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University of Groningen
Structure-function relationships in type I signal peptidases of
bacilliRoosmalen, Maarten Leonardus van
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Structure-function relationships in type I signal peptidases of
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61
CChhaapptteerr FFiivvee
DISTINCTION BETWEEN MAJOR ANDMINOR BACILLUS SIGNAL
PEPTIDASES
BASED ON PHYLOGENETIC ANDSTRUCTURAL CRITERIA
This chapter was published in:The Journal of Biological
Chemistry (2001), 276(27), 25230-25235.
555
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Chapter Five
62
DISTINCTION BETWEEN MAJOR AND MINOR BACILLUSSIGNAL PEPTIDASES
BASED ON PHYLOGENETIC AND
STRUCTURAL CRITERIA
Maarten L. van Roosmalen, Jan D.H. Jongbloed, Jean-Yves F.
Dubois, Gerard Venema, Sierd Bron andJan Maarten van Dijl
Summary
The processing of secretory pre-proteins by signal peptidases
(SPases) is essential for cell viability.As previously shown for
Bacillus subtilis, only certain SPases of organisms containing
multipleparalogous SPases are essential. This allows a distinction
between SPases that are of major andminor importance for cell
viability. Notably, the functional difference between major and
minorSPases is not clearly reflected in sequence alignments. Here,
we have successfully used molecularphylogeny to predict major and
minor SPases. The results were verified with SPases from
variousbacilli. As predicted, the latter enzymes behaved as major
or minor SPases when expressed in B.subtilis. Strikingly, molecular
modeling indicated that the active site geometry is not a
criticalparameter for classification of major and minor Bacillus
SPases. Even though the substrate bindingsite of the minor SPase
SipV is smaller than that of other known SPases, SipV could be
convertedinto a major SPase without changing this site. Instead,
replacement of amino-terminal residues ofSipV with corresponding
residues of the major SPase SipS was sufficient for conversion of
SipVinto a major SPase. This suggests that differences between
major and minor SPases are based onactivities other than substrate
cleavage site selection.
Introduction
Signal peptidases (SPases) play a key role in thetransport of
proteins across membranes in all livingorganisms. The type I SPases
are integralmembrane proteins that remove signal peptidesfrom
pre-proteins during, or shortly aftertranslocation across the
cytoplasmic membrane,thereby releasing the mature proteins from
thetrans side of the membrane (for reviews, see Refs.(159) and
(89)).
In recent years, type I SPases from many differentorganisms have
been identified. Comparison ofthese SPases showed that they can be
divided intwo sub-families: P(prokaryotic)-type andER(endoplasmic
reticulum)-type SPases (106).The P-type SPases are found in
eubacteria andorganelles of eukaryotes. In contrast, the
ER-typeSPases are typical for the endoplasmic reticular
membrane. Strikingly, a few ER-type SPases wereshown to be
present in sporulating Gram-positiveeubacteria, such as Bacillus
subtilis (106). In fact,B. subtilis was the first eubacterium in
which thepresence of both P- and ER-type SPases wasdemonstrated.
With respect to SPases, B. subtilis isnot only exceptional because
it contains both P-and ER-type SPases, but also because it is
theorganism with the largest known number of type ISPases. These
include the chromosomally encodedP-type SPases SipS, SipT, SipU and
SipV, theplasmid-encoded P-type SPases SipP1015 andSipP1040, and
the chromosomally-encoded ER-type SPase SipW (103-108). These
observationssuggested that at least some of the SPases of
B.subtilis have specialized functions. Indeed, it wasrecently
shown, that SipS, SipT and SipP1015 areof major importance for the
secretion ofdegradative enzymes and cell viability, whereasSipU,
SipV and SipW have only a minor role in
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Major and minor Bacillus signal peptidases
63
Table 5.I. Plasmids and Bacterial StrainsPlasmids Relevant
properties Reference
pGEFdSH-K83A encodes sf-SipS-His K83A of B. subtilis; 3.6 kb;
Apr (121)pGDL41 Encodes pre(A13i)-β-lactamase and SipS of B.
subtilis; replicates in E. coli and B. subtilis.
8.1 kb; Apr; Kmr(103)
pGDL46.36 pGDL48 derivative carrying the sipC (Bca) gene; Apr;
Kmr (118)pGDL48 Lacks the sipS gene and contains a multiple cloning
site; otherwise identical to pGDL41; 7.5
kb; Apr; Kmr(107)
pGDL90 pGDL48 derivative carrying the sip (Bli) gene; Apr; Kmr
This paperpGDL100 pGDL48 derivative carrying the sipT (Bsu) gene;
Apr; Kmr (105)pGDL121 pGDL48 derivative carrying the sipU (Bsu)
gene; Apr; Kmr (105)pGDL131 pGDL48 derivative carrying the sipV
(Bsu) gene; Apr; Kmr (105)pGDL140 pGDL48 derivative carrying the
sipW (Bsu) gene; Apr; Kmr (110)pM0 pGDL41 derivative; the 3' end of
the sipS gene is replaced with a multiple cloning site; Apr;
Kmr(113)
pM0V pM0 derivative carrying a SipSV fusion protein; Apr; Kmr
This paper
Bacterial strain Genotype ReferenceE. coliBL21(DE3) F- ompT
rb-mb- λDE3 (167)
B. subtilis8G5 trpC2; tyr; his; nic; ura; rib; met; ade; lacks
the sipP genes (205)8G5 ∆S like 8G5; rib+; sipS (104)8G5 ∆ST pGDL90
like 8G5; sipS sipT; contains the sip (Bli) gene This paper8G5 ∆ST
pM0V like 8G5; sipS sipT; contains the hybrid sipSV gene This
paper
protein secretion and are probably involved inspecific
non-essential processes (106;108). Forexample, SipW is specifically
required for theprocessing of two precursors, pre-TasA and
pre-YqxM, but not for cell viability (116;117). As thepresence of a
single major SPase (i.e. SipS, SipT orSipP1015) is sufficient for
growth and cell viabilityof B. subtilis, it seems that the
secretory precursorprocessing machinery of this organism
isfunctionally redundant (106;108).
In addition to an N-terminal membrane anchordomain (A), all
P-type SPases contain four well-conserved domains (B to E) (89).
These conserveddomains include residues involved in
substraterecognition and catalysis. Specifically, domain Bcontains
a strictly conserved Ser residue anddomain D a strictly conserved
Lys residue.Together, these residues form a Ser-Lys catalyticdyad
(29). The domains B to E of the P-typeSPases are conserved in the
ER-type SPases.Nevertheless, instead of a Lys residue, domain Dof
the ER-type SPases contains a strictly conservedHis residue, which
is required for catalysis. Atpresent, it is not known whether this
His residue ispart of a Ser-His catalytic dyad, or a
Ser-His-Aspcatalytic triad, as described for the classical
serineproteases (110;115).
The distinction between P-type and ER-typeSPases can readily be
made on the basis of theconserved Lys or His residues in domain D.
Incontrast, it is presently not clear which propertiesdetermine
whether a type I SPase is a major or aminor SPase of B. subtilis. A
clear definition ofthese properties is important to understand
themolecular basis for SPase substrate specificity.Therefore, the
present studies were aimed at thecharacterization of differences
between major andminor Bacillus SPases, and the identification
ofdomains in these enzymes that are critical for theirspecificity.
The results show that major and minorBacillus SPases can be
distinguished byphylogenetic analyses and that critical
informationfor their role in cell viability is provided byresidues
that are located amino-terminally of thecatalytic Ser residue.
Strikingly, molecularmodeling of the active-site of major and minor
P-type SPases of B. subtilis suggests that the activesite cleft of
the minor SPase SipV is significantlysmaller than that of the other
known Bacillus P-type SPases. Nevertheless, this difference can
notexplain why SipV is a minor SPase.
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Chapter Five
64
Materials and Methods
Plasmids, Bacterial Strains and MediaTable 5.1 lists the
plasmids and bacterial strains used. TYmedium (tryptone/yeast
extract) contained Bacto tryptone(1%), Bacto yeast extract (0.5%)
and NaCl (1%). If required,media for Escherichia coli were
supplemented withampicillin (Ap; 50 µg/ml) or kanamycin (Km; 20
µg/ml);media for Bacillus subtilis were supplemented with Km
(20µg/ml) or chloramphenicol (Cm; 5µg/ml).
Evolutionary tree computationsAmino acid sequences of Bacillus
SPases were collectedfrom the SubtiList and GenBank databases.
Alignments wereperformed with the ClustalX software (218), using
the"Gonnet 250" and the "Gonnet series" matrices as thepairwise
alignment parameters and multiple alignmentparameters,
respectively. Default gap opening and gapextension parameters were
applied. When the SPase of E. coli(Lep) was included in the
alignments, using the sameparameters, the aligned sequences showed
highly congruentareas that correspond to α-helices, β-strands, and
previouslydefined conserved domains (D) of Lep and other type
ISPases (89) (29). Therefore, the complete data set (CS),predicted
α-helices, β-strands and conserved domains wereused in the
phylogenetic analyses. Autapomorphic insertionsor deletions
(Indels) were removed from all data sets. Treereconstructions were
performed according to two differentmethods. Firstly, the Maximum
Likelihood (ML) method wasused as implemented in the PUZZLE 4.02
software (219).The VT (variable time) matrix
(http://www.dkfz-heidelberg.de/tbi/people/tmueller/paper/VT-matrix/)
wasapplied, with 4 gamma rates. One thousand replications wereused
to calculate the "Quartet Puzzling" values (QP).Secondly, the
Maximum Parsimony (MP) method was usedas implemented in the program
PAUP 4.03b(http://www.lms.si.edu/PAUP/). The MP tree
reconstructionwas done with the “Branch Swapping:
Tree-Bisection-Reconnection” (TBR) algorithm, applying 10
randomadditions of sequences. One thousand replications were usedto
calculate the bootstrap values (BP).
DNA techniquesProcedures for DNA purification, restriction,
ligation,agarose gel electrophoresis, and transformation of E.
coliwere carried out as described in Ref. (161). Enzymes werefrom
Roche Molecular Biochemicals. The polymerase chainreaction (PCR)
was carried out with Vent DNA polymerase(New England Biolabs) as
described in Ref. (113). DNA andprotein sequences were analyzed
with the PCGene AnalysisProgram (version 6.7; Intelligenetics Inc.)
and ClustalWversion 1.74 (162). B. subtilis was transformed as
described inRef. (106). Correct integration of plasmids or
resistancemarkers into the chromosome of B. subtilis was verified
bySouthern blotting, or PCR.Plasmid pGDL90, specifying Sip (Bli)
from Bacilluslicheniformis, was constructed by ligating an EcoRI-
andSalI-cleaved PCR-amplified fragment of sip (Bli), into the
corresponding sites of pGDL48. The sip (Bli)-specificfragment
was amplified with primers Lbl1 (5'-ACGCGTCGAC TATGC TGTGA CAGAC
TG-3') and Lbl2 (5'-CGGAA TTCGC AGTGC TGGCA TCA-3'), using
B.licheniformis chromosomal DNA as a template. PlasmidpM0V,
specifying a hybrid of SipS and SipV from B. subtilis,was
constructed by ligating an EcoRI- and SalI-cleaved PCR-amplified
fragment of sipV, into the corresponding sites ofpM0. The
sipV-specific fragment was amplified with primersJBV1 (5'-TGTCG
TCGAC GGTGA CAGTA TGAACCCGAC CTTCC-3') and JBV2 (5'-CGGAA TTCGC
TAGCGACGCC TCTTC AATTA GCA-3'), using B. subtilischromosomal DNA as
a template. Note that primer JBV1 isdesigned such that the
resulting SipSV fusion proteincontains the amino-terminal fragment
of SipS (residues 1-43)that includes the active site Ser43 residue,
fused to thecarboxy-terminal fragment starting at the
correspondingposition in SipV (residues 34-168).
Western Blot AnalysisPolyclonal antibodies against SipS of B.
subtilis wereprepared by immunization of rabbits (Eurogentec) with
apurified soluble form of this protein (sf-SipS-His K83A).This
protein consists of the catalytic domain of SipS, lackingresidues
2-29. Furthermore, sf-SipS-His K83A (Bsu) containsa
carboxyl-terminal hexa-histidine tag, facilitating thepurification
by metal-affinity chromatography (121). Westernblotting was
performed as described in Ref. (184). Afterseparation by SDS-PAGE,
proteins were transferred toImmobilon-PVDF membranes (Millipore
Corporation). Todetect SPases, B. subtilis or E. coli cells were
separated fromthe growth medium by centrifugation (5 min, 10.000 x
g,room temperature), and samples for SDS-PAGE wereprepared as
previously described (121;164). SPases werevisualized with specific
antibodies and horseradishperoxidase-anti-rabbit-IgG conjugates
(AmershamInternational).
Molecular modeling and molecular dynamics
simulationsThree-dimensional models of Bacillus SPases were built
onthe basis of homology with the E. coli SPase (PDB ProteinData
Bank: 1b12) using the molecular modeling programWHAT-IF
(http://www.cmbi.kun.nl/whatif/) (220). Themolecular dynamics
program GroMacs(http://md.chem.rug.nl/software.html) was used to
perform astandard energy minimization in vacuo of a
pentapeptidesubstrate in the three-dimensional model of SipS.
Results
Phylogenetic clustering of major Bacillus signalpeptidasesTo
investigate the relationships between major andminor SPases of
Bacillus species, phylogeneticanalyses were performed, applying the
MaximumLikelihood (ML) and Maximum Parsimony (MP)
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Major and minor Bacillus signal peptidases
65
Table 5.II. Summary of Quartet Puzzling and Bootstrap
values.Bootstrap values were obtained with the maximum likelihood
method (1000 replications each), and quartet puzzling valueswith
the maximum parsimony method. Values lower than 50 are not
considered significant. Values higher than 80 are shown inbold. CS,
complete sequences; α, α-helices; β, β-strands; D, conserved
domains.
Maximum Likelihood Maximum ParsimonyCS αααα ββββ D CS αααα ββββ
D
Number of variable (ML) /informative (MP) sites 236 34 82 42 159
23 59 32
SipW (1) 91 71 75 93 100 75 99 99SipV (2) 84 84 98 72 99 56 98
98SipT Bsu + SipT Bam (3) 78 80 75 80 86SipS Bsu + SipS Bam (4) 90
89 75 98 87 72SipP (5) 91 60 53 61(3) + (4) + (5) + Sip Bli 93 79
60 79 77(3) + (4) + (5) + Sip Bli + SipU 90 96 85 80(1) + SipU
62(1) + SipX Ban 70 100 99(2) + SipC Bca 66 96 54 67(3) + SipX Ban
81
methods. For this purpose, either the completesequences (CS),
conserved α-helices (α), β-strands(β) or domains (D) were used
(Table 5.II).Consistent with the fact that only few α-helices
arepresent in type I SPases (29), the ML analysis thatwas based on
α-helices resulted in a tree with apoorly resolved topology, and
the equivalent MPanalysis in 108 “most parsimonious trees”
(112steps long; CI1 excluding uninformative characters= 0.8077, RI1
= 0.7101, RC1 = 0.5833). Far betterresults were obtained when
complete sequences, β-strands, or conserved domains were used in
the MLand MP analyses (Table 5.II). In fact, the MPanalysis with
complete sequences resulted in one"most parsimonious tree" (805
steps, CI excludinguninformative characters = 0.8188, RI =
0.7152,RC = 0.5988; Fig. 5.1). One "most parsimonioustree" was
obtained when conserved β-strands wereused (283 steps, CI excluding
uninformativecharacters = 0.8618, RI = 0.8046, RC = 0.7079);and
three “most parsimonious trees” were obtainedwith the conserved
domains (134 steps, CIexcluding uninformative characters = 0.8319,
RI =0.7938, RC = 0.6753). Notably, all data sets arecongruent with
respect to the clustering of the fourbest supported groups of
Bacillus SPases: I), theSipW group; II), the SipV group; III), the
SipT ofB. subtilis (Bsu) + SipT of Bacillusamyloliquefaciens (Bam)
group; and IV), the SipSgroup (Table 5.II). As shown in Fig. 5.1,
SipT ofBacillus anthracis (Ban) seems to be more closelyrelated to
the SipW group (bootstrap percentages[BP]/quartet puzzling values
[QP] are 100/nc for
Fig. 5.1. Most parsimonious tree of known Bacillus SPases.A most
parsimonious tree of different Bacillus SPases is shown.The
calculated Maximum Parsimony (top) and MaximumLikelihood (bottom)
values are shown at junctions. The lengthof the branches reflects
the number of amino acid changesbetween different SPases, as
indicated by the bar. See Materialsand Methods for details
concerning the calculations. The clusterof major SPases is
encircled. The following SPases are shown:SipS, SipT, SipU, SipV,
SipW, SipP1015 and SipP1040 fromB. subtilis (Bsu); SipS, SipT, SipV
and SipW from B.amyloliquefaciens (Bam); SipC from B. caldolyticus
(Bca); Sipfrom B. licheniformis (Bli); and SipX and SipW from
B.anthracis (Ban). Note that SipX was originally annotated asSipT
(NCBI accession #AAF13664) but, to avoid
possiblemisinterpretations, this SPase was renamed.
CS and 100/70 for β-strands) than to the SipT(Bsu) + SipT (Bam)
group. To prevent the possiblemisinterpretation that SipT (Ban) is
related to themajor SPases SipT (Bsu) and SipT (Bam), the SipT(Ban)
protein was renamed SipX (Ban).
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Chapter Five
66
Fig. 5.2. Functional identification of major SPases.
Westernblotting analysis of B. subtilis 8G5 (BS), B. subtilis ∆S
(BS ∆S),B. subtilis ∆ST (BS ∆ST) and E. coli MC1061 (EC)
producingSip (Bli) as specified by plasmid pGDL90, or the hybrid
SPaseSipSV as specified by plasmid pM0V. As a control,
strainscontaining the empty vector pGDL48 (ev) were used.
Thepresence of SipS (Bsu), SipT (Bsu) and Sip (Bli) was
visualizedwith polyclonal antibodies against the catalytic domain
of SipS.Note that the hybrid SPase SipSV does not cross-react
withthese antibodies.
Furthermore, SipC of Bacillus caldolyticus (Bca)seems to be most
closely related to SipV (Bsu) andSipV (Bam). Most importantly, the
functionallydefined major SPases SipS, SipT and SipP1015 ofB.
subtilis cluster together (encircled in Fig. 5.1).This clustering
is supported by bootstrappercentages of 79/93 and 77/60 when
completesequences or β-strands were used for the
analyses,respectively. This observation suggests that theother
SPases in this cluster, SipP1040, SipS (Bam),SipT (Bam) and Sip
(Bli), should also be classifiedas major SPases. In contrast, all
enzymes notincluded in this cluster would be minor SPases.
Functional identification of major BacillusSPasesAs the
distinction between major and minor SPasesis based on functional
differences, we tested theoutcome of the phylogenetic analysis
incomplementation experiments with tworepresentative SPases: SipC
of Bacilluscaldolyticus (118), which clusters with the minorSPases
(Fig. 5.1), and Sip (Bli) of B. licheniformis(214), which clusters
with the major SPases. Tothis purpose, the sipC gene was expressed
in the B.subtilis strain ∆S, which lacks the sipS gene,
bytransformation with the pGDL48-derived plasmidpGDL46.36.
Subsequently, we tried to disrupt thesipT gene of the resulting
strain with a Cmresistance marker by transformation withchromosomal
DNA of B. subtilis ∆T-Cm. Eventhough this experiment was repeated
several times,no Cm resistant transformants were obtained,
Fig. 5.3. Limited cross-reactivity of SipS-specific
antibodies.Western blotting analysis of B. subtilis ∆S strains
containingplasmids for the overproduction of type I SPases of
B.subtilis. Plasmid pGDL41 was used for the overproduction ofSipS,
pGDL100 for SipT, pGDL121 for SipU, pGDL131 forSipV, and pGDL140
for SipW.
indicating that SipC of B. caldolyticus behaves as aminor SPase
in B. subtilis that cannot replace SipSand SipT. A completely
different result wasobtained in parallel experiments with Sip
(Bli). Inorder to test whether this SPase could replace SipSand
SipT of B. subtilis, the sip (Bli) gene wasamplified by PCR and
cloned. Next, B. subtilis ∆Sas transformed with the pGDL48-derived
plasmidpGDL90, which carries the sip (Bli) gene, and thesipT gene
of the resulting strain was disrupted witha Cm resistance marker by
transformation withchromosomal DNA of B. subtilis ∆T-Cm. ViableCm
resistant transformants were obtained, whichwere shown to have
disrupted sipS and sipT genes(data not shown). As shown by Western
blotting,these transformants produce Sip (Bli), which cross-reacts
with the antibodies raised against thecatalytic domain of SipS of
B. subtilis (Fig. 5.2).Taken together, these observations strongly
suggestthat Sip (Bli) behaves as a major SPase and SipC(Bca) as a
minor SPase in B. subtilis. The view thatall SPases clustering with
SipS (Bsu), SipT (Bsu),SipP1015 and Sip (Bli) behave as major
SPaseswas finally confirmed by similar complementationexperiments,
demonstrating that SipS (Bam), SipT(Bam) and SipP1040 can replace
SipS and SipT ofB. subtilis (J.D.H. Jongbloed and H.
Tjalsma,unpublished observations).
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Major and minor Bacillus signal peptidases
67
Fig. 5.4. Alignment of the substrate binding regions of known
Bacillus SPases. The conserved domains B, C, and D from
P-typeSPases of B. subtilis, B. amyloliquefaciens, B.
licheniformis, B. caldolyticus, B. anthracis and E. coli, which
contain residues thatform the S1 and S3 substrate binding regions,
were aligned. Residues, predicted to belong to the S1 or S3 pockets
are labeled with 1or 3, respectively. Residue numbers below the
alignment are derived from SipS (Bsu).
Because the antibodies raised against SipS of B.subtilis
cross-reacted with Sip (Bli) (these studies),and the major SPase
SipP1015 (108), weinvestigated whether these antibodies could
beused to discriminate between major and minorSPases of B.
subtilis. To this purpose, Westernblotting experiments were
performed with strainscontaining plasmids for the overproduction of
therespective SPases. Only SipT was shown to cross-react with the
antibodies raised against SipS, whichimplies that the major SPases
SipS, SipT andSipP1015 share at least one antigenic
determinantwhich is absent from the minor SPases SipU, SipVand SipW
(Fig. 5.3). This idea is supported by theobservation that the major
SPases SipS (Bam),SipT (Bam) and SipP1040 cross-reacted with
theantibodies against SipS (Bsu) (data not shown). Ithas to be
noted, however, that the antibodiesagainst SipS also cross-reacted
with the catalyticdomain of SipC (Bca) upon overproduction in
E.coli (data not shown), indicating that theseantibodies do not
allow the discrimination betweenmajor and minor Bacillus SPases in
general.
SPase active site modeling by homologyTo investigate whether the
active site geometries ofthe known major and minor P-type SPases of
B.subtilis are significantly different, three-dimensional models of
these SPases wereconstructed on the basis of the crystal structure
ofthe E. coli SPase as determined by Paetzel et al.(29). For this
purpose, the sequences of theseSPases were aligned with the
ClustalW Program(Fig. 5.4). SipS (Bsu) and the E. coli SPase showan
overall sequence identity of 26%, which is low
for modeling by homology. However, the fourconserved domains B
to E of these SPases show62% sequence identity. Notably, the active
site ofthe E. coli SPase is almost entirely composed ofthese four
conserved domains that are typical forall P-type SPases (29). In
what follows, we have,therefore, based our conclusions exclusively
onmodeled active site regions of Bacillus SPases. Thehomology
modeling program WhatIf was used togenerate the three-dimensional
models of variousknown SPases of bacilli. As shown for SipS of
B.subtilis (Fig. 5.5), Met44 and Leu48 (marked inblue), Val39 and
Val82 (marked in green) andLys83 form the S1 substrate binding
pocket, whileTyr37, Val54, Val73 and His80 (marked in
yellow)together with the residues marked in green formthe S3
pocket. These findings are in goodagreement with the structure of
the S1 and S3substrate binding pockets of the SPase of E.
coli(132). Furthermore, the idea that the latter residuesmake
contact with the substrate (i.e. the SPaserecognition sequence in a
precursor protein) wassupported by a molecular dynamics analysis
inwhich a pentapeptide of five Ala residues in a β-strand
conformation was modeled into the substratebinding pockets of SipS
(Fig. 5.5). Thispentapeptide was placed at the position
whichcorresponds to that of the PENEM inhibitor in thecrystal
structure of the E. coli SPase I (29).
The comparison of our models for the P-typeSPases of B. subtilis
showed that the substratebinding pockets of SipS, SipT, SipP1015
andSipP1040 were highly similar, whereas that ofSipV was
significantly smaller (Fig. 5.5).
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Chapter Five
68
Fig. 5.5. Active site models for SipS and SipV. (The
illustration on the back of the cover is a color representation of
thisfigure). Stereo pictures of residues in the modeled active site
regions of SipS (top) and SipV (bottom). Residues rendered in
spheresare likely to be involved in the formation of the S1 pocket
(Met44 and Leu48 of SipS; blue), S1 and S3 pockets (Val39 and
Val82of SipS; green) or S3 pocket (Tyr37, Val54, Val73 and His80 of
SipS; yellow) for substrate binding. The SipS model contains
apentapeptide of Ala residues, docked in the substrate binding
pocket. The P1 and P3 residues of this model substrate are shown
inorange. The active site Ser43 and Lys83 residues, as well as
Tyr81 of SipS and the corresponding Leu residue of SipV, are
shownas “ball and stick” models. The latter residues are probably
involved in substrate stabilization by interaction with P2 residues
of thesubstrate.
Conversely, the substrate binding site of SipUappeared to have a
wider S1 pocket than theequivalent sites of the other P-type SPases
of B.subtilis. Upon close examination, the volume of thesubstrate
binding pocket of SipV is relatively smallbecause the side chains
of Leu73, Ile82 andpossibly Leu54 (SipS numbering) protrude into
theS3 pocket (Fig. 5.5). The latter side chains arelarger than
those of the equivalent residues in SipSof B. subtilis (Val73,
Val82 and Val54,respectively) and other SPases (Fig. 5.4).
Takentogether, these observations indicate that the activesite
geometries of the minor SPases SipU and SipVof B. subtilis are
different from the active sitegeometries of the known major
SPases.
A SipS-SipV fusion protein is a major SPaseTo investigate
whether the active site geometry is acritical determinant for major
and minor BacillusSPases, a SipS-SipV hybrid protein (denoted
SipSV) was constructed, which is specified byplasmid pM0V.
Notably, this fusion between SipSand SipV of B. subtilis was made
at the catalyticSer residue of these SPases. Consequently,
SipSVconsists of the first 43 residues of SipS, and
thecarboxyl-terminal part of SipV. The majoradvantage of this
approach is that the active sitegeometry of SipSV is nearly
identical to that ofSipV (data not shown). Next, we tested
whetherSipSV is a major or minor SPase by introducingpM0V into B.
subtilis ∆ST, as described above forthe sipC (Bca) and sip (Bli)
genes. Strikingly,viable ∆ST transformants containing pM0V
wereobtained, showing that SipSV can replace SipS andSipT. As shown
in Fig. 5.2, SipSV is notrecognized by the antibodies raised
against SipS. Inconclusion, these observations show that SipSV isa
major SPase, and that SipV is not a minor SPasedue to the geometry
of its catalytic site but, rather,that some residues of its
amino-terminal stretch
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Major and minor Bacillus signal peptidases
69
determine SipV to belong to the class of minorSPases.
Furthermore, the antibodies against SipSdo not distinguish between
major and minorSPases.
Discussion
On the basis of their importance for cell viability,we have
previously classified the type I SPases ofB. subtilis as major
(SipS, SipT, SipP) and minorSPases (SipU, SipV, SipW) (106;108).
Thus far, itwas not clear which properties of these SPases
areimportant for this functional distinction, inparticular with
respect to the P-type SPases.Consequently, simple amino acid
sequencealignments could not be used to predict the groupto which
certain Bacillus P-type SPases wouldbelong. In the present studies,
we show for the firsttime that major and minor SPases can
bedistinguished via phylogenetic analyses.Surprisingly, the
subsequent molecular analysesdemonstrate that the distinction
between major andminor SPases does not specifically relate to
thecatalytic domain of a Bacillus P-type SPase, butrather to its
amino-terminal domain which containsthe membrane anchor. The latter
result wasunexpected, because it was recently shown byCarlos et al.
(221) that the transmembrane domainsof P-type SPases are not
important determinantsfor cleavage fidelity in vitro.
The most important outcome of the phylogeneticanalyses of the
Bacillus type I SPases is that themajor SPases form a distinct
cluster, which is well-supported by the maximum parsimony
andmaximum likelihood methods. Moreover, thesephylogenetic analyses
have predictive value, asexemplified by the complementation
experimentswith SipC (Bca) and Sip (Bli), showing that thesebehave
as minor and major SPases, respectively,when the corresponding
genes are expressed in B.subtilis. This, however, does not exclude
thepossibility that SipC is a major SPase in B.caldolyticus.
Furthermore, the phylogeneticanalyses indicate the existence of two
clusters of“minor” SPases: the SipC/SipV and the SipWclusters. The
latter cluster was identifiedpreviously as it contains the known
ER-type
SPases of bacilli (106;110). Only two BacillusSPases, SipU (Bsu)
and SipX (Ban), do not belongto the three clusters of major SPases,
SipC/SipV orSipW. This suggests that these two SPasesrepresent
possible evolutionary intermediatesbetween different clusters,
which is particularlyinteresting in the case of SipX of B.
anthracis, asthis P-type SPase might represent a link betweenthe P-
and ER-type Bacillus SPases.
The present observation that a SipSV hybridprotein, containing
the largest part of the catalyticdomain of the minor SPase SipV,
behaves as amajor SPase indicates that the catalytic domain ofthe
P-type SPases is not the most importantdeterminant for the
difference between major andminor SPases. This view is supported by
the factthat, according to our models, the active sitegeometry of
SipSV is identical to that of the minorSPase SipV. In this respect
it is important to bearin mind that the S3 substrate binding
pockets ofSipV and SipSV are relatively small compared tothose of
other P-type SPases of B. subtilis, SipU inparticular.
Nevertheless, the possibility that subtlechanges in the active site
geometry of SipSV,caused by the fusion between the SipS and
SipVmoieties, result in the conversion of a minor SPaseinto a major
SPase can presently not be excluded.The idea that the catalytic
domain is not importantfor the difference between major and minor
SPaseswould explain why the antibodies raised againstthe catalytic
domain of SipS (Bsu) can not be usedto distinguish between these
two functionallydefined groups of SPases.
What could be the role of the amino-terminalresidues of SipS in
determining its role as a majorSPase? Carlos et al. (221) have
recently providedcompelling evidence that the transmembranesegments
of type I SPases, such as SipS, are notimportant for substrate
cleavage site selection.Furthermore, we have recently shown that
themembrane anchor of SipS is not required for itsactivity (118).
Together with our present results,these observations imply that the
major-minordifference of SPases is not based on the recognitionof
residues at the -1, -3 positions, relative to thescissile peptide
bond per sé. This leaves open atleast three alternative
possibilities. First, the
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Chapter Five
70
amino-terminal residues might position thecatalytic site of a
major SPase in such a way in themembrane that it can interact with
the cleavage siteof one or more, as yet unidentified,
pre-proteinsthat have to be processed for cell viability.
Second,the amino-terminal residues might be required foran, as yet
unidentified, essential interaction of amajor SPase with
pre-protein translocases. Third,the amino-terminal residues might
target therespective SPases to topologically distinct regionsof the
membrane, such as the septa of dividingcells. Notably, the regions
preceding the active siteSer residues of Bacillus SPases, which
includetheir membrane anchor, show a relatively highdegree of
sequence variation (118). To elucidatethe role of the
amino-terminal region in SPasefunction, we are presently
investigating the role ofthe first 42 residues of SipS by
site-directedmutagenesis.
Acknowledgements
We thank Prof. Gert Vriend and Dr. Rob Veltmanfor critical
discussions on the modeling of BacillusSPases, Dr. Danilo Roccatano
en Dr. GiorgioColombo for the molecular dynamics simulations,and
Dr. Harold Tjalsma, Prof. O. Kuipers and othermembers of the
European Bacillus Secretion Groupfor stimulating discussions.
M.L.v.R. wassupported by the Dutch Ministry of EconomicAffairs
through ABON (Associatie BiologischeOnderzoeksscholen Nederland);
J.D.H.J wassupported by a grant (805-33.605) from SLW(Stichting
Levenswetenschappen); and S.B,J.D.H.J., J.Y.D., and J.M.v.D. were
supported bygrants (QLK3-CT-1999-00415 and QLK3-CT-1999-00917) from
the European Union.