditions of growth and induction Thus, we could
not experimentally verify our interpretation of the
lower kcatand higher Km. In an analysis of active site
residues in EcCM by site-directed mutagenesis, Liu
et al.[11] observed lower activity for the Q88A mutant
They proposed that the side chain of Gln88 in EcCM
hydrogen bonds with O7 of the transition state analog,
endo-oxabicyclic dicarboxylic acid (Fig 6) This
experi-mental evidence reinforces the low kcat that we
observed for 90-MtCM, which has leucine instead of
glutamine in the corresponding position
Crystal structure of *YpCM
The crystal structure of the secreted, mature, dimeric
*YpCM with a citrate ion in the active site has been
determined to 2.1 A˚ resolution, using data collected at
a single wavelength for the selenomethionine derivative
of the protein The protein crystallized in the space
group C2221 with two homodimers (A⁄ B and C ⁄ D) in
the asymmetric unit The protomers superimpose with
average rmsd values in the Ca coordinates of less than
0.8 A˚ The final model for *YpCM includes all 155
residues for chains A and C and 154 residues for
chains B and D, where the initial residue, Gln31, is
not ordered The model also includes four citrate ions,
one in each active site, 13 sulfate ions with 11
modeled at 0.5 occupancy, and 193 water molecules
[Correction added on 28 August 2008 after first online
publication: in the preceding sentence, ‘13 sulfate ion,
with 11’ was corrected to ‘13 sulfate ions with 11’] In
the Ramachandran plots, 95.1% of the residues are in
the most favored regions, 4.5% in the additional
allowed regions, and 0.4% in the generously allowed
regions The structure is all a-helical, and the protomer
has the fold of the EcCM dimer with an inserted loop
connecting the two chains Each protomer of *YpCM
has one active site, and the molecule forms a
homo-dimer In this crystal form, citrate ions from the
crys-tallization solution are present in all the active sites
This is the same fold as for *MtCM [7,14,15] The
superimposition of *MtCM on *YpCM aligns 132
resi-dues and yields an rmsd for Ca atoms of 1.8 A˚ for both
Protein Data Bank files 2F6L and 2FP2 The dimer is
also formed in the same manner as that of *MtCM
There is only 23% sequence identity over the aligned
residues As in *MtCM, the active site has residues
mainly from the N-terminal half of the chain, and the
region that would correspond to a second active site by
analogy to the EcCM dimer is closed off by a disulfide
bond In *MtCM, the disulfide bond between Cys160
and Cys193 links helices that correspond to helix 2 and helix 3 of the second EcCM protomer; in *YpCM, the disulfide bond is between the third residue of the mature protomer and the bottom of helix 1 of the second EcCM protomer, Cys33 and Cys148
Classification of MtCM
A diverse array of CMs occur in nature: AroQ class CMs such as EcCM [13], CM from Methanococcus jann-aschii[16], and allosteric CM from Saccharomyces cere-visiae (ScCM) [17]; *AroQ class CMs such as Erwinia herbicola CM (*EhCM) [16], *MtCM [5–7], and
*YpCM; and AroH class CMs such as Bacillus subtilis
CM (BsCM) [18] [Correction added on 28 August 2008 after first online publication: in the preceding sentence,
‘*Erwinia herbicola (*EhCM) [16], *MtCM [5-7], and
*YpCM; and AroH class CMs such as Bacillus subtilis (BsCM) [18]’ was corrected to ‘Erwinia herbicola CM (*EhCM) [16], *MtCM [5-7], and *YpCM; and AroH class CMs such as Bacillus subtilis CM (BsCM) [18]’] AroQ class and *AroQ class CMs function as dimers, whereas AroH class CMs function as trimers In addi-tion, ScCM has a domain for regulation of the activity
by tryptophan and tyrosine [19], whereas *MtCM [7,14] and *YpCM do not have such a regulatory domain Furthermore, structural motifs differ among the AroQ and AroH classes of CMs AroQ and *AroQ CMs exhi-bit all a-helical bundles, whereas AroH CMs contain both a-helices and b-sheets The active site in EcCM is formed by residues from all three helices of one pro-tomer and by a residue from the N-terminal long helix
of the second protomer In contrast, the active site in ScCM [17], *MtCM [7,14,15] and *YpCM is formed within a single protomer
Further subclassification of AroQ CMs on the basis
of their distinct structural prototypes was proposed by Okvist et al [14] (Fig 7) EcCM-like proteins whose catalytic site is formed with residues from both protomers are denoted as AroQa ScCM-like proteins
in which the catalytic site is formed within a single protomer with a domain for regulation of activity by tryptophan and tyrosine are denoted as AroQb Secreted CMs such as *MtCM and *YpCM, in which the catalytic site is formed within a single protomer but without an apparent regulatory domain, are denoted as AroQc A fourth subclass of CMs denoted AroQdwas proposed by Okvist et al [14], on the basis
of the primary sequence alone The structural motif of AroQdCMs resembles that of AroQa, with the notable difference of a shortened third helix that lacks two substrate-binding site residues Here we describe the first 3D structure of such a protein, MtCM
Trang 8Experimental procedures
Materials All the reagents used in this work were obtained from the specified sources [7] A selenomethionine auxotroph of
Biosciences Inc (Madison, WI, USA) The M9 salts growth medium (Cat No MD045003) for the incorporation of selenomethionine into *YpCM was purchased from Medici-lon Inc (Chicago, IL, USA)
E coli strains and plasmids
cloning the target gene and expression of the cloned gene, respectively The plasmid vector pG58 and subtilisin column were kindly provided by P N Bryan (Center for Advanced Research in Biotechnology, University of Mary-land Biotechnology Institute) Engineering of the fusion protein production vector pG58 was described by Ruan
gene product as a fusion protein with the subtilisin prodo-main The fusion protein would be bound to a resin cou-pled with a stable variant of subtilisin protease Next, equilibration with fluoride anion will trigger the cleavage
by subtilisin between the prodomain and the target protein, thus releasing the target protein in its native form, begin-ning with the initiator methionine
Cloning of Rv0948c and MT0975 genes The Rv0948c ORF for the 105 amino acid protein was
Oligonucleotide pair 1 with specific restriction recognition
TTTAAAGCGATGATGAGACCAGAACCCCCACATCA CG-3¢ (forward primer with DraI site underlined) and 5¢-CGGAATTCTTAGTGACCGAGGCGGCCCCTGCC-3¢ (reverse primer with EcoRI site underlined) Similarly, the Rv0948c ORF for the 90 amino acid protein beginning with Met16 was amplified with oligonucleotide pair 2: 5¢-GCTACGTTTAAAGCGATGATGAACCTGGAAATG CTCGAGTCC-3¢ (forward primer with DraI site underlined) and the same reverse primer Oligonucleotide pair 3 for amplification of MT0975 (217 amino acid protein – another annotation for MtCM) for cloning into pG58 was: 5¢-GCTACGTTTAAAGCGATGATGGACCGGGAGGCT TGGCG-3¢ (forward primer with DraI site underlined) and the same reverse primer as above Amplification conditions
melting of DNA, followed by 30 cycles of amplification, with
Polymerization was continued at the end for 10 min at
A
B
C
D
Fig 7 Four subclasses of AroQ CMs: the four subclasses of AroQ
CMs are shown with cartoon drawings of representative structures.
All AroQ CMs are homodimers One chain is blue and the other
chain is green The distinguishing features are emphasized by
indi-cating the active sites with red circles The regulatory sites of the
AroQbclass are highlighted with red squares The shortened third
helices of AroQd are pointed to with red arrows (A) AroQa is
EcCM (B) AroQ b is ScCM (C) AroQ c is *YpCM (D) AroQ d is
90-MtCM with the TSA from the superimposition of EcCM.
Trang 972C One hundred nanograms of M tuberculosis H37Rv
genomic DNA (generously provided by J Belisle and
P Brennan, Colorado State University) and 100 ng of
prim-ers were used in the amplification The amplified DNA
obtained with oligonucleotide pairs 1, 2 and 3 was digested
with DraI and EcoRI for cloning into the respective sites of
the pG58 plasmid A recombinant was isolated from E coli
Novablue and introduced into E coli BL21(DE3) for protein
production
Overproduction of the proteins
(105 amino acids), pG58–Rv0948c (90 amino acids) or
pG58–MT0975 (217 amino acids) recombinant plasmid was
grown in 25 mL of LB medium containing ampicillin
except for the pG58–MT0975, clone which was induced
bodies of the protein All three fusion proteins were
pro-duced in fully soluble form under these conditions
Purification of native MtCM
Cells from 1 L of induced culture of BL21(DE3) harboring
pG58–Rv0948c (encoding either the 105 amino acid protein
or the 90 amino acid protein) were suspended in 40 mL of
lysis buffer (10 mm potassium phosphate, pH 7.4, 15 mm
NaCl), to which a tablet of protease inhibitor cocktail was
added The cell suspension was passed through a French
at 48 000 g for 1 h Supernatant containing the subtilisin
prodomain–MtCM fusion protein was loaded onto a 5 mL
was washed with 60 mL of the lysis buffer at a flow rate of
1 m sodium acetate in the lysis buffer Next, the cleavage of
MtCM from the prodomain was triggered by flushing the
sodium fluoride in the lysis buffer and equilibration for
30 min The resin was washed with 25 mL aliquots of
100 mm sodium fluoride in the lysis buffer Effluent fractions
were pooled and concentrated to 5 mL in an Amicon cell
using a 5000 Da molecular mass cut-off membrane MtCM
molecular sieve chromatography on a 480 mL Sephadex G-75
superfine column, which was equilibrated and eluted with
Effluent fractions containing pure MtCM (105 amino
determi-nation and biochemical analysis The 217-MtCM was
simi-larly purified with the subtilisin column Further purification
was not pursued, as it exhibited extremely low CM activity
Cloning and expression of the *YpCM gene (y2828) in E coli
The gene y2828 from the genome sequence of Y pestis strain Kim10+ [21] was annotated as CM [Correction added on
28 August 2008 after first online publication: in the preceding sentence, ‘The gene y2828 from the genome sequence of
gene y2828 from the genome sequence of Y pestis strain Kim10+ (21) was annotated as CM’] The full-length
*YpCM gene coding sequence, including the signal peptide, was amplified by PCR using the forward primer 5¢-GG AATTCCATATGCAACCCACTCATACGCTAACAAG-3¢ (with the NdeI restriction recognition sequence underlined) and the reverse primer 5¢-CGGGATCCTTATTTTAATT
restriction recognition sequence underlined) Amplification
followed by 30 cycles of amplification, with each cycle
nanograms of Y pestis strain KIM10+ chromosomal DNA (kindly provided by R D Perry, University of Kentucky) and 100 ng of primers were used in the amplification The amplified DNA was digested with NdeI and BamHI and cloned into the respective sites of the pET15b plasmid
A recombinant was isolated from E coli Novablue and intro-duced into BL21(DE3) for protein production E coli
plasmid was grown in 100 mL of LB medium containing
Protein production was induced with 10 lm IPTG overnight
chroma-tography from the periplasmic fluid of E coli as described for *MtCM [7] The production and purification of *YpCM was scaled up for crystallization
Production and purification of selenomethionine
*YpCM
trans-formed with the pET15b–y2828 recombinant plasmid Incor-poration of selenomethionine into *YpCM was performed
according to the manufacturer’s recommendation Briefly, cells were grown in 1 L of LB medium containing ampicillin
washed twice with sterile water, and suspended in 100 mL of M9 salts medium Four 1 L volumes of M9 salts media con-taining ampicillin were inoculated with 25 mL of the culture
stage, selenomethionine was added and induced with 10 lm
sieve chromatography from the periplasmic fluid
Trang 10Crystallization of 90-MtCM
chlo-ride Crystallization conditions were surveyed by the sitting
drop vapor diffusion method using Emerald BioSystems
Wizard Screens I and II There were several hits The crystal
used for data collection was grown with a well solution of
20% poly(ethylene glycol) 400 The crystallization drops
were made with equal volumes of protein and well solution
Crystallization of *YpCM
Crystallization conditions were surveyed by the hanging
drop vapor diffusion method using the Wizard II kit from
Emerald BioSystems (http://www.emeraldbiosystems.com)
200 mm sodium chloride The original hit was with
buffer, pH 4.2) For the refined conditions, a well solution
phos-phate buffer (pH 4.2) was used, and the protein
protein, the well solution was 1.8–2.0 m ammonium sulfate
Data collection for 90-MtCM
Diffraction data were collected using a home source
was cooled to 105 K with a Rigaku Xtream 2000
cryocool-er For cryo-data collection, the crystals were mounted
through a layer of paraffin oil placed on top of the
crystal-lization drop The data were collected and processed with
Structure determination for 90-MtCM
The structure of 90-MtCM was solved by molecular
replacement using phaser [23], with the structure of PfCM
(Protein Data Bank ID: 1YBZ) The asymmetric unit of the
Molecu-lar replacement trials using a single protomer failed
was used as the search model, a solution was found The
remainder of the structure determination was carried out in
the model, and resolve [26] was used to iteratively rebuild
the model to remove bias The final refinement statistics are
shown in Table 2 coot [27] was used to view the model
graphically and to build portions not built by resolve The
stereochemistry was checked with procheck [28] and with routines inside coot
Data collection for *YpCM Preliminary data were collected on the home source described above, and cryoprotection was accomplished in the same manner as for 90-MtCM The selenomethionine data for *YpCM were collected on beamline X29A of the National Synchrotron Light Source at wavelength 0.9790 A˚ with the crystal cooled to 100 K The statistics are shown
in Table 2
*YpCM structure determination The structure of *YpCM was solved using the phasing information from the anomalous data The positions of the selenium atoms were located with shelxd [29], and the initial phases were calculated with solve [30] Two dimers in the asymmetric unit cell gives a Matthews coeffi-cient of 2.6 and a solvent content of 52.5% The initial model was built with resolve [26,31], using iterative rounds of pattern-matching, fragment identification, den-sity modification, and refinement This model included 78% of the residues and placed 71% of the side chains The noncrystallographic symmetry was used to combine the four partial chains to produce a more complete model Then, further cycles of model building and refinement were performed using xtalview [32] and refmac5 [24] The final refinement statistics are shown in Table 2 The stereochemistry was checked with procheck [28] and with
the D chain were modeled with alternative side-chain con-formations No interpretable electron density was observed for the first residues (residue 31) of chains B and D The residues between Cys148 and Asp155 are somewhat disor-dered, particularly in the C and D chains, and conse-quently have increased B-values
Other methods
CM was assayed by the method of Davidson & Hudson [34], essentially as described in our previous study [7] One microgram of MtCM protein or 200 ng of *YpCM protein were used in the assay Protein concentration was deter-mined by the Micro BCA method with BSA as the
molecular mass of the native MtCM was determined by
analyzed using an Applied Biosystems Voyager-DE STR Biospectrometry Workstation (Foster City, CA, USA) The DNA sequence of the cloned genes was confirmed by the dideoxy sequencing method [35], as adopted for the Applied Biosystems model 3130 Genetic Analyzer