Structure
Article
Structural Determinantsof Polymerization Reactivityof the P pilus Adaptor Subunit PapFDenis Verger,1,2 Rebecca J. Rose,3 Emanuele Paci,3 Greg Costakes,1 Tina Daviter,1,2 Scott Hultgren,4 Han Remaut,1,2
Alison E. Ashcroft,3,* Sheena E. Radford,3,* and Gabriel Waksman1,2,5,*1Institute of Structural and Molecular Biology, University College London, Birkbeck College, Malet Street, London WC1E 7HX, UK2School of Crystallography, Malet Street, London WC1E 7HX, UK3Astbury Centre for Structural Molecular Biology, Astbury Building, University of Leeds, Leeds LS2 9JT, UK4Department of Microbiology and Center for Women’s Infectious Disease Research, Washington University Medical School,
St. Louis, MO 63011, USA5Research Department of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK*Correspondence: [email protected] or [email protected] (G.W.), [email protected] (S.E.R.),
[email protected] (A.E.A.)
DOI 10.1016/j.str.2008.08.012
SUMMARY
P pili are important adhesive fibers involved in kidneyinfection by uropathogenic Escherichia coli. Pilussubunits are characterized by a large groove resultingfrom lack of a b strand. Polymerization of pilus sub-units occurs via the donor-strand exchange (DSE)mechanism initiated when the N terminus of an in-coming subunit interacts with the P5 region/pocketof the previously assembled subunit groove. Here,we solve the structure of the PapD:PapF complex inorder to understand why PapF undergoes slowDSE. The structure reveals that the PapF P5 pocketis partially obstructed. MD simulations show this re-gion of PapF is flexible compared with its equivalentin PapH, a subunit that also has an obstructed P5pocket and is unable to undergo DSE. Using electro-spray-ionization mass spectrometry, we show thatmutations in the P5 region result in increased DSErates. Thus, partial obstruction of the P5 pocketserves as a modulating mechanism of DSE.
INTRODUCTION
Uropathogenic E. coli (UPEC) are responsible for urinary tract
infections, notably cystitis (infection of the bladder) or pyelone-
phritis (infection of the kidney) (Hooton and Stamm, 1997;
Roberts et al., 1994). Survival and persistence of these bacteria
in the urinary tract require the expression, among other virulence
factors, of type 1 and P pili. P pili are required for the ability of
UPEC to bind Gal-a(1-4)-Gal moieties on the surface of human
kidney cells and thus are important virulence determinants of
pyelonephritis (Dodson et al., 2001; Kuehn et al., 1992; Roberts
et al., 1994; Soto and Hultgren, 1999). P pili are encoded by the
pap gene cluster and are assembled via the highly conserved
chaperone-usher pathway, involving the periplasmic immuno-
globulin-like chaperone PapD and an outer membrane usher
PapC (Barnhart et al., 2000; Dodson et al., 1993; Holmgren and
1724 Structure 16, 1724–1731, November 12, 2008 ª2008 Elsevier L
Branden, 1989; Remaut et al., 2008; Sauer et al., 2004; Saulino
et al., 1998; Slonim et al., 1992; Soto et al., 1998; Thanassi
et al., 1998). P pili consist of six types of subunits making up
a composite fiber with the Gal-a(1-4)-Gal -specific adhesin,
PapG, at the tip, a short tip fibrillum formed of 5–10 PapE subunits
joined to a more rigid helical rod formed of about a thousand
PapA subunits and finally the terminator subunit PapH, last to
be assembled (Baga et al., 1987; Bullitt and Makowski, 1995;
Gong and Makowski, 1992; Jacob-Dubuisson et al., 1993; Kuehn
et al., 1992) (Figure 1A). In addition, there are two subunits termed
‘‘adaptor subunits,’’ PapF and PapK. The adaptor subunit PapF is
located between the adhesin and the tip fibrillum, and its incorpo-
ration within the pilus is thought to induce the polymerization of
PapE and also to orientate PapG relative to the PapE fibrillum
for optimal presentation to the kidney receptor (Jacob-Dubuis-
son et al., 1993; Lee et al., 2007). The other adaptor subunit,
PapK, is found between the PapE tip fibrillum and the PapA rod
(Jacob-Dubuisson et al., 1993) (Figure 1A).
All pilin subunits adopt a truncated immunoglobulin (Ig)-like
fold where the C-terminal G b strand is lacking, thus producing
a large hydrophobic groove on the side of the protein (Choudhury
et al., 1999; Sauer et al., 1999). In a process termed donor-strand
complementation, the chaperone PapD provides the structural
information lacking in the pilus subunit by inserting one of its
b strands, the G1 b strand, into the subunit’s groove, thereby
completing the Ig fold of the subunit (Barnhart et al., 2000;
Choudhury et al., 1999; Sauer et al., 1999). Four alternating
residues in the chaperone’s G1 b strand, termed P1 to P4 resi-
dues, contact the groove in four contiguous regions of the
groove, named P1 to P4 binding regions/pockets. Pilus subunit
assembly proceeds via a noncovalent polymerization process
called donor-strand exchange (Sauer et al., 2002, 2004; Soto
et al., 1998; Vetsch et al., 2006; Zavialov et al., 2003) . All subunits,
except the adhesin PapG, possess an N-terminal extension (Nte)
peptide (see sequence alignment in Figure 1C) that is disordered
and not part of their Ig-like structure. The Nte comprises a highly
conserved array of alternating hydrophobic residues, termed
P2 to P5 residues. DSE proceeds via the formation of an interme-
diate consisting of the chaperone-subunit complex bound to the
Nte of the subunit coming next in assembly. The formation of this
td All rights reserved
Structure
Crystal Structure of the P Pilus Subunit PapF
complex is crucially dependent on the identity of the Nte P5
residue. Insertion of this P5 residue into a distinct pocket in the
subunit groove, termed the P5 pocket, initiates a zip-in-zip-out
process whereby the chaperone’s G1 strand is progressively dis-
placed by the Nte peptide, resulting in subunit-subunit interaction
and release of the chaperone (Remaut et al., 2006). Recently, the
significance of this process was confirmed by Verger et al. (2006),
who showed that PapH has an obstructed P5 pocket and is thus
unable to undergo DSE.
The defined order by which Pap subunits are assembled to
form the P pilus has recently been investigated and shown to
be strongly correlated with the interaction kinetics between
subunits’ grooves and Nte peptides, which, in turn, are dictated
by the C-terminal sequence of the Nte peptides, where the P5
residue is located (Rose et al., 2008a). In the study by Rose
and coworkers, all six chaperone-subunit complexes that the
Pap subunits can form with their cognate chaperone PapD
(PapD:PapG, F, E, K, A, and H) were challenged in vitro by
each of the five possible Nte peptides (as indicated above,
PapG does not have an Nte) and DSE was monitored using elec-
trospray-ionization mass spectrometry (ESI-MS) (Rose et al.,
2008a). Remarkably, each of the complexes reacted most
Figure 1. P Pilus Organization and Function
(A) Schematic diagram of the P pilus. Each
subunit, the chaperone, and the usher are labeled
using a one-letter code (e.g., G for PapG). For
clarity, only one protomer in the usher is shown
(ushers are dimeric). A growing and a mature P pi-
lus are shown at left and right, respectively.
(B) t50 values for each DSE reaction involving
cognate Ntes. In each case, a 10-fold molar
excess of the peptide Nte was added over the
concentration of PapD:PapX (where X denotes
the subunit under consideration) and the concen-
tration of PapD:PapX remaining was monitored as
a function of time using ESI-MS (see Experimental
Procedures). The rates of DSE, denoted here as
the time for the reaction to reach 50% completion
(t50), are shown for PapD:PapG with FNte, PapD:
PapF with ENte, PapD:PapE with KNte, PapD:PapK
with ANte, and PapD:PapA with ANte. The data
were taken from Rose et al. (2008a).
(C) Sequence alignment of the Pap subunits.
Identical residues are boxed in red, and similar
residues in purple. PapA and PapF secondary
structures are shown in orange and blue, respec-
tively, with b strands, a helices, and loops dis-
played as arrows, boxes, and lines, respectively.
The only helix in PapA is shown in red. Nte resi-
dues corresponding to the P2, P3, P4 (purple),
and P5 (red) positions are indicated. Pro32 is
boxed in magenta whereas Arg126 and Glu33
are boxed in green. Arrows in the corresponding
colors are positioned under the corresponding
boxes to help locate these residues.
rapidly when challenged by the Nte pep-
tide derived from the subunit known to
come next in the assembly of the P pilus
(termed ‘‘cognate’’ Nte), indicating that
the kinetics of groove-Nte interaction re-
capitulate the order of assembly (Rose et al., 2008a). PapG
and PapF, the two most distal subunits in the P pilus, undergo
slow DSE compared with all other subunits (Rose et al., 2008a)
(Figure 1B). The DSE reaction where PapD:PapF is challenged
by the Nte peptide of PapE (ENte) takes about 76 hr to reach
50% completion (t50 = 76 hr). DSE reaction with PapD:PapG
challenged with the Nte peptide of PapF (FNte) is even slower
with a t50 of 120 hr. In contrast, the DSE reactions for PapD:PapK
when challenged with the Nte peptide of PapA (ANte) or for
PapD:PapE when challenged with the Nte peptide of PapK
(KNte) have t50 values of 12.5 and 3.2 hr, respectively (Rose
et al., 2008a) (Figure 1B). Thus PapG and PapF are slow-ex-
changing subunits whereas the others (except PapH, which was
confirmed to be unable to undergo DSE) are fast exchanging.
From the structures of PapK, PapE, PapH, and recently PapA,
it appears that fast-exchanging subunits (PapA, PapK, and
PapE) all have an open (PapA and PapK) or disordered (PapE)
P5 pocket. Only in PapH is the P5 pocket closed or obstructed
(Sauer et al., 1999, 2002; Verger et al., 2006, 2007).
In this study, we sought to understand the slow-exchanging
behavior of the PapF subunit. By combining structural, computa-
tional, and biophysical methods, we show that the P5 pocket of
Structure 16, 1724–1731, November 12, 2008 ª2008 Elsevier Ltd All rights reserved 1725
Structure
Crystal Structure of the P Pilus Subunit PapF
PapF is partially obstructed by residues in two loops. Molecular
dynamics (MD) simulations show that these loops are flexible,
whereas the equivalent regions in the PapH structure are rigid.
Moreover, mutating contact residues between the two loops in
PapF to decrease their interaction results in faster DSE reac-
tions. These results show that intrinsic loop flexibility at and
around the P5 pocket modulates P5 pocket accessibility, which
in turn determines the rates at which subunits undergo DSE.
RESULTS
Structure of PapF and Comparisonwith Other Pap SubunitsWe first asked whether the structure of the PapD:PapF complex
could provide insights that could help understand why this com-
plex exhibits slow rates of DSE in vitro when challenged by its
cognate Nte peptide, ENte, in vitro. The full-length PapD:PapF
was thus purified but did not yield crystals because of a tendency
to aggregate at high concentration. We hypothesized that aggre-
gation of PapF may occur through DSE, and thus we sought to
prevent aggregation by mutating the conserved P4 glycine in
the Nte to Asn (Figure 1C). The P4 glycine residue in the sub-
unit-Nte complex lies on top of a bulky residue in the subunit’s
groove, and thus Asn at P4 should provide the steric clashes
that prevent the Nte from fitting into the subunit’s groove. The
resulting PapD:PapFG8N did not aggregate and yielded crystals
diffracting to 2.2 A resolution. Its structure was solved by molec-
ular replacement using the PapD:PapK structure as the search
model (see Experimental Procedures).
The PapD:PapFG8N structure is very similar to the already
known PapD:PapK, PapD:PapENtd, PapD:PapHNtd1, or PapD:
PapANtd1_G15N structures. Like in PapK, PapE, PapH, or PapA,
PapF exhibits an Ig-like fold where the seventh strand is missing
(Figure 2). Like in all other Pap complexes, the chaperone PapD
complements the PapF structure by donating its G1 strand to
complement the PapF’s fold (not shown).
When comparing the structures of PapA and PapF (Figure 2A),
two major differences are apparent in the region between
strands A2 and B (the A2-B loop) and between strands B and
C1 (the B-C1 loop). The B-C1 loop in PapF is short and disor-
dered (electron density for residues 53 and 54 is weak and these
residues could not be built), but is long and also disordered in the
PapD-bound PapA subunit of the PapD:PapA2 complex (Verger
et al., 2007) (Figures 1 and 2A). In PapA, the B-C1 loop becomes
ordered upon DSE, extending the subunit’s groove to accommo-
date the long PapA Nte. Among all Pap subunits, the only other
subunit with a long B-C1 loop is PapH, and in the structure of this
subunit, it is ordered (Verger et al., 2006) (Figure 2B).
Differences in the A2-B loop of the structure are likely to be
functionally significant as this loop forms part of the P5 pocket,
a major initiation point for DSE and a specificity-determining
DSE site (Remaut et al., 2006; Rose et al., 2008a). In PapF,
Pro32 obstructs the P5 pocket by contacting residues in the
bD1 strand (Phe81) and the E-F loop (Leu132, Gly135) (Figure 3B).
The A2-B loop is stabilized by a salt bridge between Glu33 in the
loop and Arg126 at the end of the bE strand (Figures 3A and 3B).
In Figure 3, the structure of the region around the P5 pocket
is shown in all Pap subunits for which the structure is known
(except PapE, where the regions forming the P5 pocket are dis-
1726 Structure 16, 1724–1731, November 12, 2008 ª2008 Elsevier
ordered). PapA and PapK both exhibit well-defined P5 pockets
(Figure 3C). These subunits are known to undergo DSE at very
similar rates (with a reaction midpoint [t50] of 15.2 and 12.5 hr, re-
spectively; Figure 1B). In PapE, the A2-B loop is disordered and
thus the region around the P5 pocket is presumably flexible. Re-
markably, PapE is known to be a fast-exchanging pilus subunit;
that is, it undergoes DSE at the highest rate (t50 of 3.2 hr) among
Pap subunits (Rose et al., 2008a) (Figure 1B). In contrast, in PapH
and PapF, the P5 pocket is obstructed by residues in the A2-B
loop (Thr52 in PapH and Pro32 in PapF) (Figure 2B). PapH is
unable to undergo DSE while PapF undergoes DSE at a very
slow rate (Figure 1B). Thus, there appears to be a correlation
Figure 2. Comparison of the PapF Structure with the PapA and PapH
Structures
(A) Superposition of the structures of PapFG8N (cyan) and PapANtd1_G15N
(orange). These are shown in ribbon representations with b strands indicated
as arrows and a-helices indicated as cylinders. Secondary structures referred
to in the main text are labeled. The a-helix and the A2-B and B-C1 loops are
indicated, as well as Pro32 (in stick representation).
(B) Superposition of the structures of PapFG8N (cyan) and PapHNtd1 (magenta).
Representation is the same as in panel A. Residues Pro32 of PapF, and Thr52
and Pro53 of PapH, are shown in stick representation. The a-helix and the
A2-B and E-F loops are indicated.
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Structure
Crystal Structure of the P Pilus Subunit PapF
between the level of accessibility and flexibility of the P5 pocket
and the rate of DSE: the more accessible and flexible the region
around the P5 pocket is, the faster the exchange reaction.
The A2-B Loop of PapF Is Flexible,Whereas that of PapH Is RigidFor PapF to undergo DSE, accessibility to the P5 pocket is
required. Yet, the P5 pocket of PapF is obstructed in the crystal
structure. Thus, in view of the crystal structure alone, PapF
should not be able to undergo DSE. The fact that PapF is able
to participate in the exchange reaction, albeit at a slow rate,
suggests that its P5 pocket must transition to an open state.
To test this hypothesis, MD simulations were carried out on the
PapD:PapF and PapD:PapH complex structures (Figure 4).
Each structure was stable during simulations, as indicated by
an rmsd from the crystal structure not exceeding 3 A.
The walls of the P5 pocket are formed by residues in the A2-B
loop on one side of the P5 pocket and residues in the E-F loop
and the N terminus of strand D1 on the other side of the pocket
(Figure 2B). Any opening and closing transitions in the P5 pocket
should result in fluctuations in the distances between the A2-B
loop residues and those in the E-F loop or in the N terminus of
strand D1. Thus, we monitored the following distances. In PapH
(Figure 4, top panel), we monitored the distance between Thr52
(A2-B loop) and Ile114 (strand D1), and between Thr52 and
Gly160 (E-F loop). In PapF (Figure 4, lower panel), we monitored
the distance between Pro32 (A2-B loop) and Phe81 (strand D1)
and between Pro32 and Leu132 (E-F loop). These simulations
reveal that in PapH, the distances between Thr52 and Gly160
or Ile114 do not change substantially, whereas in PapF the
distances between Pro32 and Phe81 or Leu132 fluctuate signif-
Figure 3. Comparison of the P5 Regions of
the Pap Subunits
(A and C) The structures are in surface representa-
tion. Each structure is identified with the name of
the molecule above it. In each, the location of the
conserved aromatic P4 pocket residue is indi-
cated and serves to position each panel relative
to one another. In each panel, the P5 pocket is
identified by a green circle, either in a continuous
line when the P5 pocket is accessible (PapA,
PapK) or in dashed line when it is obstructed
(PapF, PapH). Finally, for PapF and PapH, the
location of the residues mentioned in the text is
indicated.
(B) A stereo diagram of the P5 region of PapF is
shown with residues Pro32, Glu33, Arg126,
Phe81, Gly135, and Leu132 in the P5 region
(shown in sticks). The location of the P4 pocket
residue is represented in sticks and color coded
in red.
icantly. Interestingly, fluctuations in the
positioning of Pro32 relative to Phe81
and Leu132 in PapF appears to be re-
stricted to a limited number of conforma-
tional states, closed as seen in the crystal
structure and open where the P5 pocket
appears to be no longer obstructed. The
comparison of rmsd per residue between PapF and PapH is
also instructive. It clearly shows that fluctuations are much larger
in the P5 region of PapF than in the equivalent region of PapH:
Pro32, the residue in the A2-B loop obstructing the P5 pocket
in PapF, has an rmsd of 4.2 A, whereas the corresponding
Thr52 in PapH has an rmsd of 1.2 A. Overall, whereas the P5
pocket appears blocked in both the PapH and PapF crystal
structures, MD simulations suggest that the P5 pocket of PapF
becomes accessible intermittently whereas that of PapH does
not. These results thus provide an explanation as to why PapF
undergoes slow DSE and PapH does not undergo DSE at all.
Mutants Destabilizing the A2-B Loop DisplayHigher DSE RatesTo test the hypothesis that the A2-B loop flexibility is important in
controlling the rate of DSE in PapF, two mutations anticipated to
lower the stability of this loop were introduced and their effect on
DSE was monitored. Pro32 was mutated to Gly, a mutation that
would lower the contact area between the A2-B and E-F loops in
this region, and Arg126 was mutated to Ala, to remove the salt
bridge stabilizing the A2-B loop (see above). Both mutations
are expected to increase structural flexibility in the P5 pocket
region. DSE was monitored using the ESI-MS assay described
in Remaut et al. (2006) for SafA and Rose et al. (2008a) for all
Pap subunits. Because the structure determined here was of
PapFG8N, this amino acid substitution was also introduced in
the proteins. Complexes (wild-type PapD:PapF, PapD:PapFG8N,
PapD:PapFG8N_P32G, and PapD:PapFG8N_R126A) were monitored
in the absence of the attacking Nte and were shown to be stable
over the incubation period (Figure 5A). DSE reactions were initi-
ated by the addition of the Nte of PapE (ENte) and monitored over
Structure 16, 1724–1731, November 12, 2008 ª2008 Elsevier Ltd All rights reserved 1727
Structure
Crystal Structure of the P Pilus Subunit PapF
a period of 200 hr at room temperature. Control experiments
comparing wild-type PapD:PapF with the mutant PapD:PapFG8N
confirmed that the G8N mutation does not impact DSE rates
(Figure 5B). In the presence of ENte, PapD:PapFG8N undergoes
DSE with the t50 of 81.5 hr, comparable to that obtained by
Rose et al. (2008a). The double mutants PapFG8N_P32G and
PapFG8N_R126A undergo DSE at significantly higher rates than
PapFG8N with a t50 of 37.0 hr and 42.2 hr, respectively (Figure 5B).
It would thus appear that mutations expected to increase the
flexibility of the A2-B loop result in increased rates of DSE.
DISCUSSION
PapF is an important adaptor protein that is not only essential
in P pilus biogenesis, but also appears, together with PapG, to
act as a checkpoint in this process (Dodson et al., 2001;
Jacob-Dubuisson et al., 1993; Lee et al., 2007). Indeed, it was
recently shown that the in vitro DSE rates for PapG and PapF
are by far the slowest among all Pap subunits (Rose et al.,
2008a) (Figure 1B). Thus, commitment to fully-fledged pilus bio-
genesis only occurs after the two slowest subunits have reacted,
PapG with the Nte of PapF, and PapF with the Nte of the first
incoming PapE subunit. Once this is achieved, DSE proceeds
rapidly until termination by the PapH subunit.
The molecular basis for the slow DSE kinetics of the PapF
subunit was unclear. The structure of this subunit determined
here provides a molecular interpretation of these observations,
showing that PapF has a partially obstructed P5 pocket, which
appears to prevent access by the P5 residue of the attacking
PapE Nte peptide. In prior studies, we have demonstrated that
the P5 region of subunits’ grooves is an essential region serving
as the first point of contact for the Nte peptide of the subunit next
in assembly (Remaut et al., 2006). Not only does the P5 pocket of
receiving subunits act as an initiation site for DSE, but also we
have shown that this early interaction event determines, to a large
Figure 4. Molecular Dynamics Simulations
Fluctuations of distances between pairs of residues in PapH (top panel) and in
PapF (bottom panel). The residues monitored during simulations are indicated
in the inset in each panel.
1728 Structure 16, 1724–1731, November 12, 2008 ª2008 Elsevier L
extent, the specificity of the DSE reaction (Remaut et al., 2006;
Rose et al., 2008a). Among all Pap subunits, PapH is the only
subunit unable to undergo DSE. This is because it has an ob-
structed P5 pocket and, as a result, lacks a DSE-initiating site.
Thus, obstruction of the P5 pocket is a brake on DSE. In PapF,
partial obstruction of the P5 pocket would also act as a brake,
but DSE still proceeds in this case, albeit at a slow rate. This
implies that the PapF P5 pocket, in contrast to that of PapH, is
intermittently accessible for DSE initiation. This conclusion is
borne out by the MD simulations on SafA (Rose et al., 2008b)
and on the PapF and PapH subunits (this work), showing clear
differences between PapF and PapH in the region where the
P5 pocket lies: it is flexible in PapF and rigid in PapH.
The differences in flexibility between the two proteins in the P5
region are likely due to the combined effects of two structural
features: the first is the presence of the only a-helix in the Ig
fold of PapH, which is absent in PapF, and the second is the
Figure 5. DSE ESI-MS Assay of the Wild-Type and Mutant PapD:
PapF Complexes
The DSE reactions are monitored in the absence of any Nte (A) or after adding
ENte (B). Wild-type PapD:PapF is shown in black, PapD:PapFG8N in red, PapD-
PapFG8N_P32G in green, and PapD-PapFG8N_R126A in blue. Average error is
± 2.5%.
td All rights reserved
Structure
Crystal Structure of the P Pilus Subunit PapF
location of residue Pro53 in the PapH structure, a residue that
immediately follows the helix (Figure 2B). Pro53 introduces
a kink in the loop between the helix and the subsequent strand
and this kink projects Thr 52 into the PapH P5 pocket, thereby
obstructing it completely. Thr52 is thus rigidly maintained within
the P5 pocket by the combined action of the helix and Pro53. In
contrast, although PapF also contains a proline in that region
(Pro32), this proline aligns with Thr52 of PapH and not Pro53 in
the superposition of the two structures (Figure 2B). It is also
located in a long flexible region that does not contain an a-helix
like in PapH. Thus, obstruction of the P5 pocket of PapF by
Pro32 is solely due to the interaction of Pro32 with the residues
in the bD1 strand and the E-F loop, an interaction that is relatively
unstable, as the MD simulations presented here suggest. As a
result, Pro32 moves in and out of the P5 pocket. Note that the
presence of an a-helix between the bA2 and bB strands is not
unique to PapH. It is also present in PapA (Figure 2A) and in
PapK. However, in PapA and PapK, it provides a rigid foundation
for a well-defined P5 pocket (Figure 3C).
The mutational study presented here confirms our structural in-
terpretation of the observed DSE kinetics of PapF. Reducing the
contacts and electrostatic interactions between the A2B and the
EF loops residues results in faster DSE reactions. Thus, changing
the residue composition of the P5 pocket changes the DSE rates.
In a recent mutational study, Remaut et al. (2006) showed that in
the Saf system, the chaperone occupies the P5 site but appears
to do so in an equilibrium with an unbound, accessible state. Mu-
tating the chaperone’s G1 residue occupying the P5 site toa larger
hydrophobic side chain shifts the equilibrium toward the closed
state, suppresses formation of the chaperone-subunit-Nte inter-
mediate, and drastically reduces the DSE rate. Here, for the first
time, we show that mutating the P5 pocket itself results in predict-
able changes in DSE rates. Thus, this study unravels an exquisite
control system centered on the accessibility of the P5 pocket to
the attacking Nte, which modulates DSE reactions for all Pap
subunits during pilus biogenesis.
These studies altogether converge to confirm the P5 initiation
model first proposed by Remaut et al. (2006). Whether a similar
model applies to other systems where strand-zippering has
been observed (see review by Remaut and Waksman, 2006)
remains to be seen. Given the general importance of strand
addition and insertion in macromolecular interactions, thorough
mechanistic studies of these systems are overdue. However, it is
expected that they will likely share common mechanistic details
with DSE, including the need for an initiation point not only
driving the process but also contributing to specificity.
EXPERIMENTAL PROCEDURES
Plasmids
The PapD:PapF construct (pTrc-DF) was designed as described previously
(Rose et al., 2008a). The PapD:PapFG8N mutant (pTrc-DFG8N) was made
from the pTrc-DF plasmid, using oligonucleotides overlapping the sequence
encoding the G8 residue, with the glycine 8-encoding codon replaced by an
asparagine one. Similarly, the double mutants PapD:PapFG8N_P32G and PapD:
PapFG8N_R126A were prepared from the pTrc-DFG8N plasmid, using oligonucle-
otides overlapping the codon encoding P32 or R126, with the P32 or R126
codons replaced by a glycine (P32G) or an alanine (R126A), respectively.
(Stratagene QuickChange Site-Directed Mutagenesis Kit; see Table 1 for
oligonucleotide design).
Structure 16, 1724–17
Expression and Purification of PapD:PapF, PapD:PapFG8N,
PapD:PapFG8N_P32G, and PapD:PapFG8N_R126A
PapD:PapF, PapD:PapFG8N, PapD:PapFG8N_P32G and PapD:PapFG8N_R126A
were expressed and purified as previously described by Rose et al. (2008a).
Each complex was concentrated to around 10 mg/ml and some of the PapD:
PapFG8N complex was used for crystallization trials. For mass spectrometry
analysis, all the complexes were dialyzed against 5mM ammonium acetate
pH 5.5 and used at a concentration of 40 mM.
Mass Spectrometry Analysis
A 40 micromolar solution of PapD:PapF complex (wild-type or mutant) was in-
cubated with 400 mM of a peptide derived from the Nte of PapE (sequence
VDNLTFRGKLII, to which 2 lysines at the C terminus were added in order to
render the peptide soluble), in 5mM ammonium acetate buffer (pH 5.5) at
room temperature. As a control, the complex was also incubated in the
same buffer and at the same temperature in the absence of any peptide.
One-microliter aliquots were removed from the reaction mixture at given
time points and analyzed by mass spectrometry. Nano-electrospray ionization
was used, where samples (1–2 mL) are introduced into gold-plated borosilicate
vials and analyzed using a Q-Tof mass spectrometer (Waters UK Ltd.,
Manchester, UK). Data were acquired over the m/z range of 1500–5000 with
a scan speed of 2.4 s, and CsI clusters were used as an external calibrant to
ensure mass accuracy of within 0.01%. Peaks relating to the various PapD:
PapF complexes were integrated and summed. For each time point, the ratio
of these peaks to the total area of peaks in the spectrum was calculated. These
data were normalized between the maximum value (before addition of peptide)
and the baseline, and fitted using a single exponential.
PapD:PapFG8N Crystallization, Structure Determination,
and Refinement
The PapD:PapFG8N complex was crystallized at room temperature in a hanging
drop equilibrated against a reservoir solution containing 20% glycerol and
2.0–2.2 M ammonium sulfate with no buffer (original condition obtained from
solution C6, Jena Biosciences screen 6). The space group of the crystals is
P41212, with one complex per asymmetric unit (44% solvent). The cell dimen-
sions are as follows: a = 65.4 A, b = 65.4 A, and c = 166.1 A. The data for a single
crystal were processed to a resolution of 2.2 A (see Table 2). The structure was
solved by molecular replacement using PapD:PapK structure (PDB entry
1PDK) as a search model using the program AMoRe (Navaza, 2001). Succes-
sive cycles of manual rebuilding with O and conjugate gradient minimization
using crystallography and NMR system were performed (Brunger et al.,
1998; Jones et al., 1991). B factors were refined individually. Water molecules
were added in agreement with expected hydrogen bond distances and Fo �Fc density superior or equal to 3.5s. The refinement converged to the final
values of R = 23.55% and Rfree = 27.73% (20–2.2 A range; F/sF R 0.0) with
good stereochemistry (see Table 2 for additional statistics). The final model
includes all PapD main chain atoms except the last 3 residues (216–218);
the following residues in PapFG8N were missing from the electron density:
1 to 8, 53 to 54, and 98 to 114. The final model includes 2 sulfate ions and
85 water molecules.
Table 1. Oligonucleotides Used in this Study
Construct Forward primer Reverse primer
PapFG8N GTG CAG ATT AAC
ATC AGG AAC AAT
GTT TAT ATC CCC
CC
ATA TAA ACA TTG
TTC CTG ATG TTA
ATC TGC ACA TCA
GCC
PapFG8N_P32G GAT TTT GGG AAT
ATT AAT GGT GAG
CAT GTG GAC AAC
TCA CG
GAG TTG TCC ACA
TGC TCA CCA TTA
ATA TTC CCA AAA
TCA AC
PapFG8N_R126A CT TCA GTG CCC
TTT GCG AAT GGC
AGC GGG ATA CTG
AAT GG
CAG TAT CCC GCT
GCC ATT CGC AAA
GGG CAC TGA AGT
AAA GG
31, November 12, 2008 ª2008 Elsevier Ltd All rights reserved 1729
Structure
Crystal Structure of the P Pilus Subunit PapF
Molecular Dynamics Simulations
Simulations were performed using an atomistic model with implicit solvent
(Lazaridis and Karplus, 1999). The crystallographic structures of the PapD:
PapHNtd1 (PDB code 2J2Z) and PapD:PapFG8N (this work) chaperone subunit
complexes were used as initial conformations for the simulations. In PapD:
PapFG8N, the loop residues K53 and S54 missing from the model were built
as alanines. Missing residues 98–114, belonging to a long and structurally con-
served loop region among all subunits, were reconstructed by structural align-
ment based on the equivalent region of the PapK model (PDB code 1PDK).
These additional modeled regions were energy minimized, and the resulting
model showed good stereochemistry (data not shown). After minimization
and equilibration, 100 ns of Langevin dynamics at 300K with a friction coeffi-
cient of 1 ps�1 were produced for each complex.
ACCESSION NUMBERS
The coordinates of the PapD:PapFG8N complex were submitted to the Protein
Data Bank (entry code 2w07).
ACKNOWLEDGMENTS
R.J.R. was funded by a BBSRC/CASE PhD studentship and Waters UK Ltd.
We thank R. H. Bateman (Waters UK Ltd.) for continued support. The
Table 2. Data Collection and Refinement Statistics
Data collection
Data set PapD/PapFG8N
(ESRF ID14)
Resolution 20.0�2.2 A
Total/unique 138,425/19,108
reflections
Completeness (%)a 99.8 (100)
Rsym(%)b 5.3 (29.3)
I/s(I)c 9.4 (2.6)
Refinement
Resolution (A) 20.0�2.2
Number of reflectionsd 19,065 (980)
Total number of atomse 2626
R/Rfree (%)f 23.6/27.7
rmsdg Bonds (A)
0.006
Angles (�)
1.26
B values (A2)
Main chain Side chain
1.5 2.2a Completeness for I/s(I) > 0, high-resolution shell (2.32�2.2 A) in paren-
theses.b Rsym =
PjI� hIij
PI, where I = observed intensity and hIi = average
intensity from multiple observations of symmetry related reflections;
high-resolution shell in parentheses.c I/s(I), high-resolution shell in parentheses.d Total number of reflections (F/s(F) > 0.0), number in the ‘‘test set’’ in
parentheses.e Includes water molecules and sulfate ions.f Rfree was calculated on the basis of 5% of the total number of reflections
(‘‘test set’’) randomly omitted from the refinement.g Deviations from ideal bond lengths and angles and in B factors of
bonded atoms.
1730 Structure 16, 1724–1731, November 12, 2008 ª2008 Elsevier L
Q-Tof1 mass spectrometer was funded by the BBSRC. The work was sup-
ported with funds from NIH, the BBSRC, and the Wellcome Trust.
Received: July 14, 2008
Revised: August 13, 2008
Accepted: August 14, 2008
Published: November 11, 2008
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