Tài liệu Báo cáo khoa học: Structure of peptidase T from Salmonella typhimurium doc

Structure of peptidase T from
Salmonella typhimurium
Kjell Ha
Ê
kansson* and Charles G. Miller
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
The structure of peptidase T, or tripeptidase, was deter-
mined by multiple wavelength anomalous dispersion
(MAD) methodology a nd re ®ned to 2 .4 A
Ê
resolution. Pep-
tidase T comprises two domains; a catalytic domain with an
active site containing two metal ions, and a smaller domain
formed through a long insertion into the catalytic domain.
The two met al i ons, p resumably zinc, ar e s eparated by 3.3 A
Ê
,
and are coordinated by ®ve carboxylate and histidine
ligands. The molecular surface of the active site is negatively
charged. Peptidase T has the same basic fold as carboxy-
peptidase G2. When the structures of the two enzymes are
superimposed, a number of homologous residues, not evi-
dent from the sequence a lone, could be identi®ed. Com-
parison of t he active sites of peptidase T, carboxypeptidase
G2, Aeromonas p roteolytica aminopeptidase, carboxypepti-
dase A and leucine aminopeptidase reveals a common
structural framework with i nteresting similarities and
dierences in the active sites an d in the zinc coordination.
A putative b inding site for the C-terminal end of the
tripeptide substrate was found at a peptidase T speci®c
®ngerprint s equence m otif.

Keywords: tripeptidase; aminotripeptidase; metallopep-
tidase; X-ray crys tallography; M AD.
Escherichia coli and Salmonella typhimurium express s everal
intracellular enzymes capable o f hydrolyzing peptides [1].
Many of these e nzymes h ave been shown to function in the
degradation of intracellular proteins and in the catabolism
of exogenously supplied p eptides [2]. O ne of these enzymes,
peptidase T, or tripeptidase (EC 3.4.11 ) is a 409 amino-
acid metalloenzyme that hydrolyzes tripeptides a t their
N-termini [3,4]. The enzymatic activity of t he S. typhimurium
enzyme is both speci®c and unusual; dipeptides, tetrapep-
tides or t ripeptides with blocked N-termini are not cleaved.
S. typhimurium peptidase T expression is re gulated b y
FNR, a transcriptional a ctivator that re sponds to anaero-
biosis [4,5]. The aerobic expression level of peptidase T is
not suf®cient to allow this enzyme to contribute to the
utilization of exogenously supplied peptides a s amino acid
sources [3]. Under a naerobic c onditions, however, the pepT
gene is induced, leading to levels of peptidase T that allow i t
to participate in the catabolism of tripeptides [5,6]. It has
been speculated t hat this pattern of regulation may
contribute to the anaerobic utilization of amino acids as
energy sources [1].
A 45-amino-acid region of peptidase T displays similarity
to a short region in Pseudomonas sp. strain RS-16
carboxypeptidase G2 (CG2), peptidase D, and alkaline
phosphatase isozyme conversion peptidase (Iap) [4]. This
region of similarity contains two of the ®ve ligands that
coordinate the two zinc ions in the active site of CG2, for
which the three-dimensional structure is known [7]. Pepti-

dase T h as therefore b een classi®ed into the M 20 family of
proteases/peptidases [8]. A third zinc ligand, a histidine, can
be recognized as part of a HXDT motif [9], which is
conserved i n both peptidase T and CG2. While these data
indicate that peptidase T is evolutionarily related to CG2,
the lack of clear homology outside these regions, and the
unique tripeptidase speci®city of peptidase T, suggested that
the structure of the two enzymes would in part differ. We
report the three-dimensional structure of S. typhimurium
peptidase T solved by multiple wavelength anomalous
dispersion (MAD) methodology a nd re®ned to 2.4-A
Ê
resolution.
MATERIALS AND METHODS
Crystallization and data collection
Selenomethionine His-tagged peptidase T was expressed in
strain TN5619, puri®ed, c rystallized from ammonium
sulfate solu tions at pH 7.5 a nd ¯ash frozen in 50% sucrose
as previously described [ 10]. Crystals belong to s pace group
C2 with a  132.4 A
Ê
, b  46.0 A
Ê
, c  96.6 A
Ê
, b 
116.1 A
Ê
. Data were collected at 100 K at NSLS beam
station X4A (Brookhaven, NY, USA) at four different

wavelengths, and processed with
DENZO
,
SCALEPACK
and the
CCP
4 program suite [11,12].
Structure solution and re®nement
The structure was solved b y MAD methodology using 15
selenium atoms, four wavelengths and data to 2.8 A
Ê
.
Determination of selenium positions, phase and electron
density calculations and model re®nement were performed
with the
CNS
program package [13]. The model was built
manually and displayed using the graphics pro gram
O
[14].
The model was re®ned against one of the data sets processed
Correspondence to C. G. Miller, Department of Microbiology, Uni-
versity of Illin o is at Urba na-Champaign, B103 CLSL, 601 S. Good-
win Avenue, Urbana, Illinois 61801, USA. Fax: + 217 244 6697,
Tel.: + 217 244 8418, E-mail:
Abbreviations: MAD, m u ltiple wavelength anomalous dispersion;
CG2, carboxypeptidase G2; APP, Aeromonas prot e olyti ca amino-
peptidase; LAP, leucine aminopeptidase; CPA, carboxypeptidase A.
*Present address: L ab oratory of C ellular and Molecular Physiology,
August Kr ogh Institute, U niversitetsparken 13, DK 210 0, Kbh é ,

Denmark.
(Received 3 September 200 1, revised 6 November 2 001, accepted 8
November 200 1)
Eur. J. Biochem. 269, 443±450 (2002) Ó FEBS 2002
nonanomalously to 2.4 A
Ê
. Data collection and re®nement
statistics are s hown i n Tables 1 and 2. T he electron density
of the three N-terminal residues w as dif®cult to interpret. A
putative s ulfate ion, hydrogen bonded to the main-chain
amino groups of Lys3 and Leu5, was included in order to
account for all of the electron d ensity in this part of the
structure. R esidues His305±Pro306 a re not well de®ned due
to very weak electron density signals and the C-terminal
residue 409 along with the C-terminal hexahistidine tag
showed no signi®cant density at a ll. Outside t hese parts, the
polypeptide chain is g enerally well de®ned by the
2|Fo| ) |Fc| density maps. The side-chains of residues
Arg99, Asp109, Val115 and Tyr378, however, were not
de®ned beyond the Cb atom. Most of the solvent density
was relatively weak and the modeled solvent molecules have
high temperature factors. Coordinates and diffraction data
have been deposited in the Protein D ata Bank and have the
accession code 1FNO [15].
RESULTS
Overall structure
The ®nal model consists of residues M et1±Gly408 (see
Materials and methods). All 15 methionines are built in to
the s elenium atom positions determined by CNS [13]. The
sulfur atoms of Cys309 and Cys343 are within 2.0 and

2.3 A
Ê
, respectively, of the previously determined reactive
mercury sites [10]. The overall structure and the active site of
peptidase T are shown in Fig. 1. The fold of the enzyme
reveals a two-domain structure that is similar to that o f
CG2, which we did not anticipate due to the low overall
sequence similarity between the two e nzymes. T he catalytic
domain contains o ne seven-stranded mixed b sheet ¯anked
with ahelices, and a second, four-stranded antiparallel
b sheet.InCG2,thelargerofthetwob sheetsiseight-
stranded, but peptidase T residues Gly348±Glu350 (that
would have formed the eighth strand) were not recognized
as a b strand by the program
PROCHECK
[16]. The ma jor
difference between the two structures is that the
20 N-terminal residues of the mature CG2 are lacking in
peptidase T and that peptidase T has a 30 amino-acid
insertion (residues Asn97±Gln126), which contains two
additional b strands. The structure of the residues ¯anking
this in sertion also differ between the two enzymes. Another
difference is that the loop between the ®rst two helices
(Lys19±Ser27) is larger in peptidase T. Interestingly, this
loop contains a conserved proline (Pro26) with a ci s peptide
bond. When the t wo enzymes are superimposed, the
insertion o verlaps i n s pace with an insertion i n C G2, f ound
at the position of peptidase T residue Lys55. Most of the
Ser182±Ala193 a helixinCG2isbrokenupintoanirregular
structure in peptid ase T. The similarities between pepti-

dase T and CG2 extend beyond the catalytic domain to
include the second domain [7], which in the dimeric CG2
mediates the intermolecular contacts. This domai n, which is
comprised of residues Ala211±Tyr320, consists of a four-
stranded antiparallel b sheet a nd two a helices. Peptidase T
has an insertion after the ®rst a helix (at Pro246), while CG2
has an insertion at the turn between the second and third
b strand (at p eptidase T residue Thr269). Figure 2A shows
an alignment of S. typhimurium peptidase T and Pseudo-
monas sp. carboxypeptidase G2 sequences, based on the
three-dimensional structures. When the Ca positions of the
67 identical amino acids are superimposed, the rmsd is
2.4 A
Ê
(Fig. 3A).
Dimerization contacts
The peptidase T dimerization domains in two crystallo-
graphic asymmetric units make the same contacts around
the twofold axis a s i n CG2 (Fig. 1A). These consist m ainly
Table 1. Crystallographic data. Data collection a nd phasing statistics.
Data set k1 k2 (edge) k3 (peak) k4 k1
Anomalous statistics Yes Yes Yes Yes No
Wavelength (A
Ê
) 0.9879 0.9793 0.9788 0.9668 0.9879
Resolution range (A
Ê
) 20.0±2.8 20.0±2.8 20.0±2.8 20.0±2.8 20.0±2.4
Completeness (®nal shell)
a

97.7 (98.3) 98.7 (99.3) 98.7(99.2) 98.5 (98.8) 99.2 (99.2)
Total no. of observations 62 702 78 281 78 053 72 811 99 755
Unique no. of re¯ections
a
24 622 24 877 24 881 24 825 20 598
R
sym
(®nal shell) (%) 3.2(8.5) 4.2(11.1) 4.6(17.9) 3.8(8.2) 4.6 (24.6)
I/r(I) (®nal shell) 28.8 (11.2) 23.8 (11.3) 20.3 (7.1) 26.5 (13.2) 23.4 (6.0)
Phasing power, disp./anom.
b
3.5/3.5 4.7/5.1 4.1/4.4 /1.9
R
Cullis
, disp./anom.
b
0.50/0.50 0.40/0.37 0.45/0.42 /0.67
f¢obs/f¢¢obs )5.1/1.6 )7.5/5.4 )8.3/6.4 )3.8/1.7
a
A Friedel pair is considered as two unique re¯ections for the anomalously processed data.
b
24 409 structure factors were phased with a
®gure of merit of 0.79.
Table 2 . Re®nement stat istics.
Completeness of model
R
(%)
R
free
(%)

Rmsd B
ave
main/side/protein/solvent
(A
Ê
)
Protein atoms Solvent atoms Bonds (A
Ê
) Angles (°)
3119 142 22.4 26.2 0.011 1.7 39/48/43/50
444 K. Ha
Ê
kansson and C. G. Miller (Eur. J. Biochem. 269) Ó FEBS 2002
of an antiparallel b strand alignment involving residues
Ala264±Val270 (Ala268±Ala270 in CG2), and an antipar-
allel a helical coiled-coil together with the segments sur-
rounding this helix (Lys228±Thr253). Thus, the two
dimerization domains together make up a continuous,
eight-stranded antiparallel b sheet in both peptidase T and
CG2.
Active site
There is one strong solvent electron d ensity signal in the
active site, as previously noted [10], and a second atomic site
was revealed in the |Fo| ) |Fc| map. Due t o the homology
of the active site residues and the similar active site
structures of peptidase T , C G2 and Aeromonas p roteolytica
aminopeptidase (APP) [7,17], they have both been inter-
preted as zinc ions, and the positions of the two ions and the
geometry of the amino-acids c oordinating them are s imilar
in the three enz ymes. The weak d ensity of the second zinc

ion is probably due to low occupancy, as no zinc salt was
included in the crystallization solution. With full occupancy,
the r e®ned temperature factors were 37 and 112 A
Ê
2
,
respectively (the average solvent molecule temperature
factor was 50 A
Ê
2
). Despite the high temperature factor,
the chemical environment and similarity with the CG2
active site suggest that they are both zinc ions. The well-
de®ned, more strongly bound zinc (Zn501), is coordinated
by His78, Asp140 and Glu196, while the second zinc ion
(Zn502) is coordinated by Asp140, Glu174 and His379
(Fig. 1B). The av erage temperature factor for the side-chain
ligands of the well-de®ned zinc ion is 26 A
Ê
2
, while the values
for Glu174 and His379 are h igher ( 60 A
Ê
2
). The distance
Fig. 1. Ribbon re presentation and zinc l igands
of peptidase T. (A) Ribbon representation [34]
of peptidase T, v iewed along the crystallo-
graphic two-fold symmetry axis. b Strands are
showninblueiftheybelongtothecentral

b sheetofthecatalyticdomain,greenifthey
belong to the four-stranded bsheetofthe
dimerization domain, and ligh t blue if they
belong the smaller bsheets of the catalytic
domain. aHelices but not 3
10
helices are
shown in r ed. A second, symmetry related
molecule is s hown i n gray. (B) T he zinc ligands
of p eptidase T . The i nteraction between hi sti-
dine and aspartate in the H XD T motif an d
the cis peptide bond b etween Asp140 and
Asp141 are shown.
Ó FEBS 2002 Structure of peptidase T (Eur. J. Biochem. 269) 445
between the two zinc ions is 3.3 A
Ê
. No zinc bound water was
found; the c losest zinc±water contact is 3 .1 A
Ê
(Zn502). The
absence of a zinc bound water could be due to the l ow
occupancy of the second zinc site or to the limited resolution
of the data. Superimposition o f the two m etal ions and t he
Ca atoms o f the ®ve ligand amino-acids of p eptidase T a nd
the other two enzymes results, in either case, i n a n rmsd of
0.5 A
Ê
.
Zinc ligand motifs
The ®rst of the metal coordinating amino acids, His78, is

found in an HXDT motif, where X is a h ydrophobic amino
acid. In p eptidase T, residue X is a valine ( Val79), which is
buried in a hydrophobic cluster. T he polypeptide makes a
sharp turn at t his position, terminating a b strand (Fig. 1B).
As a r esult of this turn, both the main chain carbonyl and
the side chain carboxylate group of the Asp80 residue are
within hydrogen bond distance to the Nd1atomofHis78
(3.0 and 3.3 A
Ê
, respectively). The second of the metal ion
coordinating amino acids is Asp140, which interacts with
both metal ions. This residue is followed by another aspartic
acid residue through a cis peptide bond in peptidase T, CG2
and AP P. This cis peptide bond breaks a helix at the
N-terminal end and positions the two aspartic acids closer in
space than they would otherwise have been. The third metal
ion ligand, Glu174, is p receded by G lu173, which has been
suggested to act as a base in the catalytic mechanism of APP
[18]. In both peptidase T and CG2, but not in APP, these
two glutamic a cids are p receded by an aspartic acid residue,
although its side chain conformation differs in the two cases.
The part of the polypeptide running up to the fourth ligand,
Asp196, adopts similar c onformations in the three enzymes.
In CG2, the following residue, a proline, makes van der
Fig. 2. Sequence alig nments and visualized
electrostatic surface potential of the active s ite
of p eptidase T . (A) Sequence alignment of
Pseudomonas sp. strain R S-16 carboxypepti-
dase G2 (CG2) a nd Salmonella typhi murium
peptidase T based o n piecewise superimposi-

tion of local s tructure elements. T he sequence
of pe ptidase T is given i n lines of 50 a mino-
acids. CG 2 residues t hat do n ot have ho mo-
logues in peptidase T are written above the
alignment. Peptidas e T secondary structure
elements a re indicated with the same colour
scheme as in Fi g. 1A. The zinc ligands are in
yellow boxes, and identical residues a re
marked with an asterisk (*). (B) The electro-
static surface potential of the active site o f
peptidase T visualized by the p rogram
GRASP
[35]. Re gions of positive potential are shown in
blue and negative pote ntial in red. A p-iodo-
D
-phenylalanine hydroxamate m olecule,
superimposed fr om the e xperimentally
determined APP complex [18], a nd a sulfate
ion are shown in g reen. The insert, viewed
from the top, shows the conserved RGGTDG
®ngerprint motif i n black beneath a
semitransparent s urface.
446 K. Ha
Ê
kansson and C. G. Miller (Eur. J. Biochem. 269) Ó FEBS 2002
Waals interactions with Ala140±Asp141 (homologous to
Ala139±Asp140 in peptidase T ). The situation is similar in
APP but different in S. typhimurium peptidase T, where the
following three residues a re glycines. The ®rst two of these
glycines are conserved in most of the available p eptidase T

sequences. The ®fth ligand, His379, is m ore dif®cult to
identify from the sequence alone, but is preceded by a
hydrophobic residue (Tyr, Phe, Ile or Met) in all three
enzymes and has a glutamate four or ®ve residues on the
C-terminal side. This glutamate hydrogen bonds to the main
chain amino groups of Leu137, Gly138, Ala139, and
Asp140 in the loop preceding the important active site
residues Asp140±141 which are connected by the cis peptide
bond. This interaction probably c ontributes to the s tability
of the active site framework. This HXXX(X)E motif is
conserved not only in peptidase T and CG2 but also in APP
and in the other more distantly related proteins, as discussed
below.
Fig. 3. Stereo representation of CG2 and LAP superimposed onto peptidase T. (A) Stereo representation of CG2 (thin blue) superimposed on
peptidase T (thick black) using the identical ho molo gous residues indicate d i n Fig. 2A. (B) Stereo view of the superimposed active sites of LAP (thin
red) and peptidase T (thic k black) s howin g the zinc ions and zinc ligand s of both p roteins as well as amastatin bound to LAP a s d iscu ssed in the text .
Ó FEBS 2002 Structure of peptidase T (Eur. J. Biochem. 269) 447
Substrate binding
Structural investigation of enzyme±ligand complexes is
usually one of the most useful m ethods for obtaining an
understanding of the active sites of enzymes. However, no
peptide a nalogue inhibitor is yet known for peptidase T.
Instead, a putative substrate bindin g site has been m apped
based on our knowledge of the structures of leucine
aminopeptidase (LAP) and APP [18,19]. It has been pointed
out that, e ven though the active sites of LAP and APP
appear to be d issimilar, the two zinc ions and the zinc-
bound water o f L AP can be superimposed on the zinc ions
and the zinc-bound water of APP [20]. We superimposed the
active site of APP on LAP in this way but because the

peptidase T structure h as no zinc-bound water, it was
superimposed on APP using the zinc ions and the C a atoms
of the amino-acid zinc ligands. Comparison w ith t he LAP±
amastatin complex [19] and the APP±phenylalanine
hydroxamate complex [18] indicate the location of the S1
pocket in p eptidase T. The active s ite of peptidase T and its
electrostatic surface potential are shown in Fig. 2 B. The
negatively charged S1 pocket is, in addition to the zinc
ligands and the sequence motifs already mentioned, lined
both with residues that are conserved in the peptidase T
family, such as Asn370 and Thr356, and with nonconserved
residues, e .g. G lu204, Gly197 and Ile352. To predict t he S1 ¢
and S2¢ subsite locations is more dif®cult, but an extended
tripeptide substrate molecule would run over the cleft that
separates the two domains. There are several conserved
residues in this region, e.g. Arg353, Gly354 and Gly355,
although the side chain of the arginine, in its present
conformation, is too far a way to be able t o interact with a
bound tripeptide. A solvent molecule that was interpreted as
a sulfate ion due to its relatively strong density and bulk size,
is hydrogen bonded to the conserved r esidues Arg280,
Tyr319, Gly355 as well as to His223 from a symmetry-
related molecule. The active site of LAP with a bound
amastatin molecule is shown superimposed o n the pepti-
dase T a ctive site in Fig. 3B.
DISCUSSION
Polypeptide fold and zinc ligands
The overall str uctural fold of peptidase T reveals homology
with the c atalytic domains of CG2, APP, LAP a nd
Carboxypeptidase A (CPA) [7,17,21±23]. The hexameric

LAP contains a second, N-terminal, domain involved in
subunit contacts. CG2, on the other hand, is dimeric and
has a 110 amino-acid insertion that forms a second domain
that is responsible for the dimeric interchain contacts. This
second d omain is also present i n p e ptidase T, which w e d id
not anticipate from the sequence. APP and CPA, are mono-
meric enzymes consisting of a s ingle domain. Although
CPA has a s ingle c atalytic zinc ion, the other four enzymes
have two zinc ions in their active sites. The active sites of
APP, CG2 and peptidase T have homologous zinc ion
ligands and are more closely related to each other than to
LAP and CPA. The similarity between the structure of t he
second domain in CG2 and peptidase T extends to the
interchain contacts around the twofold crystallographic axis
of the two structures . This suggests that peptidase T, as
CG2 i s a dimer. Peptidase T from various sources has been
subjected to gel ®ltration chromatography in order to
determine if it forms oligomers. Lactobacillus he lveticus
peptidase T was reported to be a trimer [24], Lactococcus
lactis and Pediococcus pentosaceus peptidase T behave as
dimers [25,26], Bacillus subtilis and S. typhimurium pepti-
dase T h ave been reported to e lute as monomers [4,27],
while E. coli peptidase T had an apparent molecular mass
of 80 000 Da [28]. The oligomeric state of S. typhimurium
peptidase T has been reinvestigated and the results indicate
that it is a dimer (D. Broder & G. Miller, unpublished
observations).
The amino-acid sequences of LAP and CPA are not
similar and the three-dimensional structures of these
proteins are too different f rom that o f p eptidase T to a llow

a meaningful superimposition. It is interesting, however, to
compare the topological positions of the active site residues.
By topological or homologous position we mean the
position in the sequence with reference to the strands (i.e.
before, on, or after a certain strand) that make up the central
mixed b sheet found in the catalytic domain of all of these
enzymes. The appearan ce of metal ligands in topologically
similar positions in b sheets of the same c onnectivity clearly
indicates a divergent evolutionary relationship. It has been
reported that the zinc ligands of LAP and APP a re found in
structurally nonequivalent position s [17]. W e ®nd, however,
that two of the amino acids that coordinate the two zinc ions
(His78 and Asp140) in peptidase T are indeed found in
topologically similar positions in both LAP and CPA.
Moreover, Glu196 in peptidase T a nd His196 in CPA are
also in homologous positions. Hence, all three amino acids
that coordinate the strongly bound zinc ion i n p eptidase T,
CG2 and APP are homologous to the three amino acids that
coordinate the single zinc i on in CPA. A similar conclusion
based on extensive sequence comparisons between and
within the CPA and CG2 families was recently reported [29].
In addition, one of the ligands for t he more weakly bound
zinc ion in peptidase T, Glu174, is in a position that is
topologically similar to t he zinc ligand Glu334 i n LAP. T he
zinc ligands in the different enzymes are aligned in Fig. 4.
The e volutionary relationship b etween peptidase T, CG2
and APP is also indicated by the presence of the c onserved
HXDT motif [9]. T he conformation of thes e residues results
in a forked h ydrogen bond between the histidine Nd1atom
and the side chain carboxylate and main chain carbonyl

group of the aspartate. T his probably e nsures that N d1and
not N e2 i s protonated, enhancing the electronegative
character of t he latter, which coordinates to the strongly
bound metal atom in t he active site. Interestingly, the
corresponding residue in CPA, His69, coordinates to the
zinc ion via its N d1 atom, while the Ne2 atom is hydrogen
bonded to a n aspartic acid residue, albeit from a different
part of the structure. The role of the t hreonine residue in the
HXDT motif is less obvious, but the environment of this
Fig. 4. Alignment o f homologous z inc ion ligands in p eptidase T, CG2,
APP, LAP and CPA.
448 K. Ha
Ê
kansson and C. G. Miller (Eur. J. Biochem. 269) Ó FEBS 2002
hydrophilic side chain seems t o b e a ccessible t o the solvent.
In some presumably related e nzymes, e .g. p eptidase D [30],
this residue is hydrophobic. The zinc ligand Asp140 in
peptidase T and t he homologous residues in C G2 and A PP
are linked to the subsequent amino-acid through a cis
peptide bond. In terestingly, there is a lso a cis peptide bond
near the active site in CPA, but the positions and
orientations of these peptide bonds vis-a
Á
-vis thezincions
as well as their locations in the amino-acid sequence are very
different in the two cases. There is for example no cis peptide
bond after G lu72 in CPA, which would correspond to
Asp140 in peptidase T. The role of these cis peptide bonds
remains obscure.
Substrate binding site and speci®city

The different domain organization in LAP, CPA, APP,
CG2 and peptidase T may re¯ect different ways to
discriminate against longer polypeptides. In A PP and
CPA, the N-terminal ( APP) or the C-t erminal (CPA) end
of the s ubstrate binds into a pocket and the absence of
additional steric hindrance enables the enzyme to cleave
polypeptides of varying size [31,32]. In peptidase T and
CG2, however, t he presence o f the dimerization domain
may restrict t he size of the substrate on t he C-terminal si de
of the scissile bond. Interestingly, CG2, which releases
C-terminal glutamic acid residues from peptides and from
folic acid and folic acid analogues such as m ethotre xate, has
slightly less space available and is more positively charged in
this part of the cleft. In LAP ®nally, t he size of the substrate
is restricted by controlled access to the active sites caused by
the assembly into hexamers. It is not obvious why pepti-
dase T in c ontrast to CG2 r equires a free N-ter minal amino
group in its substrates. It seems possible that the negative
charge around the S1 subsite may provide a favorable
interaction with a free N-terminal amino group. This
negative charge may also prevent dipeptides from entering
the active site, as there would be electrostatic repulsion
between the C-terminal carboxylate group and this part of
the enzyme. APP, which also has a negatively charged active
site, does not cleave peptides with a negatively charged P1
side chain, and displays lower activity towards dipeptides
and peptides with a negatively charged group in P1¢ position
[31,32]. This suggests that there is a penalty for having
negative charge on the substrate too close t o the
N-terminus. I n CG2, on the other hand, the presence of a

positively charged region closer to the active site, in part
caused by Arg324, creates a binding site for C-terminal
glutamic acid residues in the S1 ¢ binding site. I t should be
noted in this context, that the activity p ro®le of p eptidase T
does vary among different species [24±26,28].
While APP, CG2 and peptidase T have the same
polypeptide fold and similar active sites, a ligand complex
structure has been reported only for APP. The position of
this ligand and comparison of the zinc ions and the
binding of am astatin t o L AP suggest the location o f t he S1
subsite. As peptidase T cleaves a variety o f tripeptides,
albeit at different rates, the interactions between substrate
side chains and enzyme are probably not very speci®c.
However, the position of a putative sulfate ion in the
peptidase T structure suggests a possible binding site for
the substrate C -terminal c arboxylate g roup, within 10 A
Ê
of
the zinc ions. The sulfate ion is hydrogen bonded to four
amino-acid residues. Two of these, Arg280 and Gly355,
are conserved not only within the peptidase T sequences,
but in CG2 as well. In peptidase T , Gly355 is found in a
highly conserved X
1
X
2
RGGTGD motif, where X
1
and X
2

in most cases are P and I, respectively. This characteristic
motif distinguishes peptidase T from the other peptidases
of the MH clan, and may serve as a ®ngerprint motif. The
third of the sulfate binding amino acids, Tyr319, is
homologous to Arg324 in CG2 (Fig. 2A). This residue
has been s uggested to interact with the C-terminal
glutamate residue of CG2 substrates [7]. The fourth amino
acid, His223, from a symmetry-related molecule, is also
conserved in CG2. However, in CG2 this residue is more
than 13 A
Ê
away from the zinc ions and hence too remote
to interact with the substrate.
A catalytic mechanism has been suggested for LAP [20],
in which a bicarbonate bound to Arg336 acts in the same
way as Glu151 in APP [18] by promoting deprotonization of
the zinc bound water. The homologous and conserved
residue in peptidase T , G lu173, is indeed found in the same
relative structural position, as is Glu175 in CG2, strength-
ening the arguments of Stra
È
ter et al .[20].
The structure of S. typhimurium peptidase T not only
provides a framework for understanding the unusual
speci®city of this enzyme but also yields potentially useful
information concerning the very l arge family of proteins
related to peptidase T. The presence of three sequence
motifs that might de®ne this family has been pointed out
[33]. The proteins identi®ed by these motifs include
peptidases (peptidase T, CG2, yeast carboxypeptidase S)

and other enzymes p otentially involved in protein break-
down (mammalian aminoacylases), enzymes involved in
the h ydrolysis of a cylamino-acid intermediates in amino-
acid biosynthesis (DapE and A rgE), and many ORFs of as
yet unknown function. Representatives of the family are
found in all domains of life. The p reviously proposed
motifs involve the regions around the ®rst three zinc
ligands. We suggest that a fourth conserved motif
HXXX(X)E corresponding to the ®fth zinc ligand can be
added a s a further identifying feature of t his l arge family of
proteins.
ACKNOWLEDGEMENTS
We thank T. Knox f o r continu ous assistance, C. Ogata (NSLS X4A)
for X-ray assistance, A. Wang for access to computer facilities and
D. Broder for constructing strain TN5619 and for stimulating
discussions. This w ork w as supported by a grant (AI10333) from the
National Institute for Allergy and Infectious Diseases to C. G. M.
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