Prolyl isomerization as a molecular timer inphage infectionBarbara Eckert, Andreas Martin, Jochen Balbach & Franz X Schmid

Prolyl cis-trans isomerizations are intrinsically slow reactions and known to be rate-limiting in many protein folding reactions.Here we report that a proline is used as a molecular timer in the infection of Escherichia coli cells by the filamentous phage fd.The phage is activated for infection by the disassembly of the two N-terminal domains, N1 and N2, of its gene-3-protein,which is located at the phage tip. Pro213, in the hinge between N1 and N2, sets a timer for the infective state. The timer isswitched on by cis-to-trans and switched off by the unusually slow trans-to-cis isomerization of the Gln212-Pro213 peptidebond. The switching rate and thus the infectivity of the phage are determined by the local sequence around Pro213, and canbe tuned by mutagenesis.

The cis-trans isomerizations of X-Pro (where X is any amino acid)peptide bonds (prolyl isomerizations) are slow reactions that occur inthe time range of minutes at 25 1C (refs. 1–4). Their importance asrate-determining steps in protein folding reactions is well character-ized5,6. Although conformational folding can be extremely fast7,8, it isoften retarded 41,000-fold by the isomerizations at prolyl residuesthat are cis in the native state, but predominantly trans in the unfoldedstate5,6,9. Prolyl isomerizations can be catalyzed by prolyl isomerases.These enzymes are involved not only in the catalysis of folding6,9 butalso in regulatory processes10,11.

Here we investigated how a prolyl residue with an unusually slowtrans-to-cis isomerization acts as a molecular device for timing theinfection of E. coli cells by the filamentous phage fd. The infection ismediated by the phage gene-3-protein (G3P), which is located at thephage tip (Fig. 1a). G3P is anchored in the phage coat by itsC-terminal domain, and the N2 and N1 domains12,13 are used forsequential interactions with the target cell during infection14,15. In thelatent state of the phage, N1 and N2 are tightly associated (Fig. 1b),which ensures a high long-term stability of G3P but renders the phageincompetent for infection. In a stepwise process, this resting form ofthe phage first binds with a surface-exposed region of the N2 domainto the tip of the bacterial F pilus16. This activates G3P by exposing thebinding site for TolA, which is the ultimate phage receptor at the cellsurface17,18. The TolA-binding site is located on the N1 domain at theN1-N2 interface18 and thus is protected in the closed form of G3P(Fig. 1b). In the course of phage infection the open, binding-competent form must persist until the N1 domain reaches theC-terminal domain of TolA (TolA-C) (Fig. 1a)17,19,20.

During the in vitro refolding of the N1-N2 fragment of purifiedG3P, the individual domains fold rapidly, but domain assembly in thefinal step is very slow and shows a time constant (t) of 6,200 s

(25 1C)21. This domain closing reaction is limited in rate by the trans-to-cis isomerization of the Gln212-Pro213 peptide bond in the hingebetween the two domains. It locks G3P in a stable but inactive form22

(Fig. 1b). Here we asked whether this unusually slow, proline-limiteddomain movement in G3P is also important for the function of theprotein during phage infection. We find that in vivo Pro213 isomer-ization is used as a timer that controls the lifetime of the open,infectious form of G3P. The timer is switched on by cis-to-trans andswitched off by the trans-to-cis isomerization of the Gln212-Pro213peptide bond. The switching rate is determined by the local sequencearound Pro213.

RESULTSPhage infectivity and Pro213 isomerization are correlatedThe rate of isomerization of the peptide bond between residues 212and 213 during the final step of the in vitro refolding of purified N1-N2 fragment is determined by Pro213 itself and by the adjacentresidues Gln212 and Pro214. The P213G mutation accelerates thisreaction 26-fold22, and the mutations to alanine of the flankingresidues Gln212 and Pro214 increase the rate of domain dockingtwo- and three-fold, respectively (Table 1).

To examine whether Pro213 and its flanking residues are alsoimportant for the phage infectivity in vivo, we created three librariesof phage variants in which the codons for position 212, 213 or 214 ofG3P were randomized, respectively. After ten successive rounds ofinfection and phage propagation only triplets coding for glutamine atposition 212 and for proline at positions 213 and 214, as in the wild-type phage, were found (Supplementary Methods online).

Gln212, Pro213 and Pro214 of G3P might be important forinfection in vivo because they determine the rate of domain closing(and thus the lifetime of the open form of G3P). If so, the mutations

Published online 5 June 2005; doi:10.1038/nsmb946

Laboratorium fur Biochemie und Bayreuther Zentrum fur Molekulare Biowissenschaften, Universitat Bayreuth, D-95440 Bayreuth, Germany. Correspondence should beaddressed to F.X.S. (fx.schmid@uni-bayreuth.de).

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at these positions, which increase the rate of domain closure, shoulddecrease the infectivity of the corresponding phage variants. Com-parative infections of separate E. coli cultures are not well suited todetermine relative phage infectivities. They can vary substantiallybecause the expression of the F pili, the primary receptors, dependsstrongly on the growth conditions of E. coli23. To avoid theseambiguities, we allowed two phage variants to compete against eachother for infection of a single E. coli culture and then identified thesurviving phage by colony PCR. To generate unique priming sites forthe two phage variants, we exploited the degeneracy of the geneticcode for serine. At two Ser-Ser positions alternative isocoding six-nucleotide sequences were introduced into the gene 3 of the phage,and PCR with the corresponding complementary oligonucleotides wasused to determine the identity of the phage variant present in theinfected cells after a competition.

In the first experiment, the wild-type phage with a Pro213 in G3Pcompeted against a mutated phage with a Gly213. The P213Gsubstitution reduces the time constant of domain closing 26-fold(Table 1)22. Four successive rounds of competitive infections withwild-type and P213G phage were carried out (Fig. 2). The P213Gphage variant with the fast domain closing reaction vanished withinthree rounds. In experiments starting with varying ratios of the twophage variants, the P213G phage was also outcompeted within a fewinfection cycles (data not shown).

The P213G mutation weakens the interactions between the domainsN1 and N2 in G3P, which could also explain why the mutated phagelost in the competition with the wild-type phage. The N1-N2 part ofG3P unfolds in two transitions. The first transition reflects thedissociation of the domains, which is linked with the unfolding ofthe N2 domain. The second transition reflects the unfolding of theN1 domain21,24. The P213G mutation influences the domain inter-actions and thus decreases the midpoint of the first transition (TM)from 48.1 1C to 44.3 1C (Table 1). A similar destabilization of thedomain interaction is caused by the G153D mutation, but thisreplacement does not affect the rate of the Pro213-limited domainclosing reaction (Table 1). Therefore, the wild-type phage was exposedto a competition with the G153D phage. In this competition both thewild-type and the G153D phage persisted for four rounds with a slightadvantage for the wild-type phage (Fig. 2). Even after ten infectionrounds, 15% of the G153D phage was present (data not shown). In thedirect competition between the P213G and G153D phage, the P213G

phage vanished within three rounds (Fig. 2).Thus, the slightly weaker domain interactionsbrought about by the P213G or G153Dmutations did not cause the observed differ-ences in infectivity.

The prolyl isomerase cyclophilin 18 accel-erates the isomerization at Pro213, but not atGly213 (ref. 22). Therefore it reduced thedifference in the rate of domain closingbetween the wild-type phage with Pro213and the mutated phage with Gly213 andaccordingly decreased the advantage of thewild-type phage over the Gly213 mutant inthe competitive infections (Fig. 2). Unlike amutation, the catalysis by cyclophilin doesnot change the stability of G3P. The decreasedinfectivity relative to the P213G mutant isthus best explained by the acceleration ofthe Pro213 trans-to-cis isomerization in thewild-type G3P.

The Q212A and P214A mutations accelerate the trans-to-cis iso-merization at Pro213 two- and three-fold, respectively (Table 1), andthe corresponding phage variants disappeared within four rounds inthe competitions with the wild-type phage (Fig. 2). They disappearedalso in the competitions with the G153D mutant, but theyoutcompeted the P213G mutant (Supplementary Fig. 1 online).Together, these results show that phage infectivity does not correlatewith the strength of the domain interactions in G3P, but, in all cases,correlates inversely with the rate of trans-to-cis isomerization of thepeptide bond between residues 212 and 213, and thus with the lifetimeof the open form of G3P.

Phage inactivation by Pro213 trans-to-cis isomerizationEscherichia coli strains without F pili are also infected by filamentousphage20, but infectivity is reduced from B1011 colony-forming unitsper ml (c.f.u. ml�1) to 105–107 c.f.u. ml�1 (Table 1). In these strains,pilus-mediated domain disassembly is not possible. Therefore, theinteraction of N1 with TolA, and thus the infection of E. coli, dependson spontaneous or forced domain-opening reactions in G3P. Allmutations at or near Pro213 loosen the domain interactions andincrease the rate of domain opening. The P213G mutation, inparticular, accelerates domain opening B50-fold22. This explains theincreased infectivities of the phage mutants toward pilus-free cells(Table 1, native phage titers).

Outer membrane C

M

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CytoplasmInner membrane

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TolAbinding site

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binding siteTolA

F pilus

Phage

Phage

CT

g3pN1

N1

N2′

N2′

N2 hinge

N2 hinge

a b

Figure 1 Function of G3P in phage infection. (a) The initial steps of phage infection. Binding of the

N2 domain of G3P to the tip of an F pilus is followed by domain opening and binding of N1 to the

C-terminal domain of TolA. (b) Structure of the N1-N2 fragment of G3P18. Domain N1 is red,

the globular part of domain N2 (N2¢) is blue, the hinge subdomain of N2 is green and cis-Pro213 is

yellow. The binding sites for the F pilus and for the C-terminal domain of TolA are indicated. The figure

was prepared by using PDB entry 2G3P13 and MolMol33.

Table 1 Stabilities, rates of domain closing and infectivities of the

phage variants

TM

(1C)aDomain closing

t (s)bPhage titer

(c.f.u. ml�1 � 10�5)c

Variant of G3P Native Denatured

Wild type 48.1 6,200 1.2 1,400

G153D 44.0 6,200 8.4 1,600

Q212A 44.5 3,800 8.8 1,500

P214A 44.3 1,900 17 1,200

P213G 44.3 240 160 170

aTM is the midpoint of the first thermal transition of the N1-N2 fragment of G3P. It reflectsthe cooperative domain dissociation and unfolding of N2. bTime constant of domain closing.cThe phage titers are given as colony-forming units (c.f.u.) for the infection of E. coli cellswithout F pili. The phages were denatured by incubation at 65 1C for 10 min. The error rangesare 70.2 1C for TM, 75% for t, and 750% for the phage titers.

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In vitro, the domains of G3P can be dissociated by denaturant-induced or thermal unfolding (Table 1). We therefore incubated thewild-type phage for 10 min at 65 1C and then infected E. coli cellswithout F pili at 25 1C. Thermally induced domain opening increasedthe infectivity of wild-type phage 1,200-fold, and all phage variantswith a Pro213 (the G153D, Q212A and P214A variants, Table 1)showed similarly high infectivities. The same activation was obtainedwhen the G3P on the phage was unfolded by 5 M guanidiniumchloride (GdmCl) before infection. For the P213G variant, theincrease in infectivity after unfolding could not be followed becausedomain closing after the refolding jump (Table 1) is faster than thedead time of the phage infection experiments (300 s).

Next, we examined how long the activated state of the wild-typephage persists after the unfolding-mediated domain opening. In theseexperiments G3P was first unfolded by exposing the phage particles to5.0 M GdmCl, after which refolding and domain association wereinitiated by dilution to 0.5 M GdmCl (at 25 1C). Samples werewithdrawn at various times to determine infectivity toward E. colicells without F pili. The inactivation followed the same time course asthe trans-to-cis isomerization of Pro213 (Fig. 3a). It was reversible,and a second unfolding by 5.0 M GdmCl reopened the domains andrestored the high phage infectivity.

When G3P is refolded in the presence of the TolA-C domain, thetwo proteins interact transiently. The N1 domain of G3P refoldsrapidly and binds to TolA-C. This formation of complex leads toa strong increase in the Forster resonance energy transfer betweenthe tryptophan residues of N1 and AEDANS-labeled TolA-C(Supplementary Fig. 2 online). Later in folding, TolA-C is displacedfrom its binding site when the tight intramolecular interactionsbetween N1 and N2 are established. This displacement reactionshows the same rate as the trans-to-cis isomerization at Pro213, andboth follow the time course of phage inactivation (Fig. 3a). Thissuggests that Pro213 trans-to-cis isomerization controls TolA bindingand consequently phage inactivation.

Mutations at either side of Pro213 modify the isomerization rate. Inthe purified N1-N2 protein, the Q212A and P214A mutations decreasethe time constant of isomerization from 6,200 s to 3,800 s and 1,900 s,respectively (Table 1). The inactivation kinetics of the correspondingphage mutants show the same alterations. The P213G mutationaccelerates isomerization 26-fold, and accordingly phage inactivationoccurs in the 300 s dead time of the experiment (Fig. 3b).

Catalysis by prolyl isomerases provides the strongest evidence for aproline-limited process, as these enzymes accelerate specifically prolylisomerizations6,11. The phage inactivation is well catalyzed by theprolyl isomerase cyclophilin 18 (Fig. 3c). This completes the evidencethat the trans-to-cis isomerization of Pro213 controls the lifetime ofthe activated, highly infectious form of the phage. Even though theswitching rate at Pro213 is strongly enhanced by a prolyl isomerase, itis unlikely that this catalysis is physiologically relevant.

Isomerization rate determined by local sequenceFischer and co-workers measured by NMR spectroscopy the rates ofX-Pro isomerization in the acetylated pentapeptides Ac-Ala-X-Pro-Ala-Lys-NH2 as well as the rate of Ala-Pro isomerization in the peptideAc-Ala-Pro-Pro-Ala-Lys-NH2 (refs. 3,4). These data showed thatGln-Pro isomerizes 2.3-fold slower than Ala-Pro, and Ala-Pro-Pro

P213G

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1 2Infection cycle Infection cycle

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1 Phage inactivation Phage inactivation

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TolA displacement0.8

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Figure 3 Loss of phage infectivity follows proline-limited domain closing. All measurements were carried out in 0.5 M GdmCl, pH 7.0, at 25 1C after a ten-fold dilution from 5.0 M GdmCl. (a) Coincidence of phage inactivation, trans-to-cis isomerization at Pro213 and TolA-C displacement. The dashed line shows

the fit of a monoexponential function (t ¼ 5,600 s) to the time course of TolA-C displacement. The kinetics of inactivation and Pro213 isomerization result

in t values of 5,700 s and 6,200 s, respectively. (b) Inactivation kinetics of phages with mutations in G3P at or adjacent to Pro213. The kinetics of

inactivation of the Q212A and P214A phage give t ¼ 3,700 s and t ¼ 2,030 s, respectively. (c) Catalysis of phage inactivation by cyclophilin 18. In the

control, unfolding by GdmCl was omitted. The analyses (continuous lines) give t values of 6,200 s, 3,700 s and 1,030 s in the presence of 0, 10 and

50 nM cyclophilin18, respectively.

Figure 2 Competitions between different phage variants for the infection of

E. coli. The phage carried different substitutions in G3P as indicated in the

panels (WT, wild type; Cyp18, cyclophilin 18). For the first infection, phage

aliquots with equal infectivity toward E. coli XL1-Blue were mixed. Four

rounds of infection, phage propagation, isolation and reinfection were carried

out. The fractions of cells infected with the two phage variants after each

round are shown. The error bars represent 75% confidence limits for the

experiments where 48 individual clones were analyzed.

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isomerizes 3-fold slower than Ala-Pro-Ala, in good agreement with theinfluence of the corresponding mutations around Pro213 in therefolding of G3P (Table 1).

To determine the rate of isomerization in the presence of both aglutamine before and a proline after Pro213, a pentapeptide repre-senting the local sequence around Pro213 (Ac-Ala-Gln-Pro-Pro-Val)was examined by time-resolved NMR spectroscopy under the sameexperimental conditions as the folding and infection experiments. Thecis-trans isomerization kinetics of the Gln-Pro bond could be followedby the intensities of the NH resonances of Ala1 and Gln2 (Fig. 4). Theapparent time constant of 395 s for cis-trans equilibration of thepeptide agrees well with the value expected from the isomerizationrates in the pentapeptides4. Moreover, it is very similar to the timeconstant of 350 s for the equilibration at Pro213 in the unfolded N1-N2 fragment22. The measured rate is equal to the sum of the rateconstants for the cis-to-trans and the trans-to-cis reactions. Togetherwith the equilibrium constant K ¼ [cis] / [trans] ¼ 0.06 (ref. 22), thisgives t ¼ 6,980 s for the trans-to-cis isomerization in the peptide, ingood agreement with the inactivation rate of the phage (Fig. 3a). Thissuggests that the unusually low isomerization rate in G3P and the longlifetime of the activated state of the phage are indeed determined bythe local sequence around Pro213.

DISCUSSIONThe cis-trans isomerization of Pro213 is used as a simple devicefor timing phage infection. It has often been suggested that, beyondthe well-established role in protein folding, prolyl isomerizationcan be used as a conformational switch to regulate processessuch as transmembrane signaling, protein phosphorylation andthe cell cycle10,25–30. For phage infection, the prolyl isomerization inG3P is used as both a timer and a switch. With a cis-Pro213 thetimer is switched off, and in this state the phage is stable and robustbut not infectious. The timer is turned on when the N2 domain ofG3P binds to the F pilus. This binding weakens the N1-N2 domaininteractions, presumably by local unfolding, and thus relieves theconformational tension that keeps Pro213 in the cis state. Like aspring, Pro213 reverts to the more stable trans state, and thus thetimer is set. The lifetime of the ‘on’ state is encoded directly in the localsequence around Pro213. It ensures that the trans-to-cis reaction isappropriately slow and that G3P remains in the active conformationlong enough to complete infection by binding to TolA, the ultimatephage receptor. This simple setup of the proline timer presumablyreflects the limited possibilities for regulation outside the cell. Intra-

cellular proline timers are probably modulated by coupling withcovalent protein modifications, protein-protein interactions or cata-lysis by prolyl isomerases.

METHODSThe expression, purification and modification of the proteins used are

described in Supplementary Methods.

Folding experiments. The heat-induced unfolding transitions of the N1-N2

fragment of G3P and its variants were measured at a protein concentration of

4 mM in 100 mM potassium phosphate, pH 7.0, by the decrease in circular

dichroism at 230 nm and analyzed as described24. The time constants of

domain closing for the different variants of the N1-N2 fragment of G3P were

determined in vitro at 25 1C in 100 mM potassium phosphate, pH 7.0, by a

kinetic two-step unfolding assay for native molecules as described21. The

kinetics of the release of TolA-C during the refolding of 1 mM N1-N2 fragment

of G3P was measured as described in Supplementary Figure 2.

Competition between wild-type and mutated phage for the infection of

E. coli. For all experiments we used a derivative of the fCKCBS phage31, which

contains the gene for chloramphenicol acetyl transferase and G3P with a

modified linker between the N2 and C-terminal domains32. Phage were isolated

from E. coli culture medium by polyethylene glycol precipitation. To determine

the infectivity of wild-type and mutated phage 5 ml of phage suspension

(B1011 c.f.u. ml�1) was added to 495 ml E. coli XL1-Blue (A600 D 2) and

incubated at 37 1C for 30 min. The number of infected cells and thus the

number of infectious phage were determined by plating serial dilutions

on dYTcam agar. Suspensions of the different phage variants with

equal infectivity were mixed in a 1:1 ratio and 5 ml was added to 495 ml

E. coli XL1-Blue (A600 D 2) and incubated at 37 1C for 5 min. After

centrifugation the remaining phage was removed with the supernatant, and

the pelleted infected cells were resuspended in 5 ml dYTcam and propagated for

8 h. Four rounds of infection, phage propagation, preparation and reinfection

were carried out and the fraction of cells infected with the respective phage was

determined after each round. To distinguish between the different competing

phage variants we used colony PCR and primers specific for silent mutations

that were introduced into the codons for Ser172 and Ser173 or Ser207 and

Ser208, respectively, of G3P. After each round of infection 48 individual clones

were assayed.

Infection of F pili–deficient E. coli HB2156 cells. Escherichia coli HB2156

(provided by P. Holliger, MRC Centre for Protein Engineering)20 was infected

in the presence of 50 mM CaCl2 with native or denatured phage. Phage was

denatured by incubation at 65 1C or in 5.0 M GdmCl for 10 min. Phage

suspension (25 ml, B1014 c.f.u. ml�1) was added to 450 ml E. coli HB2156

(A600 D 2) and 25 ml 1 M CaCl2 and incubated at 37 1C for 5 min. Afterwards

the remaining phage was removed by centrifugation and infected cells were

washed and resuspended in 100 ml dYT. After incubation at 37 1C for 30 min,

serial dilutions were plated on dYTcam agar to determine the number of

infectious phage.

To determine the rate of domain closing in vivo, phage were denatured

in 5.0 M GdmCl for 10 min. Refolding was initiated by a ten-fold dilu-

tion with 100 mM potassium phosphate, pH 7.0, at 25 1C. Then, after various

times of refolding, 25 ml aliquots were withdrawn and added to 450 ml

E. coli HB2156 (A600 D 2) and 25 ml 1 M CaCl2. Infection and serial dilu-

tions to determine the number of infectious phage were carried out as

described above. To measure catalyzed refolding, the same procedure was

carried out with 10 or 50 nM cyclophilin 18 present in the refolding step at

0.5 M GdmCl.

Prolyl isomerization in a model peptide derived from G3P. The cis-trans

equilibration of the pentapeptide Ac-Ala-Gln-Pro-Pro-Val-OH was determined

after a fast ten-fold dilution from dry TFE plus 0.6 M LiCl into 20 mM

potassium phosphate. The integral of the amide proton resonance of Ala1 and

Gln2 at 8.3 p.p.m. was used for monitoring the time course of prolyl

isomerization. The measurements were taken at 25 1C in a Bruker DRX500

NMR spectrometer; the final peptide concentration was 5 mM in 20 mM

potassium phosphate, 60 mM LiCl, 10% (v/v) TFE, 10% (v/v) D2O, pH 5.0.

3

2.9

2.8

2.7

NM

R in

tens

ity (

a.u.

)

Time (s)0 300 600 900 1,200

Figure 4 The slow isomerization at Pro213 is determined by the local

sequence. Cis-trans equilibration of the pentapeptide Ac-AQPPV-OH, as

derived from the intensity of the amide proton resonances of Ala1 and

Gln2 after a ten-fold dilution of the peptide in dry TFE plus 0.6 M LiClwith 20 mM potassium phosphate, pH 7.0 (open circles). The continuous

line represents a double-exponential curve fit. The fast phase (t ¼ 23 s)

reflects the cis-trans-isomerization of the Pro3-Pro4 bond, the slow phase

(t ¼ 395 s) that of the Gln2-Pro3 bond. a.u., arbitrary units.

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Accession codes. BIND identifiers (http://bind.ca): 263477, 263478.

Note: Supplementary information is available on the Nature Structural & MolecularBiology website.

ACKNOWLEDGMENTSWe thank C. Unverzagt for help with peptide synthesis, M. Zeeb for help withNMR measurements, G. Fischer for a sample of cyclophilin 18, P. Holliger forE. coli HB2156 and C. Lehner, W. Schumann, B. Westermann and the membersof our group for comments on the manuscript. This work was supported by theDeutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 11 March; accepted 6 April 2005

Published online at http://www.nature.com/nsmb/

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