Received 3 September 2001, revised 6 November 2001, accepted 8 November 2001 region of similarity contains two of the five ligands that coordinate the two zinc ions in the active site
Trang 1Structure of peptidase T from Sa/monella typhimurium
Kjell Häkansson* and Charles G Miller
Department of Microbiology, University of Ilinois at Urbana-Champaign, Urbana, IL, USA
The structure of peptidase T, or tripeptidase, was deter-
mined by multiple wavelength anomalous dispersion
(MAD) methodology and refined to 2.4 A 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 metal ions, presumably zinc, are separated by 3.3 A,
and are coordinated by five 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 alone, could be identified Com- parison of the active sites of peptidase T, carboxypeptidase G2, Aeromonas proteolytica aminopeptidase, carboxypepti- dase A and leucine aminopeptidase reveals a common structural framework with interesting similarities and differences in the active sites and in the zinc coordination
A putative binding site for the C-terminal end of the tripeptide substrate was found at a peptidase T specific fingerprint sequence motif
Keywords: tripeptidase; aminotripeptidase; metallopep-
tidase; X-ray crystallography; MAD
Escherichia coli and Salmonella typhimurium express several
intracellular enzymes capable of hydrolyzing peptides [1]
Many of these enzymes have been shown to function in the
degradation of intracellular proteins and in the catabolism
of exogenously supplied peptides [2] One of these enzymes,
peptidase T, or tripeptidase (EC 3.4.11.-) is a 409 amino-
acid metalloenzyme that hydrolyzes tripeptides at their
N-termini [3,4] The enzymatic activity of the S typhimurium
enzyme is both specific and unusual; dipeptides, tetrapep-
tides or tripeptides with blocked N-termini are not cleaved
S typhimurium peptidase T expression is regulated by
FNR, a transcriptional activator that responds to anaero-
biosis [4,5] The aerobic expression level of peptidase T is
not sufficient to allow this enzyme to contribute to the
utilization of exogenously supplied peptides as amino acid
sources [3] Under anaerobic conditions, however, the pepT
gene is induced, leading to levels of peptidase T that allow it
to participate in the catabolism of tripeptides [5,6] It has
been speculated that 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 (ap) [4] This
Correspondence to C G Miller, Department of Microbiology, Uni-
versity of Illinois at Urbana-Champaign, B103 CLSL, 601 S Good-
win Avenue, Urbana, Illinois 61801, USA Fax: + 217 244 6697,
Tel.: + 217 244 8418, E-mail: charlesm@life.uiuc.edu
Abbreviations MAD, multiple wavelength anomalous dispersion;
CG2, carboxypeptidase G2; APP, Aeromonas proteolytica amino-
peptidase; LAP, leucine aminopeptidase; CPA, carboxypeptidase A
* Present address: Laboratory of Cellular and Molecular Physiology,
August Krogh Institute, Universitetsparken 13, DK 2100, Kbh O,
Denmark
(Received 3 September 2001, revised 6 November 2001, accepted 8
November 2001)
region of similarity contains two of the five 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 has therefore been classified into the M20 family of proteases/peptidases [8] A third zinc ligand, a histidine, can
be recognized as part of a HXDT motif [9], which is conserved in 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 specificity 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 and refined to 2.4-A resolution
MATERIALS AND METHODS
Crystallization and data collection Selenomethionine His-tagged peptidase T was expressed in strain TN5619, purified, crystallized from ammonium sulfate solutions at pH 7.5 and flash frozen in 50% sucrose
as previously described [10] Crystals belong to space group
116.1 A 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 ccp4 program suite [11,12]
Structure solution and refinement
The structure was solved by MAD methodology using 15 selenium atoms, four wavelengths and data to 2.8 A Determination of selenium positions, phase and electron density calculations and model refinement were performed with the cNs program package [13] The model was built manually and displayed using the graphics program o [14] The model was refined against one of the data sets processed
Trang 2
Table 1 Crystallographic data Data collection and phasing statistics
Completeness (final shell)* 97.7 (98.3) 98.7 (99.3) 98.7(99.2) 98.5 (98.8) 99.2 (99.2)
1/o() (ñnal shell) 28.8 (11.2) 23.8 (11.3) 20.3 (7.1) 26.5 (13.2) 23.4 (6.0)
* A Friedel pair is considered as two unique reflections for the anomalously processed data.” 24 409 structure factors were phased with a figure of merit of 0.79
Table 2 Refinement statistics
Protein atoms Solvent atoms (%) (%) Bonds (A) Angles (°) ( Ả)
nonanomalously to 2.4 A Data collection and refinement
statistics are shown in Tables | and 2 The electron density
of the three N-terminal residues was difficult to interpret A
putative sulfate ion, hydrogen bonded to the main-chain
amino groups of Lys3 and Leu5, was included in order to
account for all of the electron density in this part of the
structure Residues His305—Pro306 are not well defined due
to very weak electron density signals and the C-terminal
residue 409 along with the C-terminal hexahistidine tag
showed no significant density at all Outside these parts, the
polypeptide chain is generally well defined by the
2|Fo| - |Fc| density maps The side-chains of residues
Arg99, Aspl09, Vall15 and Tyr378, however, were not
defined 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 Data Bank and have the
accession code IFNO [15]
RESULTS
Overall structure
The final model consists of residues Metl—Gly408 (see
Materials and methods) All 15 methionines are built into
the selentum atom positions determined by CNS [13] The
sulfur atoms of Cys309 and Cys343 are within 2.0 and
2.3 A, 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 of
CG2, which we did not anticipate due to the low overall
sequence similarity between the two enzymes The catalytic
domain contains one seven-stranded mixed { sheet flanked
with œhelices, and a second, four-stranded antiparallel
Bsheet In CG2, the larger of the two Bsheets is eight-
stranded, but peptidase T residues Gly348-Glu350 (that would have formed the eighth strand) were not recognized
as a Bstrand by the program PROCHECK [l6] The major 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 flanking this insertion also differ between the two enzymes Another difference is that the loop between the first two helices (Lys19-Ser27) is larger in peptidase T Interestingly, this loop contains a conserved proline (Pro26) with a cis peptide bond When the two enzymes are superimposed, the insertion overlaps in space with an insertion in CG2, found
at the position of peptidase T residue Lys55 Most of the Ser182—Ala193 o helix in CG2 is broken up into an irregular structure in peptidase 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 domain, which is
comprised of residues Ala211—Tyr320, consists of a four- stranded antiparallel B sheet and two o helices Peptidase T has an insertion after the first « helix (at Pro246), while CG2 has an insertion at the turn between the second and third Bstrand (at peptidase 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 Co positions of the
67 identical amino acids are superimposed, the rmsd is 2.4 A(Fig 3A)
Dimerization contacts
The peptidase T dimerization domains in two crystallo- graphic asymmetric units make the same contacts around the twofold axis as in CG2 (Fig 1A) These consist mainly
Trang 3Fig 1 Ribbon representation and zinc ligands
of peptidase T (A) Ribbon representation [34]
of peptidase T, viewed along the crystallo-
graphic two-fold symmetry axis B Strands are
shown in blue if they belong to the central
B sheet of the catalytic domain, green if they
belong to the four-stranded B sheet of the
dimerization domain, and light blue if they
belong the smaller B sheets of the catalytic
domain « Helices but not 3,9 helices are
shown in red A second, symmetry related
molecule is shown in gray (B) The zinc ligands
of peptidase T The interaction between histi-
dine and aspartate in the HXDT motif and
the cis peptide bond between Asp140 and
Asp141 are shown
of an antiparallel Bstrand alignment involving residues
Ala264—-Val270 (Ala268—Ala270 in CG2), and an antipar-
allel 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
CG?2
Active site
There is one strong solvent electron density signal in the
active site, as previously noted [10], and a second atomic site
was revealed in the |Fo| — |Fc| map Due to the homology
of the active site residues and the similar active site
structures of peptidase T, CG2 and Aeromonas proteolytica
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 coordinating them are similar
in the three enzymes The weak density 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 refined temperature factors were 37 and 112 A?, respectively (the average solvent molecule temperature factor was 50 A’) Despite the high temperature factor, the chemical environment and similarity with the CG2 active site suggest that they are both zinc ions The well- defined, more strongly bound zinc (Zn501), is coordinated
by His78, Asp140 and Glul96, while the second zinc ion (Zn502) is coordinated by Asp140, Glul74 and His379 (Fig 1B) The average temperature factor for the side-chain ligands of the well-defined zinc ion is 26 A’, while the values for Glul74 and His379 are higher (60 A’) The distance
Trang 4446 K Hakansson and C G Miller (Eur J Biochem 269)
© FEBS 2002
Fig 2 Sequence alignments and visualized electrostatic surface potential of the active site
of peptidase T (A) Sequence alignment of
GD CG2 43 VIKTLEK LYNIETGT AEG IAAAGNFLEA BLKNILGF TỰ
PepT 1 MDKLLERFLH YVSLDTQSKS GVROQVPSTEG QMKLLRLLKQ QLEEMGLVNI
X42 «ŒŒŒ4Œ43222^2z2x4Œ0Œ%( tem)
SAGLVVG RGG YLKG ILAKA
ce2 B2 DNIVG KIKG KNLLLMS iD TV PFRVEG
PepT 51 GTLMA TLPANVEGDI PAIGFISHVD TSPDFSGKNV NPQIVENYRG
GPGI CG2 132 DKAY xã RD DKGGNAVILH *+ + + €
PepT 101 GDIALGIGDE VLSPVMFPVL HOLLGQTLIT TDGKTLLG) DKAGVAEIMT
«=&—œqgggøaooeee GVR FGSRDLIQEEAK PTSAGDE CG2 152 TUK LỤK BY D YGTITYLEN TRE GS LAD YVLSF
PepT 151 ALAVLKGNPI PHGDIKVAPT PD IGKGAK HPDVEAFPGAQ WAYTVDGGGV
eeeees sose
A CG2 20B KLSLGTSG 1 ẬYVOQVNI TK ASHAGA PEL GYNALVEASD LVLRT
PepT 201 GELEFENFNA ASVNIKIVGN NVHPGTAKGV MVNALSLAAR IHABVPADEA
BeonrrTaUGG
252 MNIDDKAK GNVSNIIP
CG2 NLR FNWTI AKA ÄSATLNADVE YARNEDEDAA MKTLEERAQO
PepT 251 PETTEGYEGF YHLASMKGTV DRAEMHYIIR DFDRKOFEAR KRKMMEIAKK
- 5
309 KK NAG EGGK
ce2 LPEAD VKVIVTRGRP AF KLV! DKAVA YY KEA \GGTLGVE
PepT 301 VGKGLHPDCY IELVIEDSYY NMREKVVEHP HILDIAQQAM RDCHITPEMK
® —esyp- “e8 4422/x.xexœó
RT SUGI K
CG2 355 E GGGTDAAY AALSGKPVIE x*i4* + * + PGFGYNS DAEYVDISAI = + * PRRLYMAARL *
PepT 351 PIRGGTDGAQ LSFMGLPCPN LFTGGYNY KHEPYTLEGM EXAVOVIVRI
CC
GK
CG2 408 TMDLGA
AELTAKRGO
between the two zinc ions is 3.3 A No zinc bound water was
found; the closest zinc—water contact is 3.1 A (Zn502) The
absence of a zinc bound water could be due to the low
occupancy of the second zinc site or to the limited resolution
of the data Superimposition of the two metal ions and the
Ca atoms of the five ligand amino-acids of peptidase T and
the other two enzymes results, in either case, in an rmsd of
0.5 A
Zinc ligand motifs
The first of the metal coordinating amino acids, His78, is
found in an HXDT motif, where X is a hydrophobic amino
acid In peptidase T, residue X is a valine (Val79), which is
buried in a hydrophobic cluster The polypeptide makes a
sharp turn at this position, terminating a B strand (Fig 1B)
As a result of this turn, both the main chain carbonyl and
Pseudomonas sp strain RS-16 carboxypepti- dase G2 (CG2) and Salmonella typhimurium peptidase T based on piecewise superimposi- tion of local structure elements The sequence
of peptidase T is given in lines of 50 amino- acids CG2 residues that do not have homo- logues in peptidase T are written above the alignment Peptidase T secondary structure elements are indicated with the same colour scheme as in Fig 1A The zine ligands are in yellow boxes, and identical residues are marked with an asterisk (*) (B) The electro- static surface potential of the active site of peptidase T visualized by the program GRASP [35] Regions of positive potential are shown in blue and negative potential in red A p-iodo- p-phenylalanine hydroxamate molecule, superimposed from the experimentally determined APP complex [18], and a sulfate ion are shown in green The insert, viewed from the top, shows the conserved RGGTDG fingerprint motif in black beneath a
semitransparent surface
the side chain carboxylate group of the Asp80 residue are within hydrogen bond distance to the N61 atom of His78 (3.0 and 3.3 A, 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 APP 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, Glul74, is preceded by Glu173, 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 acids are preceded 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 conformations in the three enzymes
In CG2, the following residue, a proline, makes van der
Trang 5
Asp 196 Gly 197
lu174 (dimer)
cự Asp19 Gly 197
h
Lge
sp140
His223 Glu174 (dimer)
is379
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 homologous residues indicated in Fig 2A (B) Stereo view of the superimposed active sites of LAP (thin red) and peptidase T (thick black) showing the zinc ions and zinc ligands of both proteins as well as amastatin bound to LAP as discussed in the text
Waals interactions with Alal40—-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 are glycines The first two of these
glycines are conserved in most of the available peptidase T
sequences The fifth ligand, His379, is more difficult to
identify from the sequence alone, but is preceded by a
hydrophobic residue (Tyr, Phe, Ie or Met) in all three
enzymes and has a glutamate four or five residues on the
C-terminal side This glutamate hydrogen bonds to the main
chain amino groups of Leul37, Gly138, Alal39, and
Asp140 in the loop preceding the important active site residues Asp140—141 which are connected by the cis peptide bond This interaction probably contributes to the stability
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
Trang 6448 K Hakansson and C G Miller (Eur J Biochem 269)
Substrate binding
Structural investigation of enzyme-—ligand complexes is
usually one of the most useful methods for obtaining an
understanding of the active sites of enzymes However, no
peptide analogue inhibitor is yet known for peptidase T
Instead, a putative substrate binding site has been mapped
based on our knowledge of the structures of leucine
aminopeptidase (LAP) and APP [18,19] It has been pointed
out that, even though the active sites of LAP and APP
appear to be dissimilar, the two zinc ions and the zinc-
bound water of LAP 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 has no zinc-bound water, it was
superimposed on APP using the zinc ions and the Ca atoms
of the amino-acid zinc ligands Comparison with the LAP-
amastatin complex [19] and the APP-phenylalanne
hydroxamate complex [18] indicate the location of the SĨ
pocket in peptidase T The active site of peptidase T and its
electrostatic surface potential are shown in Fig 2B The
negatively charged SI 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 Glu204, Gly197 and Ile352 To predict the S1’
and S2 subsite locations is more difficult, 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 away to be able to 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 residues 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 on the pepti-
dase T active site in Fig 3B
DISCUSSION
Polypeptide fold and zinc ligands
The overall structural fold of peptidase T reveals homology
with the catalytic domains of CG2, APP, LAP and
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 domain is also present in peptidase T, which we did
not anticipate from the sequence APP and CPA, are mono-
meric enzymes consisting of a single domain Although
CPA has a single catalytic 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 the
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
CG? is a dimer Peptidase T from various sources has been
© FEBS 2002
subjected to gel filtration chromatography in order to determine if it forms oligomers Lactobacillus helveticus 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 have been reported to elute 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 from that of peptidase T to allow
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 appearance of metal ligands in topologically similar positions in B sheets of the same connectivity clearly indicates a divergent evolutionary relationship It has been reported that the zinc ligands of LAP and APP are found in structurally nonequivalent positions [17] We find, 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 and His196 in CPA are also in homologous positions Hence, all three amino acids that coordinate the strongly bound zinc ion in peptidase T, CG2 and APP are homologous to the three amino acids that coordinate the single zinc ion 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 the more weakly bound zinc ion in peptidase T, Glul74, is in a position that is topologically similar to the zinc ligand Glu334 in LAP The zinc ligands in the different enzymes are aligned in Fig 4 The evolutionary relationship between peptidase T, CG2 and APP is also indicated by the presence of the conserved HXDT motif [9] The conformation of these residues results
in a forked hydrogen bond between the histidine N61 atom and the side chain carboxylate and main chain carbonyl group of the aspartate This probably ensures that N61 and not Ne2 is protonated, enhancing the electronegative character of the latter, which coordinates to the strongly bound metal atom in the active site Interestingly, the corresponding residue in CPA, His69, coordinates to the zinc ion via its Ndl atom, while the Ne2 atom is hydrogen bonded to an aspartic acid residue, albeit from a different part of the structure The role of the threonine residue in the
HXDT motif is less obvious, but the environment of this
Fig 4 Alignment of homologous zinc ion ligands in peptidase T, CG2, APP, LAP and CPA.
Trang 7hydrophilic side chain seems to be accessible to the solvent
In some presumably related enzymes, e.g peptidase D [30],
this residue is hydrophobic The zinc ligand Asp140 in
peptidase T and the homologous residues in CG2 and APP
are linked to the subsequent amino-acid through a cis
peptide bond Interestingly, there is also a cis peptide bond
near the active site in CPA, but the positions and
orientations of these peptide bonds vis-d-vis the zinc ions
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 Glu72 in CPA, which would correspond to
Asp140 in peptidase T The role of these cis peptide bonds
remains obscure
Substrate binding site and specificity
The different domain organization in LAP, CPA, APP,
CG2 and peptidase T may reflect different ways to
discriminate against longer polypeptides In APP and
CPA, the N-terminal (APP) or the C-terminal (CPA) end
of the substrate 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, the presence of the dimerization domain
may restrict the size of the substrate on the C-terminal side
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 methotrexate, has
slightly less space available and is more positively charged in
this part of the cleft In LAP finally, the 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 contrast to CG2 requires a free N-terminal amino
group in its substrates It seems possible that the negative
charge around the SI 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 PI’ position
[31,32] This suggests that there is a penalty for having
negative charge on the substrate too close to the
N-terminus In 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 SI’ binding site It should be
noted in this context, that the activity profile of peptidase 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 amastatin to LAP suggest the location of the S1
subsite As peptidase T cleaves a variety of tripeptides,
albeit at different rates, the interactions between substrate
side chains and enzyme are probably not very specific
However, the position of a putative sulfate ion in the
peptidase T structure suggests a possible binding site for
the substrate C-terminal carboxylate group, within 10 A 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;X,RGGTGD motif, where X, and X>
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 fingerprint motif The third of the sulfate binding amino acids, Tyr319, is homologous to Arg324 in CG2 (Fig 2A) This residue has been suggested 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 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, Glu173, is indeed found in the same
relative structural position, as is Glul75 in CG2, strength-
ening the arguments of Strater et al [20]
The structure of S typhimurium peptidase T not only provides a framework for understanding the unusual specificity of this enzyme but also yields potentially useful information concerning the very large family of proteins related to peptidase T The presence of three sequence motifs that might define this family has been pointed out [33] The proteins identified by these motifs include peptidases (peptidase T, CG2, yeast carboxypeptidase S) and other enzymes potentially involved in protein break- down (mammalian aminoacylases), enzymes involved in the hydrolysis of acylamino-acid intermediates in amino- acid biosynthesis (DapE and ArgE), and many ORFs of as yet unknown function Representatives of the family are found in all domains of life The previously proposed motifs involve the regions around the first three zinc ligands We suggest that a fourth conserved motif HXXX(X)E corresponding to the fifth zinc ligand can be added as a further identifying feature of this large family of
proteins
ACKNOWLEDGEMENTS
We thank T Knox for continuous 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 work was supported by a grant (AI10333) from the National Institute for Allergy and Infectious Diseases to C G M
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2 Yen, C., Green, L & Miller, C.G (1980) Degradation of intra- cellular protein in Salmonella typhimurium peptidase mutants
J Mol Biol 143, 21-33
3 Strauch, K.L & Miller, C.G (1983) Isolation and characteriza- tion Salmonella typhimurium mutants lacking a_tripeptidase (peptidase T) J Bacteriol 154, 763-771.
Trang 8450 K Hakansson and C G Miller (Eur J Biochem 269)
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