Structure of a widely conserved type IV pilus biogenesis
factor that affects the stability of secretin multimers
Melissa B. Trindade1#, Viviana Job1#, Carlos Contreras-Martel1,
Vladimir Pelicic2, and Andréa Dessen1*
1Institut
de Biologie Structurale Jean-Pierre Ebel, UMR 5075 (CEA,
CNRS, UJF, PSB); 41 rue Jules Horowitz, F-38027 Grenoble
2Department
of Microbiology, Imperial College London, London
SW7 2AZ, UK
*to whom correspondence should be addressed
email : andrea.dessen@ibs.fr
phone : (33)4-38-78-95-90
fax : (33)4-38-78-54-94
#
these authors contributed equally
Keywords : bacterial virulence, type IV pilus, secretin, crystallography, 3D
structure
1
SUMMARY
Type IV pili (Tfp) are arguably the most widespread pili in bacteria, whose
biogenesis requires a complex machinery composed of as many as 18 different
proteins. This includes the conserved outer membrane-localized secretin,
which forms a pore through which Tfp emerge on the bacterial surface.
Although, in most model species studied, secretin oligomerization and
functionality
requires
the
action
of
partner
lipoproteins,
structural
information regarding these molecules is limited. In this work, we report the
high resolution crystal structure of PilW, the partner lipoprotein of the type
IV pilus secretin PilQ from Neisseria meningitidis, which defines a conserved
class of Tfp biogenesis proteins involved in the formation and/or stability of
secretin multimers in a wide variety of bacteria. The use of PilW’s structure as
a blueprint reveals an area of high sequence conservation in homologous
proteins from different pathogens that could reflect a possible secretin
binding site. These results could be exploited for the development of new
broad-spectrum antibacterials interfering with the biogenesis of a widespread
virulence factor.
2
INTRODUCTION
A common theme in bacterial pathogens concerns the use of long hairlike filaments, collectively known as pili, as colonization factors. There exists a
wide variety of different pili, which are currently grouped according to their
mechanisms of assembly 1, but type IV pili (Tfp) seem by far the more
disseminated. Tfp, elongated, flexible filaments, are found in both Gramnegative and Gram-positive bacteria, and are key virulence factors in species
as diverse as enteropathogenic Escherichia coli (EPEC), pathogenic Neisseria
species, Pseudomonas aeruginosa, Salmonella typhi and Vibrio cholerae 2; 3; 4; 5; 6; 7; 8.
One of the main distinctive features of Tfp is their capacity to be retracted,
which has been characterized at the level of a single filament in N. gonorrhoeae,
and they have been shown to generate substantial mechanical forces 9. This
promotes some of the additional Tfp-linked properties such as competence
for DNA transformation, biofilm formation, bacterial aggregation, and a form
of locomotion called twitching motility 10; 11.
Tfp are mainly composed of pilin subunits and are defined by two
distinct subtypes known as type IVa and type IVb, the former being by far the
most widespread and accounting for most of the bewildering diffusion of Tfp
noted above
12.
N. meningitidis, our model organism, expresses type IVa pili
and is, together with P. aeruginosa, the prototypal species for this Tfp subtype.
Systematic genetic studies in these two species have defined the complete sets
3
of genes encoding proteins specifically dedicated to Tfp biogenesis 13; 14 which
are virtually identical and composed of 15 and 18 proteins, respectively.
However, the exact functions of a majority of these proteins remain unknown
and consequently Tfp biogenesis is a poorly understood process.
A systematic genetic analysis performed in N. meningitidis showed,
however, that Tfp biogenesis can be resolved into discrete steps onto which
each of the Pil proteins could be mapped 15. This provided evidence that most
Tfp biogenesis proteins are actually dispensable for pilus assembly and serve
to counteract PilT-mediated retraction of the fibers and to make them
functional 15. A single protein, the secretin PilQ, was found to be essential for
the late step in Tfp biogenesis during which the filaments, which are
assembled in the periplasm, emerge onto the cell surface
15; 16.
PilQ forms
multimers that are essentially pores in the outer membrane through which
pili are thought to emerge
15; 17.
The GSP (general secretion pathway) secretin
superfamily, to which PilQ belongs, also includes membrane-associated,
oligomeric channels of the type II (T2SS) and type III secretion systems (T3SS)
18,
as well as the secretin from the f1 filamentous phage 19. Secretins form SDS-
, detergent-, and heat-resistant dodecamers
20; 21; 22; 23.
Electron microscopy
images of PilQ have revealed it to assemble in doughnut form, with a large
cavity at the center, which could be the site through which the polymerized
Tfp fiber travels 17; 20; 24; 25.
4
There is now growing evidence for the existence of a subcomplex of
Tfp biogenesis proteins in the outer membrane, which is centered around the
secretin
and
contains
other
proteins
which
are
key
for
secretin
multimerization, stability, and/or correct localization onto the outer
membrane. For example, the Klebsiella oxytoca T2SS secretin PulD requires
PulS for proper outer membrane association; in its absence, PulD remains
associated to the inner membrane
23; 26.
The T3SS secretin MxiD from Shigella
flexneri requires MxiM for outer membrane recognition and oligomerization
27;
28,
while in Yersinia enterocolitica, YscW ensures outer membrane
localization and multimerization of another T3SS secretin, YscC 29. All of these
proteins are known as pilotins, or pilot proteins. Secretins involved in Tfp
biogenesis also rely on other proteins for stability of the multimers, such as
BfpG, TcpQ and PilW in EPEC, V. cholerae and N. meningitidis, respectively,
but they apparently do not need them for proper localization into the outer
membrane
13; 30; 31.
The latter protein, PilW, is of particular interest due to the
fact that it is widespread, being found in bacteria expressing type IVa pili. It
apparently plays the same role in all of these organisms since, in its absence,
no secretin multimers can be detected in species of Proteobacteria as distant
as N. meningitidis and Myxococcus xanthus, where it is known as Tgl
13; 21; 32; 33.
In addition, as demonstrated in N. meningitidis, PilW is one of the proteins
that serve to counteract PilT-mediated retraction of the fibers and makes the
fibers functional
13.
Altogether, this suggests that there could be an outer
5
membrane complex of Pil proteins involved in the late stages of Tfp
biogenesis, which includes at least PilQ and PilW, that may interact directly.
To date, one structure of a secretin partner protein has been reported:
that of the Shigella flexneri pilotin MxiM
structure of a domain of Neisserial PilP
27.
34
It should be noted that the NMR
is also available, but this protein is
now known not to affect the stability of PilQ multimers 35. Here, we report the
high resolution crystal structure of PilW from N. meningitidis, and show that it
is totally distinct from that of the aforementioned molecules. The structure
reveals that the N-terminal and C-terminal regions both fold into
tetratricopeptide (TPR) domains, whose boundary is stabilized by a disulfide
bridge. Mapping of sequence similarities between PilW and its orthologs onto
the PilW superhelical surface reveals a key region within the concave face of
the TPR superhelix that could be essential for secretin stabilization, and thus,
Tfp functionality. This information could be further exploited for the
development of antibacterials that target the Tfp biogenesis machinery.
6
RESULTS AND DISCUSSION
Characterization, crystallization, and structure solution of PilW
PilW is an outer membrane-associated protein that is essential for Tfp
biogenesis in N. meningitidis. It is conserved in all Gram-negative bacteria
harboring type IVa pili and harbors an N-terminal lipoprotein signal peptide
sequence; thus, PilW is likely to be a lipoprotein which, upon cleavage of a
signal peptide of 19 residues, remains attached to the outer membrane
through a lipid on the Cys20 side chain
13.
Secondary structure prediction
programs and protein domain database analyses (ScanProsite, InterProScan,
SMART) predicted the presence of three sets of tetratricopeptide (TPR) motifs
(res 36-69, 70-103, and 141-174), which could potentially form a TPR fold
encompassing most of N-terminus of the molecule; the fold of the C-terminus
of the molecule could not be predicted.
A recombinant form of PilW, which lacked the N-terminal signal
sequence (residues 1-19), was expressed as a cleavable MBP fusion and
formed a covalent, disulfide-bonded dimer (through the interaction of the
Cys20 residues from different monomers). In order to identify the optimal
conditions for crystallization, the stability of PilW with respect to thermal
denaturation in different buffer and concentration conditions was analyzed
using fluorescence spectroscopy (see Materials and Methods). The PilW
7
covalent dimer crystallized at pH 5 in the presence of LiCl2, but poor data
quality prevented us from solving the structure by any conventional phasing
methodologies. Nevertheless, treatment of the covalent dimer with trypsin
yielded a stable, monomeric form of the molecule in which the 7 N-terminal
residues of the mature protein (thus including Cys20), had been removed, as
confirmed by electrospray mass spectrometry and N-terminal sequencing.
Crystals of monomeric PilW (residues 28-253) were in space group P6222,
diffracted X-rays at the ESRF synchrotron to 1.6 Å, and contained 1 molecule
per asymmetric unit. In view of the high quality diffraction of these new
crystals, the structure could be solved using the MIR technique with 2.0 Å
data collected on an in-house generator from a seleno-methionylated crystal
and a native sample. Four seleno-methionine sites were easily identified
through employment of this technique, which allowed automatic building of
a large part of the molecule. Molecular replacement was subsequently
employed in order to phase the highest resolution data. Data collection and
structure refinement statistics are shown in Table 1.
8
A superhelix with a positive backbone
PilW is comprised of thirteen anti-parallel -helices that fold into six
TPR motifs; the motifs are organized as to form a super-helical structure (Fig.
1). PilW’s helices 5 and 7, which lie within TPR motifs 3 and 4, are
interconnected by a disulfide bridge (Cys115-Cys150), pointing to the
necessity of stabilizing the two halves of the TPR superhelix.
TPR motifs are present in a wide range of proteins, and are involved in
biological processes as diverse as transcriptional control and protein folding,
in which they often mediate protein–protein interactions and the assembly of
multiprotein complexes
36.
The arrangement of several linked TPR motifs in
tandem forms a superhelical assembly, generating both concave and convex
surfaces. To date, most of the known TPR structures have been solved from
eukaryotic sources, and have been shown to bind their cognate proteins either
through the concave face of the TPR structure 37 or by employing inter-helical
turns
38.
Recently, 3D structures from bacterial TPRs have become available,
and have revealed that TPRs can employ both concave and convex surfaces
for cognate protein recognition
39.
In the case of PilW, the concave region is
decorated with highly conserved amino acids (see below), while the convex
side is highly charged. Interestingly, the convex region is reminiscent of a
‘backbone’ decorated by a line of basic residues that span the entire length of
the protein (Fig. 2a). This could be indicative of the fact that PilW employs its
9
convex region for interaction with other Tfp biogenesis factors, or the
negatively-charged outer membrane. Within the concave region (Fig. 2b), a
small enclave of positive charges is formed by Lys 179, Arg 183, and Lys 218.
Tfp biogenesis lipoproteins with a common fold
Fig. 3 displays a structure-based sequence alignment of PilW orthologs
from diverse Proteobacteria known or suspected to harbor type IVa pili.
These proteins have been shown to be essential for Tfp biogenesis in several
model organisms such as N. meningitidis, P. aeruginosa and M. xanthus
13; 21; 40.
Bioinformatic analyses for these molecules identify the presence of TPR
motifs in all of them (not shown). These proteins share several major
homology regions within the TPR superhelix, as evidenced by the analysis of
residues highlighted in red boxes in Fig. 3, which are either identical or highly
conserved in at least four of the aligned sequences. Notably, a large number
of these residues are hydrophobic, and, in the PilW structure, are located
within the interface of different helices. Thus, intra-helical stabilization is
provided by the formation of hydrophobic interactions, as suggested for other
TPR folds
41.
This analysis suggests not only that these proteins display
similar structures, but that the aforementioned residues probably play
comparable roles within the different proteins. This hypothesis is
strengthened by the recent solution of the structure of PilF from P. aeruginosa,
10
which also folds into a TPR superhelix
42,
similarly to PilW (rms deviation =
2.14 Å over 171 atoms; PDB code 2FI7).
PilW represents the first TPR structure stabilized by a disulfide bridge.
This is likely to be a general feature for most of the Tfp biogenesis proteins of
this class since the two corresponding cysteine residues (Cys115-Cys150) are
conserved in most of the sequences shown in Fig. 3. However, in some
orthologs, such as PilF
42
and Tgl from P. aeruginosa and M. xanthus
respectively, these cysteines have been replaced by residues with
hydrophobic side chains, which suggests that that a non-polar surface may
also be involved in stabilization of this specific region; indeed, in the structure
of PilF
42,
these residues face each other, being located within a highly
hydrophobic area. Nevertheless, Tgl from M. xanthus carries 4 other cysteine
residues in addition to the Cys in the lipoprotein signal sequence (residues
171, 191, 201, 241, identified with green asterisks in Fig. 3). Hence, it is
conceivable that, within a potential Tgl superhelix, the C-terminal half of the
molecule is further stabilized by at least one disulfide bond.
A potential insight into function
PilW is one of the few proteins involved in Tfp biogenesis for which
considerable functional information is available. As shown by a systematic
genetic analysis in N. meningitidis, this protein is not essential for Tfp
11
assembly per se but is rather involved at late step of Tfp biogenesis during
which, together with an important subset of Pil proteins, it counteracts pilus
retraction and makes the filaments perfectly functional
affects the stability of secretin multimers
13,
13.
In addition, PilW
an effect which was also
confirmed in another piliated species, M. xanthus
21.
These findings are
suggestive of specific interactions between PilQ and PilW (or Tgl), and point
to a likely outer membrane protein complex involved in the late stages of Tfp
biogenesis.
The structure of PilW, presented here, provides some clues as to its
unique functional properties. First, it clearly strengthens the notion that PilW
is not a pilot protein. The TPR superhelix fold strikingly differs from that of
the only pilot protein whose structure is available. Indeed, MxiM, which is the
pilotin of the MxiD secretin in the type III secretion secretin of Shigella 28, folds
into a "cracked" -barrel capable of binding ligands within a central
hydrophobic cavity
27.
Second, the highly charged convex side of PilW could
mediate interactions with a negatively-charged partner protein or even with
the outer membrane. Third, and possibly the most relevant observation, relies
on the fact that three of the residues which are absolutely conserved within all
sequences presented in Fig. 3, Asn109, Tyr137 and Asn146, are located in
proximity to each other within the concave region of the TPR superhelix (Fig.
4), on 5 and 7. Moreover, Asn109 is located within the Asn-Asn-Xhyd-GlyX-Xhyd-Leu motif (where Xhyd represents a hydrophobic amino acid), which is
12
highly conserved (blue background in Fig. 3). In PilW, Asn108 plays the role
of central residue in a hydrogen bonding network involving Asn109 and
Asn146; notably, this role is also played by Asn residues in all sequences
displayed on Fig. 3, with the exception of Tgl from M. xanthus; in the latter
molecule, this role seems to be played by a Thr residue, which could provide
a single hydrogen bond through its O group. It is of interest that most of the
aforementioned residues are clustered on the interface between TPR motifs 3
and 4, precisely the region that is stabilized by the disulfide bridge (Figs. 3
and 4). These observations suggest that the constellation of residues on 5
and 7 may be of particular importance for PilW function, and their
clustering within the concave area formed by the TPR superhelix indicates
that this could represent a protein-binding region. Notably, employment of
protein-binding
pocket
predictive
tools
such
as
PASS
(http://www.ccl.net/cca/software/UNIX/pass/pass_jcamd.html) also point
to this region as a potential partner-recognition site.
The identity of the potential partner that binds to this region is difficult
to predict at this stage due to the multiple properties associated with PilW in
N. meningitidis
13,
which are for the most part still to be confirmed in other
piliated species. However, the confirmation in M. xanthus that Tgl is also
necessary for the stability of PilQ mutlimers
21
points to the secretin as the
most likely protein recognized by the above protein-binding region. A surface
representation of PilW in which residues which are identical (red) and similar
13
(orange) to Tgl is shown in Fig. 5, and reveals that the concave region of the
TPR superhelix is highly conserved between these two proteins; in addition to
the residues described above for all PilW orthologs, other potentially relevant
amino acids within this region also include the Pro138-Thr139-Pro140 motif
that immediately follows Tyr137 (highlighted in light blue in Fig. 3), all
located within 7, and the loop which precedes it. These results suggest that
PilW and Tgl (and maybe PilF) may recognize their cognate secretins in a
similar fashion and thus act as chaperones for secretin multimers. However,
the multiple functions associated with it indicate that it would be reductive to
consider PilW only as a chaperone of secretin multimers, although it could
not be excluded at this point that PilW mediates these functions indirectly,
possibly through its contact with PilQ.
In conclusion, with the three-dimensional structure of PilW now at
hand, a detailed structure/function analysis of this protein is possible. Due to
the wide conservation of this protein in bacteria expressing type IVa pili and
its key role in Tfp biogenesis, such an analysis will certainly shed light on an
insufficiently understood biological process. In addition, the common
constellation of residues within the PilW superhelix identified in this work
could represent a novel, tractable target for the development of potential
antibacterials which could interfere with Tfp biogenesis without having to
cross the inner bacterial membrane in order to exert their action. This in turn
14
could have far-reaching economic consequences due to the wide distribution
of Tfp in human, animal and plant pathogens.
15
MATERIALS AND METHODS
Cloning, expression and purification of PilW
The region of the N. meningitidis pilW gene coding for amino acids 20253 of the full-length protein was amplified using conventional PCR
methodologies and cloned into pCRII-TOPO (Invitrogen) using E. coli TOP10
(Invitrogen), to generate plasmid pYU17. After digesting pYU17 by EcoRI and
HindIII, the fragment was subcloned in the pMAL-p2X vector (New England
Biolabs) digested with the same enzymes.
pYU18 was transformed into E. coli BL21 (DE3) cells. Protein
expression was induced in Luria Broth with 0.5 mM IPTG at 30oC overnight.
Cells were harvested by centrifugation and lysed by sonication in 20 mM TrisHCl pH 7.4, 200 mM NaCl, 1 mM PMSF, 0.1 M aprotinin, 1 M leupeptin,
and 1 M pepstatin. The supernatant was cleared by centrifugation and
applied to an amylose column (New England BioLabs, USA) pre- equilibrated
in lysis buffer. Sample elution was performed with a 10 mM maltose step,
which was followed by treatment with 4 units of Factor Xa per milligram of
protein. The cleaved protein was re-loaded onto an amylose column, and
unbound samples were loaded onto a Superdex 75 column (GE Healthcare,
Sweden). The purified PilW product contained 4 vector-derived residues
(ISEF) followed by residues 20-253 corresponding to the mature PilW protein.
16
Pooled, concentrated fractions were employed in crystallization trials as
described below.
Subsequently, in order to facilitate purification, a histidine tag was
introduced C-terminally to the signal peptide-encoding region of the malE
gene by site-specific mutagenesis using the Quick Change II kit (Stratagene).
This construct was transformed into E. coli BL21(DE3) cells and expression
was induced as described above. After the lysate was cleared by
centrifugation, the supernatant was applied to a Ni2+ Sepharose column in
lysis buffer (with 20 mM imidazole), and protein was eluted with a step
imidazole gradient. Samples were treated with trypsin at a 1:1000 ratio for 3
hrs; the reaction was stopped by the addition of 1 mM PMSF, after which the
protein was reloaded onto a Ni2+ Sepharose column, used to trap the
histidine-MBP tag. The flow through was subsequently loaded onto a
Superdex 75 column in 50 mM Tris pH 8.0, 100 mM NaCl. The eluted peak of
PilW (28-253) was subsequently submitted to crystallization trials (below).
Selenomethionine-substituted PilW was overexpressed in the same E.
coli BL21(DE3) strain in minimal medium supplemented with thiamine (0.2
mg/mL) leucine, (50 mg/L), valine (50 mg/L), isoleucine (50 mg/L), lysine
(100 mg/L), phenylalanine (100 mg/L), threonine (100 mg/L) and
selenomethionine (60 mg/L). Purification was performed as described above
17
for the native molecule. MALDI-TOF analyses revealed the full substitution of
four methionines.
18
Thermal stability assays
PilW’s stability with respect to thermal denaturation was analyzed
using fluorescence spectroscopy in order to identify optimal conditions for
crystallization and biochemical analyses. Protein samples were incubated
with SYPRO® Orange dye and heated between 20°C and 100°C; the dye binds
to hydrophobic regions of a protein that become exposed during
denaturation, and a fluorescent signal is measured 43. Samples were prepared
in various pH, NaCl, and protein concentrations, and tested in 96-well thinwall PCR plates (BioRad) in a total volume of 25 µL. Protein samples whose
concentration ranged from 0.1 to 4 mg/mL) were prepared in 0.1 M buffers of
pH values ranging from 4.0 to 10.0, with NaCl concentrations of 10, 100, 250,
500 mM, 1M, and 2 M. 2.5µL of 50x SYPRO Orange (Molecular Probes)
sample were added to each mix. Assays were performed using an IQ5 realtime PCR detection system (Bio-Rad) over a temperature range of 20°C to
100°C, with an increment of 0.2°C/step. At each step, excitation was
performed at 470 nm, while emission of SYPRO® Orange fluorescence was
monitored at 570 nm by employing a camera
43.
The melting temperature
(Tm) values of the protein in each condition were determined graphically
from the first derivative of the fluorescence curves. These results suggested
that PilW was unstable at a pH value below 5.3, and at a concentration value
above 5 mg/ml. Crystallization trials were performed with protein samples at
a maximal concentration of 2.5 mg/mL in the drop.
19
Crystallization, data collection, and structure solution
Crystals of PilW (20-253) were obtained by the hanging-drop vapor
diffusion method in 100 mM citrate pH 5.2, 1 % polyethylene glycol 6000, and
800 mM LiCl2, but as described in the Results section, could not be exploited
for structure solution. Subsequently, crystals of PilW (28-253) were also
obtained by the hanging-drop vapor-diffusion method by mixing 1L of the
protein sample with 1L of well solution at 20°C. Crystals grew in 100 mM
Tris pH 8.5, 800 mM Li2SO4, 10 mM NiCl2 and were cryoprotected by brief
immersion in mother liquor containing increasing concentrations of glycerol
(up to 15%), after which they were flash-cooled in the nitrogen stream. A first
highly redundant native data set was collected at the European Synchrotron
Radiation Facility (ESRF) ID14-EH3 beamline (Grenoble, France), using 0.931
Å wavelength (Native-1). A second one was collected in-house with Cu K
radiation (1.54 Å) using an ENRAF-NONIUS rotating-anode generator
(Native-2). Finally, a selenium-derivatized crystal was also collected in-house
using Cu K radiation.
Diffraction images were indexed and scaled with the program XDS
44,
and merged with the CCP4 program suite 45. SHELXD 46 was employed in the
identification of the four expected Se sites, (∆f” 1.139 electrons) by employing
both the Native-2 and seleno-methionine datasets. Heavy atom position
20
refinement and phasing were performed with SHARP
47; 48,
while density
modification was performed with the programs SOLOMON 49 and DM 50, and
automatic model building was performed ARP/wARP 6.1 51. Once the model
was traced in the 2.0 Å native data set, the molecular replacement program
PHASER 52 was used to calculate phases for Native-1. Subsequently, COOT 53
was employed in cycles of manual model building, while cycles of restrained
refinement were performed with REFMAC 5.2
54
as implemented in CCP4
program suite 45. Stereo-chemical verification was performed by PROCHECK
55
and secondary structure assignment was performed by DSSP
56.
Data
collection and structure refinement statistics can be found in Table 1. Figures
were generated with PyMol (http://www.pymol.org).
21
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25
FIGURE LEGENDS
Fig 1. Schematic arrangement and tertiary fold of PilW. (A) PilW consists of a
signal peptide (SP), a lipoprotein-binding Cys residue (shown in red),
and six TPR motifs. The region included in the crystal structure is
shown in a red box. (B) PilW folds into a right-handed, superhelical
assembly composed of 13 helices that comprise 6 TPR domains: TPR1
(1, 2), TPR2 (3, 4), TPR3 (5, 6), TPR4 (7, 8), TPR5 (9, 10),
and TPR6 (11, 12). TPR domains 3 and 4 are stabilized by a disulfide
bridge.
Fig. 2 Surface potential of PilW. The PilW superhelix displays a highly
charged surface, and is notably decorated by basic residues along its
backbone. The two views (panels A and B) differ by 180o.
Fig. 3. Sequence alignment of PilW orthologs. Bacteria include Neisseria
meningitidis, Aeromonas hydrophila, Vibrio cholerae, Yersinia enterocolitica,
Xylella fastidiosa, Pseudomonas aeruginosa, and Myxococcus xanthus.
Identical residues are highlighted with a red background, while similar
residues are shown in red. Positions where there are four or more
identical residues are shown in red boxes. The cysteines that participate
in the formation of the disulfide bridge between TPRs 3 and 4 are
marked with a green background. The conserved residues that line 5
26
are marked in blue, while the Tyr-Pro-Thr-Pro motif, which could
potentially play a role in PilW (N. meningitidis) and Tgl (M. xanthus)
functionalities, is shown in light blue. The figure was prepared by
aligning all sequences with CLUSTALW, and subsequently employing
ESPript
57
in order to display the structure of PilW onto the sequence
block.
Fig. 4. The concave region of the TPR superhelix is highly conserved.
Asn108, Asn109, Tyr137 and Asn146 are located within 5-7, within
TPRs 3 and 4.
Fig. 5. PilW and Tgl display high similarity within their concave regions.
Surface representation of PilW, where residues which are identical (red)
and similar (orange) to Tgl are shown. The two proteins, whose
functions could be very similar, display a high level of sequence
identity and similarity within the concave region of the superhelix.
27
ACKNOWLEDGEMENTS
The coordinates of PilW have been deposited in the Protein Databank with code
XXXXX and are available pre-relase from andrea.dessen@ibs.fr. The authors would
like to thank the ESRF ID14-EH3 beamline staff for help with data collection, Julien
Offant and RoBioMol (LIM, IBS) for help with thermal stability assays, Bernard Dublet
and the IBS mass spectroscopy facility (LSMP) for electrospray analyses and JeanPierre Andrieu (LBM, IBS) for N-terminal sequencing. This work was partly supported
by INSERM and the Agence Nationale de la Recherche (grant JC05_44953 to VP).
28
FIGURES
Figure 1
29
30
Figure 2
31
Figure 3
32
Figure 4
33
Figure 5
34