Molecular Mechanism of Holin Transmembrane Domain I in Pore

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Molecular Mechanism of Holin Transmembrane Domain I in Pore Formation and Bacterial Cell Death Muralikrishna Lella, Soumya Kamilla, Vikas Jain, and Radhakrishnan Mahalakshmi ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00875 • Publication Date (Web): 23 Dec 2015 Downloaded from http://pubs.acs.org on January 2, 2016

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ACS Chemical Biology

Molecular Mechanism of Holin Transmembrane Domain I in Pore Formation and Bacterial Cell Death Muralikrishna Lella,1 Soumya Kamilla,2 Vikas Jain2,* and Radhakrishnan Mahalakshmi1,* 1

Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of

Science Education and Research, Bhopal, India-462023. 2

Microbiology and Molecular Biology Laboratory, Department of Biological Sciences, Indian

Institute of Science Education and Research, Bhopal, India-462023. Corresponding author: [email protected]; [email protected].

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Abstract: Bacterial cell lysis during bacteriophage infection is timed by perfect orchestration between components of the holin-endolysin cassette. In bacteria, progressively accumulating holin in the inner membrane, retained in its inactive form by anti-holin, is triggered into active hole formation, resulting in the canonical host cell lysis. However, the molecular mechanism of regulation and physical basis of pore formation in the mycobacterial cell membrane by D29 mycobacteriophage holin, particularly in the nonexistence of a known anti-holin, is poorly understood. In this study, we report, for the first time, the use of fluorescence resonance transfer measurements to demonstrate that the first transmembrane domain (TM1) of D29 holin undergoes a helix ↔ β-hairpin conformational interconversion. We validate that this structural malleability is mediated by a centrally positioned proline, and is responsible for controlled TM1 self-association in membrana, in presence of a proton gradient across the lipid membrane. We demonstrate that TM1 is sufficient for bacterial growth inhibition. The biological effect of D29 holin structural alteration is presented as a holin self-regulatory mechanism and its implications are discussed in the context of holin function. Keywords: Chameleonic peptide,

conformational

interconversion,

fluorescence, lipid micelle.

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membrane protein,

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INTRODUCTION The fatal host cell lysis step during bacteriophage infection is one of the most precisely programmed events, coordinated between the mechanical membrane disruption by a holeforming membrane protein termed holin and the peptidoglycan-degrading enzyme endolysin.1-3 In the canonical holin-endolysin cassette of λ phage, holin progressively accumulates in the bacterial inner membrane in a YidC– and signal recognition particle– independent manner,3-5 where it is believed to be retained in the inactive form by antiholin. The onset of membrane depolarization drives holin assembly into holes that are large enough for endolysin release.3-11 It is believed that local membrane depolarization at holin-enriched lipid rafts would exponentially propagate throughout the bacterial inner membrane and result in the formation of >300 nm diameter holes.4, 12-14 Our current understanding of holin function and regulation is derived primarily from holins and antiholins of coliphages.4, 9, 15, 16 We now know that the number of transmembrane (TM) α-helical segments for coliphage holins and antiholins can vary from 1-4.3, 17 However, whether similar structural and functional factors are involved in the mechanism of holin regulation in mycobacteriophages (Mϕ), has not been studied. Currently, >4000 Mϕ are documented, of which >600 have been sequenced.18 Of particular interest is the lytic Mϕ D29, which is the predator for Mycobacterium tuberculosis, among other mycobacteria. Mϕ D29 possesses a putative holin sequence coded by the gp11 gene, and is predicted to possess only two TM segments.19-21 Unlike coliphages, an antiholin sequence is conspicuously missing in the Mϕ D29 genome. This raises concerns on how D29 phage achieves holin regulation and timed host cell lysis. The molecular mechanism of regulation and physical basis of pore formation is poorly understood in the Mɸ system. A previous finding from our laboratory showed that the C-terminal domain of holin regulates host lysis.22 Our studies also demonstrated that the first transmembrane domain of D29 Mϕ holin could undergo a conformational switch from a helical form to an extended structure.23 This opened further questions on the regulational nature and functional implications of such a conformational conversion during holin assembly. We now report that the first TM (TM1) domain of D29 Mϕ holin is functionally important and sufficient to cause host cell death. We further establish that the central proline facilitates membrane association and internally regulates TM1 assembly in membrana.

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RESULTS AND DISCUSSION Conformational switch of TM1 driven by the central proline shows micelle-independent behavior. Prediction of the TM1 domain sequence of D29 Mϕ maps to 23 residues between E7I29.23 This is the ideal length for either a single-span transmembrane helix stabilized by internal hydrogen bonds, or an antiparallel β-sheet with sticky edge strands. We previously showed that a synthetic version of this helix, with the sequence KIRETLYYVGTLVPGILGIALIWGGIDANH2 (code: TM1PG), displays conformational interconversion between a helical structure at high DPR (detergent-to-peptide ratio) of LDAO (lauryldimethylamine oxide; critical micelle concentration (CMC) ≈ 1-2 mM), to a β-sheet rich structure in low DPR.23 This structural variation was facilitated by the presence of a central flexible Pro-Gly segment in the sequence (shown with bold letters in the sequence). To address the significance of this central proline, we replaced Pro in [...]LVPGIL[...] of TM1PG to alanine or α-aminoisobutyric acid (Aib, U) in peptides [...]LVAAIL[...] (code: TM1AA) and [...]LVUGIL[...] (code: TM1UG), respectively. We anticipated that Ala and Aib would promote the formation of a helical structure24 in the sequence. We also used D-proline25 to nucleate a β-hairpin structure in [...]LVDPGIL[...] (code: TM1dPG). Further, to examine the influence of peptide behavior in different micellar systems, we chose LDAO and DPC (n-dodecylphosphocholine; CMC = 1.1 mM). These detergents possess similar 12-C hydrophobic tails, micellar properties and support membrane protein structure,26 but differ in their headgroups, i.e., zwitterionic in LDAO versus polar in DPC. In addition, DPC is an excellent mimetic of the native phosphocholine membrane.27 We synthesized all peptides using Fmoc chemistry (Supplementary Information Methods; Supplementary Figure S1), and refolded the peptides in various DPRs of LDAO or DPC. We assessed the change in secondary structure content of the four TM1 analog peptides using far-UV circular dichroism (CD) spectroscopy (Supplementary Figure S2). The dependence of TM1 structure on temperature and varying concentrations of LDAO from 4 – 100 mM, or DPC from 5 – 50 mM (all detergent concentrations are above the CMC), is shown in Figure 1 and Supplementary Figures S2-S3. The CD value (ME208 or ME215) do not change considerably for TM1dPG and TM1UG, i.e., the two peptides display defined secondary structures of β-hairpin and helix, respectively, with temperature or DPR. However, TM1AA, which is structurally similar to TM1UG, shows a considerable loss in ME208, in both LDAO and DPC. Hence, TM1AA loses 4


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helicity, upon heating. This occurs due to aggregation of TM1AA upon heat-mediated structural collapse. In contrast, TM1PG shows a progressive gain in helical content with increasing LDAO (4-40 mM) or DPC (5-10 mM) concentration and temperature (Figure 1 and Supplementary Figures S2-S3). We thus conclude that the central proline facilitates conformational interconversion and DPR-dependent structural reversibility in TM1.

FRET experiments along with CD analysis confirm reversible Pro-regulated switch from helix to structured β-hairpin and not β-sheet oligomers. Steady state Förster Resonance Energy Transfer (FRET) measurement serves as an ideal tool to monitor spatial changes in the conformation that would indicate whether TM1PG undergoes a structural interconversion from helix→β-sheet, helix→β-hairpin, or remains as a kinked helix. We used the intrinsic fluorescence of the indole side chain of Trp as the donor and Alexa Fluor® 350 or dansyl chloride tagged to the N-terminus of the peptide as acceptor. The peptides were correspondingly named as A- or D-, respectively; for example, TM1PG with an Nterminal Alexa Fluor® 350 label is notated as A-TM1PG. We first confirmed that the presence of a bulky chromophore does not affect the conformational features of the four peptides, using extensive spectroscopic measurements (details are in the Supplementary Notes section of Supplementary Information; also see Supplementary Figures S4-S5). Ideally, the donor-acceptor FRET pair is separated by ~34 Å in a helical structure. However, in the β-hairpin conformation, they will be positioned in close proximity thus facilitating Trp fluorescence decay via FRET to Alexa Fluor® 350 or the dansyl group (Figure 2A). Hence, TM1dPG, with a β-hairpin structure, should display the highest FRET efficiency, while TM1AA and TM1UG, being helical in nature, should demonstrate lowest FRET. All intermediate structures that TM1PG can adopt, including the DPR-dependent structural conversion that we observe, should give rise to intermediate FRET efficiency. The results of FRET experiments are summarized in Figure 2C-D for LDAO and Supplementary Figure S8 for DPC. The dependence of FRET intensity on the micelle concentration (DPR) is shown in Figure 2B and in Supplementary Figures S6-S7. We observe an increase in FRET efficiency in TM1PG in lower DPR, which establishes lowering of the distance separating the donor-acceptor pair. This can only arise if TM1PG undergoes conversion from

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helix→β-hairpin, thus bringing the donor and the acceptor in close proximity. The conformational switch as judged from the FRET data for TM1PG thus concur with the CD data. We next confirmed that the observed FRET in TM1PG is indeed intramolecular, occurring due to the conformation interconversion, and not intermolecular; intermolecular FRET may happen in the case of peptide aggregates. We therefore measured the FRET signal from the labeled peptide while titrating it with the unlabeled sample (details in Supplementary Information). A non-linear dependence is anticipated if peptide aggregation influences our FRET results. Additional contributions from intermolecular FRET in peptide aggregates display a nonlinear dependence on the labeled peptide concentration (see Supplementary Notes section of Supplementary Information). We observe a linear dependence of FRET on the concentration of labeled peptide (Figure 2E-F and Supplementary Figure S8). Hence, TM1PG is largely monomeric when refolded in micelles, even in low DPR. The FRET data, when considered along with anisotropy and lifetime measurements for TM1PG (see Supplementary Figures S4-S5), thus allow us to conclude that TM1PG shows negligible aggregation or oligomerization in solution. The structural interconversion of TM1PG is between a helical and monomeric β-hairpin form.

Colorimetric and fluorescence capture analysis of peptide-membrane interaction suggests a superior association for a non-helical structure. In the mycobacterial cell, holin inserts itself into the inner membrane after synthesis in the cytosol,9 and its membrane insertion requires the TM domains.22 We therefore examined whether the peptide structure predisposed its ability for membrane association and insertion. To study membrane insertion rate, we employed a colorimetric assay using polydiacetylene (PDA) vesicles.28-31 PDA has been used as colorimetric sensors for various environmental stimuli.32, 33 In PDA vesicles, a color change from blue (λmax= 640 nm) for empty PDA vesicles, to red (λmax= 520 nm) is observed when the lipid is modified by external agents (see Supplementary Notes section of Supplementary Information for details of the mechanism of color change in PDA vesicles). In our experiments, a red color is obtained when the delocalized electrons in the conjugated framework are perturbed by peptide or DPC interaction (Figure 3A). The rate of this color change depends on the peptide insertion and association rate with PDA, as well as on lateral diffusion between PDA and the peptide (Supplementary Figure S9).

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We studied peptide-membrane association both in PDA vesicles and in PDA-doped DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) vesicles; DMPC is a widely accepted mimetic of lipid membranes,31, 34 and provides us with a mimetic of in vivo bilayers. Results of our colorimetric response for peptide insertion and diffusion in membranes are summarized for peptide-DPC interaction with PDA vesicles in Supplementary Figure S9a and for DMPC/PDA vesicles in Supplementary Figure S9b. While the membrane association step is rapid (completed in Ala/DPro/Aib mutants. We also show that proline internally regulates assembly of TM1 in the membrane. Through this study, we demonstrate that the initial formation of ‘leaky’ membrane occurs at very slow rate over several minutes, and can cause local disruption of the proton motive force. Such local membrane depolarization can result in rapid insertion of TM1 ensuing membrane disruption. While in membrana oligomerization is not affected in the β-hairpin structure, the membrane disruption rate is slower. Our studies with PDA vesicles suggest that all four peptides show similar association with the membrane, but display notable differences in their in membrana association process that causes membrane disruption (PLB measurements). The presence of the central Pro-Gly considerably distorts the formation of a well-defined helical structure, as observed previously for transmembrane helices.35 A consequence of structural malleability is likely to be the observed sluggishness in the pore formation and membrane disruption properties of TM1PG, as observed in our PLB measurements. This initial slow association may, therefore, be important for holin function. The well-structured perfect helical form of TM1 (TM1UG), obtained upon substitution of Pro to Aib in the peptide, shows rapid membrane insertion followed by immediate membrane disruption even in the presence of the proton motive force. The structural malleability and functional behavior of this peptide can readily be compared to the reports wherein Aibcontaining peptides have shown increased cell permeability and substantial antiviral activity.36 13



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This provides important insights into why a helical structure was not chosen for TM1, and why a central proline was conserved at this position across various Mϕ holins.23 Alanine possesses a moderately high propensity to adopt helical structure in membrane.37, 38 Our data with TM1AA clearly suggest that despite the central di-Ala segment, TM1AA does not achieve the helical content seen for TM1UG; in the latter case, Aib imposes helicity in the scaffold, due to its restricted ɸ-ψ values.24 The answer for the lowered helical content of TM1AA lies in the increased secondary structure propensity of the flanking amino acids for extended ɸ-ψ values.39 Indeed, the membrane break is abolished when the N-terminal residues are swapped with the Cterminal segment of TM1 (data not shown). Our data support non-ideal helical structure of TM1 as perfectly suited for rapid membrane insertion (as seen from our data for TM1PG in Figure 4), which is followed by the proline-mediated conformation switch in membrana (Figures 1-2). It is also worthwhile to discuss the behavior of TM1dPG, which adopts a pre-formed βhairpin in solution. Such β-hairpin structures can be assembled in the membrane and may be capable of complete membrane break. Similar assemblies can be related to the β-amyloid oligomers observed in the case of Alzheimer’s disease. These oligomers can form ion channellike arrangement that leads to increase in pore size through an increase in the number of assembled monomer.40 The β-barrel – like topology for these structures has been confirmed by molecular dynamics simulations,41,

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atomic force microscopy43 and planar bilayer44

experiments. However, in the case of TM1 peptides, the several instances of peptide aggregation seen for TM1dPG could have served as an evolutionary pressure against selecting a pre-formed hairpin structure for the first transmembrane domain. The native sequence of TM1PG is perfected for conformational interconversion, wherein the central PG segment, can facilely transition from a largely helical structure that supports membrane insertion to a β-hairpin structure for association and pore formation in the membrane. A pre-formed β-hairpin is likely to be avoided in the cytoplasm during synthesis, to prevent premature protein aggregation. As seen for our TM1dPG peptide, such structures are inactive and do not cause membrane disruption. Our in vitro studies with the synthetic peptide translate well to the in vivo results. Here again, expression of the first transmembrane domain of holin is sufficient to cause bacterial cell death. However, the process is slow, and proceeds over several hours. The presence of the C-terminal segment enhances the lysis time to below 30 min. Our recently reported data with in vivo expression of holin in E. coli further support this 14


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observation.22 Additionally, in line with our findings, a membrane association role for centrally placed proline residues has been seen in other TM segments.45 We propose concerted pore-formation mechanism from our findings (Figure 7), where TM1 adopts a largely helical structure prior to membrane insertion. Subsequently, structural malleability can give rise to controlled in membrana association of the monomeric peptide, with a concerted dilution in the local lipid concentration. Change in the lipid-to-protein ratio with increasing holin synthesis can now trigger a conformational switch in TM1, promoting pore formation. Such holin assembly and its sudden precipitation event leads to membrane depolarization, which causes membrane disruption through mechanisms that resemble holin activity in various coliphages.3-16 This is evident from the delayed onset in membrane break when a pH gradient is imposed in our PLB experiments. The depolarized state promotes rapid holin oligomerization in the membrane and leads to the observed membrane disruption (refer to Figure 4). It has not escaped our notice that the reversible nature of TM1 structure can provide holin with convenient regulation; the in vivo implication of this is currently unclear. Our studies clearly demonstrate for the first time that the physical process of pore formation by mycobacteriophage D29 holin is encoded in the first transmembrane domain of this protein. TM1, along with the C-terminal domain of holin, may regulate holin function and execute timed lysis of the mycobacterial host. The TM1 sequence from residues K4-A31 adopts a predicted β-hairpin structure by the Behairpred algorithm46 and shows a mixed β-sheet (for the N-terminal residues) and α-helical content (for C-terminal segment) by PSIPRED47 (data not shown). In context of the full-length holin protein, TM1 is predicted to adopt an α-helix by PSIPRED (data not shown). This suggests that the TM1 sequence is capable of adopting both secondary structures. Protein sequences that can adopt both α-helix and β-sheet structures have been observed earlier, and are called ‘chameleonic’ sequences. The longest sequence with this property in proteins is a stretch of 11residues,48 and in peptide systems, reversible 17-residue49 and 14-residue50 sequences composed entirely of natural amino acids have been reported. Other model systems that exhibit alternative structural interactions under the influence of metals,51, 52 pH,53 and light54 are known. To the best of our knowledge, this is the first study to report the longest peptide sequence (of 23 residues) that exhibits a reversible structural conversion from a well-folded helix to a β-hairpin structure

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within micelles. It would now be possible to engineer the TM1 sequence as a potent antimycobacterial peptide for bacterial cell death.

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METHODS Peptide synthesis and labeling with fluorescent probes. All peptides were synthesized using Fmoc chemistry on a Rink Amide AM resin with a 0.63 mmol/g loading capacity, using dry DMF as the medium. Deprotection of Fmoc was achieved using 20% piperidine and the progress of the reaction was monitored using Kaiser test and mass spectrometry.23, 55 Final peptides were generated using the cleavage cocktail (TFA: water: phenol: ethanedithiol: thioanisole in the ratio 85:5:5:2.5:2.5), followed by cold ether precipitation, and verified by mass spectrometry. Onresin labeling of the fluorophore (Alexa Fluor® 350 or dansyl chloride) at the N-terminal residue was achieved using HOBt or DIPEA in DMF. All labeling reactions were carried out at least twice and confirmed by mass spectrometry. Labeling efficiency was calculated using labeled peptide absorbance at fluorophore λmax and unlabeled: labeled peptide ratios for all reactions were maintained at ~1.0:0.5. Details are provided in the Supplementary Information. Peptide folding and circular dichroism experiments. Desired quantity of peptide in the powder was dissolved in 100 mM LDAO (lauryldimethylamine oxide; critical micelle concentration (CMC) ≈ 1-2 mM) or 100 mM DPC (n-dodeyclphosphocholine; CMC = 1.1 mM) micelles prepared in 50 mM sodium phosphate buffer pH 7.2, and were subjected to repeated cycles of heating and vortexing to promote peptide folding.23 All biophysical experiments were carried out using 0.022-0.024 mM samples, unless otherwise specified. The detergent concentrations were maintained above the CMC for both LDAO and DPC, to ensure that both detergents were predominantly in the micellar form during all our spectroscopic measurements. Quantification was achieved using a molar extinction coefficient of 8408 M-1 cm-1 at 280 nm. CD spectra were acquired in various micellar conditions at 25 °C, using a 1 mm path length quartz cuvette at scan speeds of 100 nm/min. Data were integrated over three acquisitions and converted to molar ellipticity values using reported methods.23,

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Thermal denaturation and

recovery measurements were carried out between 5-95 °C and 95-5 °C, respectively, at 5 °C steps. Details are in the Supplementary Information. Fluorescence and anisotropy measurements. Steady state Förster Resonance Energy Transfer (FRET) measurements were carried out using Trp excitation at 280 nm (2 nm slit width) and emission spectra were recorded between 295-550 nm (3 nm slit width). Inter- and intra-molecule FRET was demarcated by titrating unlabeled peptide into labeled peptide samples to achieve stepwise dilutions and final unlabeled : labeled ratios of 1:1, 1:0.8, 1:0.6, 1:0.4, 1:0.2, 1:0. Data 17



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were normalized against Trp emission intensities, and acceptor intensity at λmax were plotted (Alexa Fluor® 350 λmax= 442 nm and dansyl chloride λmax= 500 nm). Details are in the Supplementary Information. Colorimetric assay using PDA and DMPC/PDA vesicles. Polydiacetylene (PDA) and DMPC/PDA (DMPC is 1,2-dimyristoyl-sn-glycero-3-phosphocholine) vesicle preparation is provided in the Supplementary Information. Vesicles were prepared at a concentration of 1 mM in 2 mM Tris-HCl buffer pH 8.5. 0.1 mM peptide stocks in 50 mM DPC, 50 mM sodium phosphate buffer pH 7.2 was added to the vesicle at a 1:20 dilution. The % CR (percent colorimetric response) was calculated from absorbance values at 520 nm and 640 nm (see Supplementary Information for details). Direct fluorescence measurements were also carried out using Trp, and the steady state fluorescence intensity change as well as change in anisotropy was monitored. All data were fitted to exponential functions and rates were derived. Details are in the Supplementary Information. Pore formation measurement using planar lipid bilayers. Black lipid membranes were generated using DiPhPC (diphytanoyl phosphatidylcholine) on a planar lipid bilayer workstation in which the membrane bilayer was painted across a 150 µm aperture generated in the septum of a Delrin cup. A constant 10 mV voltage was applied in both cis and trans sides of the chamber, pre-filled with 25 mM sodium phosphate buffer pH 7.2 containing 0.5 M KCl. ~0.011 mM peptide was added to the cis chamber containing 1 ml buffer, and the measured current was recorded using a 50 Hz filter, sampling frequency of 10 kHz, and digitized. Opening and closing event frequency was calculated throughout the recording and converted to conductance (G) in nS, using the formula G = [observed current in pA] / 10 mV. To achieve a pH gradient across the membrane, 25 mM sodium phosphate buffer pH 6.2 was filled in the trans chamber; the other conditions were retained as described above. Details are in the Supplementary Information. In vivo expression, spot assay, and bacterial growth curve analysis. E. coli BL21(DE3) strain carrying the various holin constructs (both full-length and the TM1 region) in pET21b vector were grown at 37 °C in LB broth containing 100 µg/ml of ampicillin, with constant shaking at 200 rpm. The cells were induced for protein production by the addition of 1 mM IPTG, when the optical density of the culture at 600 nm (OD600) reached ~0.6. Bacterial growth was monitored with time by drawing cultures at specific time intervals and measuring OD600. Spot assays were carried out by plating 4 µl of culture at various time points after induction, at specific dilutions, 18


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on LB-agar plate containing 100 µg/ml ampicillin. Plates were imaged after an overnight incubation at 37 °C.

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ACKNOWLEDGEMENTS M.L. and S.K. thank IISER Bhopal for research fellowships. R.M. is a Wellcome Trust /DBT India Alliance Intermediate Fellow. This work is supported by the Department of Biotechnology award number BT/07/IYBA/2013-20 to V.J.

Notes The authors declare no conflict of interest.

Supporting Information Supporting Information available. This material can be accessed free of charge via the Internet.

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FIGURE LEGENDS Figure 1. Dependence of the secondary structure content on DPR. Changes in the far-UV CD spectrum (molar ellipticity at 208 nm; ME208, in deg cm2 dmol-1) were monitored at various temperatures (T, °C) and LDAO concentrations (see Supplementary Figure S3 for DPC data). Peptides with a well-defined secondary structure of α-helix (TM1UG) or β-sheet (TM1dPG) (right panels) do not display considerable changes in ME208 with temperature or DPR. However, TM1PG, which undergoes a DPR-dependent conformational interconversion from an extended to a helical form, shows an increase in secondary structure (higher negative ME208 values; top left panel), as the temperature or DPR is increased. On the contrary, TM1AA (bottom left graph) shows a loss in structure in low DPRs (4-20 mM LDAO; orange, yellow and green bar graphs). Behavior of all peptides is similar in both LDAO and DPC (Supplementary Figure S4), indicating that the nature of the micelles does not alter the conformational preference of these peptides. Figure 2. Monitoring conformational switch in TM1PG using FRET. (A) Schematic representation of conformational interconversion predicted to occur in TM1PG and the use of FRET to probe this conformational interconversion. Helical structure shows low FRET efficiency, due to larger donor (Trp, W) – acceptor (Alexa Fluor® 350; A) distance. (B) Experimentally observed increase in acceptor fluorescence (shown here for Alexa Fluor® 350) for A-TM1PG in various LDAO concentrations. Acceptor fluorescence intensity is normalized against Trp fluorescence. All other data are provided in Supplementary Figures. S6-S7. (C and D) Summary of the dependence of acceptor fluorescence (Alexa Fluor® 350, panel C; dansyl chloride, panel D) across the four peptides, with increasing LDAO concentration. Note how the highest FRET is observed in A-TM1dPG, which retains a beta-sheet structure across all DPRs. FRET efficiency is lowest for A-TM1AA and A-TM1UG. Only A-TM1PG shows a rapid increase in FRET efficiency as the peptide migrates from a helical structure in high LDAO concentration (100 mM) to a β-hairpin form in low (4 mM) LDAO. (E and F) Linear dependence of FRET efficiency on the concentration of labeled peptide (Alexa Fluor® 350, panel E; dansyl chloride, panel F) in 4 mM LDAO, suggesting that the observed FRET is not a result of peptide aggregation. Aggregates would give rise to intermolecular FRET, which will display a non-linear 26


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dependence to the labeled peptide concentration. See Supplementary Notes section of Supplementary Information for details. Data for 100 mM LDAO is provided in Supplementary Figure S8. Also see text for details. Figure 3. Membrane association rates of TM1 studied colorimetrically using PDA. (A) Representative plate images showing TM1PG interacting with PDA vesicles in DPC, monitored over 24 h. Empty PDA vesicles (first row) retain their blue color and remain unchanged, while the addition of TM1PG induces a color change from blue to red, with time. The rate of change of color is faster at higher DPC concentrations. (B and C) Effect of TM1PG addition to PDA vesicles monitored using tryptophan anisotropy (B) and fluorescence (C) measurements. Complete data are provided in Supplementary Figures S9-S11. The initial anisotropy of TM1PG in DPC (concentration provided above each panel) is shown as blue square. The arrow indicates the sudden reduction in Trp anisotropy when TM1PG is added to PDA vesicles. Trp anisotropy and fluorescence increase gradually with time (shown in B and C). The rate of increase was obtained by fitting anisotropy and fluorescence data to an exponential function (red solid lines). Two rates (k1 and k2) were obtained from fits to a double exponential function. In some cases, the data could be explained by a single exponential function and one rate (k) was obtained. The rates are summarized in (D) and (E) for PDA and (F) and (G) for DMPC/PDA vesicles. (D) and (F) are rates from Trp anisotropy, while (E) and (G) are rates from fluorescence intensity. Rates were the fastest for TM1PG (D) in PDA vesicles. Similarly, fast association rate was seen for TM1PG and TM1dPG (in 10 mM DPC) in DMPC/PDA vesicles. The corresponding rates derived from fluorescence are similar for all peptides. Hence, association with the membrane constrains the conformational flexibility of the indole side chain without affecting its local environment. This occurs when Trp is positioned near the phosphocholine headgroup or lipid solvated at the membrane interface, after TM1 inserts in the membrane. Figure 4. Planar lipid bilayer (PLB) experiments establish TM1 oligomerization and pore formation in membrana. (A) Schematic representation of peptide association into the membrane and pore formation. Peptide refolded in micelles is added to the cis chamber. It then inserts into the membrane and forms pores. Addition of empty micelles at similar concentrations do not show any change in the observed current. (B) TM1PG exhibits association with the membrane 27



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and leads to membrane disruption within seconds. A single complete recording is shown here, as an example (Also see Supplementary Figure S12). In the recording, a current of ~0 pA indicates a stable membrane at +10 mV. Increase in current indicates membrane leakage, with a current of 1000 pA corresponding to complete membrane disruption in our experimental set-up. (C) Four illustrative instances of TM1PG association with the membrane. Each peptide-membrane and/or peptide-peptide association within the membrane that results in increased membrane permeation by ions is marked by discreet stepwise increase in the measured current from 0 pA – 1000 pA. (D) Frequency of occurrence of step sizes of various conductance (G = [current]/[voltage]). Here, voltage = 10 mV. High frequencies for TM1PG were obtained at conductance of ~0.4 nS and a minor instance centered at ~0.8 nS. These were derived from fits of the conductance bins (grey bars; fits are shown as solid lines). The highest frequencies for the other peptides are conductance values of ~0.5 nS and ~1.3 nS for TM1dPG, ~0.3 nS and ~1.3 nS for TM1AA and ~0.9 nS and ~2.4 nS for TM1UG. (E) Membrane association and disruption times for the various peptides in the absence of a pH gradient. Membrane association (0-200 pA) of TM1 is largely similar across the four peptides (graph on left), with a marginally slower rate for TM1PG. The time taken to achieve membrane disruption (200-1000 pA), highlighted as the rapid break in the planar bilayer, shows a clear demarcation for the four TM1 analogs (middle graph). The overall process follows the order (slowest) TM1PG > TM1dPG > TM1AA > TM1UG (fastest) at a holding voltage of +10 mV, and is most diverse for TM1PG (~75-600 s). Figure 5. Planar lipid bilayer (PLB) experiments for TM1 pore formation in the presence of a pH gradient. (A) Schematic representation of PLB experimental set-up, in which the peptide was added to the cis chamber filled with buffer of pH 7.2. To mimic the initial stage of holin assembly in the plasma membrane of the bacterial cell, we have maintained a pH gradient between the cis (pH 7.2) and trans (pH 6.2) chambers. (B), (C) and (D) show representative recordings for peptides TM1PG, TM1AA and TM1UG, respectively. In the presence of a pH gradient, peptide oligomerization within the membrane is slow, and delayed membrane break is observed (compare (B) with Figure 4B). This suggests that when there is proton gradient between the intracellular space and periplasmic space, holin assembly in the plasma membrane is very sluggish. (E) Representative data for TM1AA highlighting instances where membrane disruption was not observed. Accumulation of the TM1AA peptide in membrana increases the 28


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observed current (see recording between 20-27 min). However, instead of complete membrane disruption and increase in current to 1000 pA, the membrane is re-formed (indicated by arrow). This may occur due to dissociation of the membrane-bound peptide from the membrane. (F) Membrane association and disruption time measured (as described in Figure 4E) for the TM1 peptides, in the presence of a pH gradient. When a proton gradient is maintained between the two chambers, the peptide-membrane association, and subsequent membrane break, is drastically slowed, from seconds to minutes. Distribution of the time T1 taken for membrane association of each peptide and to attain a current of 200 pA is shown in the left panel. Time (T) recorded for all TM1 variants to disrupt the DiPhPC membrane is shown in the right panel. TM1dPG shows inconsistent results (indicated with an arrow in both graphs). In some experiments, we did not observe any peptide-membrane association; instead, we observed precipitation of the peptide in the cis chamber. This may be due to lowered peptide-membrane association affinity in the presence of a pH gradient. Additionally, in the case of TM1AA, ~40% of membrane association events (T1) did not result in membrane disruption (T). Instead, we obtained membrane reformation, as illustrated in (E). The number of T1 and T events observed is indicated above each graph. In all experiments, the applied voltage was maintained at 10 mV. Figure 6. Effect of full-length holin and its TM1 region on bacterial growth and survival. The effect of the wild-type PG and the AA mutant of both HolFL (full-length holin) and TM1* were studied in vivo by expression in E. coli BL21(DE3) cells. (A) Bacterial growth was monitored by measuring the optical density of the culture at 600 nm. The recording was carried out at every 20 min, after addition of IPTG into the culture medium at time zero. Uninduced (UI) controls are also shown for comparison. Standard deviation was calculated from three independent experiments. Further, spot assay (shown in (B)) was carried out with these constructs. Bacterial cells expressing various proteins were spotted at three different dilutions (indicated on the left of the agar plate picture) and at different time points (in minutes, mentioned on the top of the panels). The constructs are indicated (top) with the respective mutations (left). Figure 7. Model for pore formation by Mϕ D29 holin TM1. The first transmembrane domain of holin is translocated to the bacterial inner membrane, after its synthesis in the cytosol. Upon sufficient holin accumulation, in membrana oligomerization of TM1 ensues. The local change in 29



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lipid-to-protein ratio triggers the conformational switch of holin, and a β-hairpin structure is formed by TM1. Further association of TM1 β-hairpins in the membrane gives rise to the formation of pores that cause membrane depolarization and triggers lysis of the host cell. Data from in vivo measurements of HolFL constructs (see Figure 6) clearly indicates that the Cterminal domain also has a regulatory role and functions by enhancing pore formation or stabilizing the oligomeric structure.

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Figure 1. Dependence of secondary structure on DPR. 60x54mm (300 x 300 DPI)



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Figure 2. Monitoring conformational switch in TM1PG using FRET. 76x42mm (300 x 300 DPI)


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Figure 3. Membrane association rates of TM1 studied colorimetrically. 80x46mm (300 x 300 DPI)



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Figure 4. PLB experiments establish TM1 oligomerization and pore formation in membrana. 127x161mm (300 x 300 DPI)


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Figure 5. PLB experiments for TM1 pore formation in the presence of a pH gradient. 63x29mm (300 x 300 DPI)



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Figure 6. Effect of full-length holin and its TM1 region on bacterial growth and survival. 87x114mm (300 x 300 DPI)


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Figure 7. Model for pore formation by Mφ D29 holin TM1. 22x7mm (300 x 300 DPI)



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TOC graphic 27x8mm (600 x 600 DPI)


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Molecular Mechanism of Holin Transmembrane Domain I in Pore

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