The unique sites in SulA protein preferentially cleaved
by ATP-dependent Lon protease from
Escherichia coli
Wataru Nishii
1
, Takafumi Maruyama
1
, Rieko Matsuoka
1
, Tomonari Muramatsu
2
and Kenji Takahashi
1
1
School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Japan;
2
Biophysics Division, National Cancer
Center Research Institute, Chuo-ku, Tokyo, Japan
SulA protein is known to be one of the physiological
substrates of Lon protease, an ATP-dependent protease
from Escherichia coli. In t his study, we i nvestigated the
cleavage speci®city of Lon protease toward SulA protein.
The enzyme w as shown to cleave  27 peptide bonds in
the presence of ATP. Among them, six peptide bonds
were cleaved preferentially in the early stage of digestion,
which represented an apparently unique cleavage sites
with mai nly Leu and Ser r esidues at the P
1
,andP
1
¢

two types of substrates in vivo. One type of the substrates
includes abnormal proteins such as those with i mproper
polypeptide length o r tertiary structure. Their degradation
should contribute t o the quality control of i ntracellular
proteins. Another involves physiological substrates, such a s
SulA, kN, RcsA, CcdA and Pem1, which are short-lived
regulatory proteins, whose speci®c and rap id degradation is
crucial for normal cell g rowth [2±7]. SulA i s one of the most
physiologically important substr ates among the second
type. The protein is transcriptionally induced by environ-
mental stresses, such as UV irradiation, and prevents
premature segregation of damaged DNA into daughter cells
during DNA repair processes [8,9]. Induced SulA prevents
the self-assembly of FtsZ protein, leading to the inhibition
of cell division (®lamentation) [10].
The substrate recogn ition mechanism of Lon protease
has not yet been well clari®ed. The cleavage sites by the
enzyme in v itro have been reported for kN [5] and C cdA [3]
proteins, oxidized in sulin B c hain and g lucagon [5], a nd
several ¯uorogenic substrates [11]. In these proteins and
peptides, the cleavages occurred mainly a fter hydrophobic
residues, in spite that not all such sites were cleaved. So far,
however, no more consensus features have been reported in
the p rimary or higher-order structures of the substrates.
There has been little s tudy on the cleavage sites, particularly
for SulA, by Lon p rotease. This is presumably because
recombinant SulA was reportedly rather insoluble and/or
unstable [12,13].
In the present study, we were able to prepare SulA in
a soluble form and investigated its cleavage sites by Lon

The expression vector pMAL-p-SulA was a generous gift
from S. Sonezaki (Kyushu Institute of Technology, Tobata,
Japan). Using the vector, MBP-SulA was expressed in
E. coli DH5 cells, puri®ed by amylose-resin chromatogra-
phy and treated with factor Xa to generate MBP, SulA3±
169 and SulA23±169 as described previously [12]. After
digestion of 500 lg of SulA, generated SulA3±169 and
SulA23±169 were separately puri®ed to homogeneity by
using a p reparative disc SDS/PAGE apparatus (Nihon Eido
Co., Ltd, Tokyo, Japan). The puri®ed SulA3±169 and
SulA23±169 solutions (2.7 mL and 4.5 mL, respectively)
were then dialyzed against 2 L of 20 m
M
Tris/HCl, pH 8.0,
at 4 °C for 4 days with three changes of the buffer. After
concentration of the protein solutions to 300 lLbyan
ultrafreeÒ-15 centrifugal ®lter device (Millipore Co.), 80%
glycerol was added to t hem to a ®nal concentration of 20%.
The ®nal concentrations of SulA3±169 and SulA23±169
were 1.65 mgámL
)1
and 0.775 mgámL
)1
, respectively. MBP
was puri®ed by using AKTA e xplorer 10S with a HiPrep 26/
60 Sephacryl S-300 HR column (Amersham Pharmacia
Biotech, Ltd).
SDS/PAGE analysis
SulA3±169 and MBP (15 lg each) were separately incubat-
ed at 37 °Cwith3lgofLonin25lLof50m

M
MgCl
2
,0.5m
M
ATP
and 0±0.05% SDS, were incubated at 37 °C for 1 h. The
reaction was stopped b y addition of 100 lLof1%SDSand
1.2 mL o f 0.1
M
sodium borate, pH 9.1 and then the
¯uorescent intensity (excitation, 335 nm; emission, 410 nm)
was measured.
Identi®cation of the peptide fragments by LC-MS
SulA3±169 samples (each 150 lg) were incubated f or
appropriate periods with 30 lg of Lon protease in 250 lL
of 50 m
M
Tris/HCl, pH 8.0, containing 15 m
M
MgCl
2
,with
or without 4 m
M
ATP. The reaction was stopp ed by
addition of 35 lL of 50% trichloroacetic acid to each
reaction mixture. The r eaction mixture was then centrifuged
and 100 lL of the supernatant was applied to a LCQ
TM

2
D
program [14,15], the secondary structure of the
protein w as estimated from the spectrum to be 29% in
a helix, 15% in b sheet and 56% in random loop structure,
which w ere similar to those (34% in a helix, 19% in b sheet
Fig. 1. Far-UV CD spectra of SulA3±169 (solid line) and SulA23±169
(broken line). The C D spectrum w ere measured u sing a 0.1- cm cuvette
at 37 °C at a protein concentration of 5.4 l
M
in 20 m
M
Tris/HCl,
pH 8.0, containing 20% glyc erol.
452 W. Nishii et al. (Eur. J. Biochem. 269) Ó FEBS 2002
and 47% in random loop structure) predicted from the
primary structure by the pro®le f ed neural network systems
from Heiderberg (PHD) [16,17] (see below). SulA23±169
was a lso prepared in the same way and its CD s pectrum w as
almost the same as that of SulA3±169 (Fig. 1).
The SulA3±169 preparation might possibly contain a
small amount of SDS that had not been completely
removed b y dialysis. In that case, t he remaining SDS
should interfere with the activity of Lon protease. W e
therefore investigated the effect of SDS on the activity of
Lon protease toward a ¯uorogenic substrate, suc-Phe-Leu-
Phe-4MbNA. Table 1 shows that the activity of the enzyme
was inhibited by a low concentration of SDS (about 50%
inhibition in the presence of 0.0025% SDS). On the other
hand, an extensive degradation of SulA3±169 by the enzyme

presence of ATP and after 3 h of incubation in the absence
of ATP w ere also analyze d in the same way. The yields of
the fragments were estimated b y amino-acid analyses. The
results are shown in Fig. 4. Twenty-seven cleavage sites were
identi®ed with the sample incubated for 30 min in the
presence of ATP. During incubation for 3 min in t he
presence of ATP, preferential cleavages occurred at six
peptide bonds: Leu57-Gly58, Leu67-Thr68, Leu73-Ser74,
Ala80-Ser81, Leu94-Ser95 and Leu158-Ser159, which were
hydrolyzed over 5% (Fig. 4 and Table 2). It was remark-
able that these cleavage sites contained mainly Leu and Ser
at the P
1
and P
1
¢ positions, respectively, representing an
apparent consensus in the primary structure.
The other cleavage sites contained various residues at the
P
1
positions (Table 2). The cleavage occurred almost
exclusively after nonch arged amino acids, s uch as Ala,
Val, Met, Thr, Ser, Leu, Phe, Gln and Gly (20 of the
21 sites), where hydrophobic residues were predominant.
However, no apparent consensus residues at other than P
1
positions were found except that Ser appeared to be
preferred at the P
1
¢ position: seven out of 21 Ser residues

ATP, between certain hydrophobic residues, such as Ala,
Leu, Met and Phe, and Ser and that cleavages occurred at
sites other than the major sites of cleavage that occurred in
the presence of ATP, except for the cleavage of Leu158-
Ser159.
DISCUSSION
In the present study, SulA3±169 was used exclusively as the
substrate protein for Lon protease. P reviously, it w as
reported that the pre-MBP-SulA fusion protein was well
soluble in a queous solution, but that the free SulA p rotein
(SulA3±169) separated from the fusion p rotein b y factor Xa
digestion was rather insoluble [12]. In the present study,
however, we could prepare a soluble form of SulA3±169,
cleaved from the fusion protein, by preparative SDS/PAGE
followed by extensive dialysis. SulA3±169 appeared to have
been properly refolded during the preparation procedure
used. SDS was found to strongly inhibit Lon protease. This
is in sharp contrast with the case of the proteasome, another
ATP-dependent protease, which is known to be activated by
certain concentration ( 0.04%) of SDS [18]. As Lon
protease degraded SulA extensively, t he detergent is thought
to have been removed suf®ciently from SulA 3±169 by
dialysis.
The CD spectrum of SulA3±169 showed that the protein
had a signi®cant amount of secondary structures, and the
secondary structure contents were almost identical with
those predicted from the known amino-acid sequence.
These results suggested that the SulA3±169 protein had
essentially the same s econdary structures with the native
Fig. 3. Separation of degradation products of SulA by reverse-phase

degradation by Lon protease.
In the p resence of A TP, Lon protease hydrolyzed
SulA3±169 extensively, whereas MBP, used as a control,
was not cleaved at all. This is consistent with the report
that, when the pre-MBP-SulA fusion protein was used as
the substrate, only the SulA portion was degraded by Lon
protease in an ATP-dependent manner [12]. When the
digest of SulA3±169 was analyzed by SDS/PAGE, inter-
mediate protein bands, with molecular masses at least over
10±12 kDa, were scarcely detected. This may indicate that
the initial cleavage at a certain peptide bond is followed by
further extensive degradation of the initial cleavage prod-
ucts. Indeed, the initial rapid and preferential cleavages
were o bserved at a limited number of peptide bonds,
including Leu67-Thr68, Leu57-Gly58, Ala80-Ser81,
Leu158-Ser159, Leu73-Ser74, and Leu94-Ser95. Interest-
ingly, these peptide bonds are all located in the central
region of the polypeptide chain except for Leu158-Ser159,
which is in the C-terminal region. The central region was
reported to be important for t he activity of SulA as a cell-
division inhibitor, including essential residues, Arg62,
Leu67, Trp77 a nd Lys87, for the inhibitory activity and
to presumably constitute a surface for protein±protein
interaction [13]. It is tempting to assume that these initial
cleavage sites are strategically placed mainly in the central
region of SulA so that the cleavage at any of these sites
would lead to rapid inactivation of the protein. As for the
C-terminal region, it is interesting to note that the
C-terminal 20 residues were suggested to be important
for the recognition by Lon protease [13] and that the

Cleavage
site
P
5
P
4
P
3
P
2
P
1
¯ P
1
¢ P
2
¢ P
3
¢ P
4
¢ P
5
¢
Fast W Q L W L67 T68 P Q Q K
L L Q Q L57 G58 Q Q S R
E W V Q A80 S81 G L P L
A T R Q L158 * S159 G L K I
P Q Q K L73 S74 R E W V
Q I S Q L94 S95 P C H T
Medium R P V S A150 * S151 S H A T

absence of ATP. It may be worthy o f note that the central
region of SulA is especially rich in secondary structures. In
the case of C cdA degradation, it was also suggested that the
enzyme might disrupt the secondary structure o f the protein
in an ATP-dependent manner [3]. Although the major
cleavage sites were topologically different i n the presence and
absence of ATP, amino-acid residue speci®city at the
cleavage sites w ere e ssentially the same with or without
ATP. The ATP-independent cleavage a t both t erminal
regions will not impair t he physiological function of SulA
2
as
these regions were reported to be dispensable for its activity
in viv o [13].
The f act that in the presence of ATP the initial cleavage at
a certain peptide bond appeared to be followed by further
extensive degradation of the initial cleavage products
suggests the possibility that Lon protease may be a kind
of processive enzyme, like the 20S proteasome and C lpAP
protease [22±24]. Indeed, such a possibility h as been
discussed previously [25]. The oligomeric structure of Lon
protease [26], somewhat resembling those of 20S protea-
some and ClpAP, is consistent with this supposition,
although further studies are necessary to draw a de®nite
conclusion in this regard.
As for the amino-acid residue speci®city of Lon protease
toward SulA, it is notable that all the P
1
positions relative to
the scissile bonds were occupied by uncharged amino-acid

This study was supported in part by grants-in-aid for scienti®c research
from the Ministry of E ducation, Science, Sports and Culture of Japan.
We gre atly t hank D r Shuji Sonezaki (Department o f A pplied
Chemistry, Faculty of Engineering, Kyushu Institute of T echnology,
Tobata) for providing the pMAL-p-SulA plasmid.
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