Structural Interconversion of Holin Transmembrane Domain I

Structural Interconversion of Holin Transmembrane Domain I is Dictated by a Single Proline: A FRET-based Analysis and its Functional Importance in Pore Formation.

• Muralikrishna Lella,^† Soumya Kamilla,^‡ Vikas Jain^‡,* and Radhakrishnan Mahalakshmi^†,*

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ABSTRACT: Mycobacterial cell lysis during the lytic D29 bacteriophage infection is timed by perfect orchestration amongst/between components of the holin-endolysin cassette. In Gram-negative 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 inner membrane by D29 holin, particularly in the nonexistence of an 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 abolished in the conformationally rigid substitution mutants containing Ala, ^DPro, or Aib. Using electrophysiology measurements coupled with calorimetric vesicle assays, we demonstrate that due to the conformational switch, native TM1 exhibits sluggish self-association in membrana, while its rigid variants show accelerated lipid bilayer disruption. The biological implications of D29 holin structural alteration is presented as a holin self-regulatory mechanism and its implications are discussed in the context of data-driven peptide-based therapeutics.

The fatal host cell lysis step during bacteriophage infection is one of the most precisely programmed events, coordinated by the mechanical membrane disruption by a hole-forming membrane protein termed holin and the peptidoglycan-degrading enzyme endolysin.¹ In the canonical holin-endolysin cassette, holin accumulates in the bacterial inner membrane and is retained in the inactive form until membrane depolarization drives holin assembly into holes that are large enough for endolysin release.^1b,2 It is believed that ion leak through pinholes formed in holin-enriched lipid rafts would result in local membrane depolarization, which would exponentially propagate throughout the bacterial inner membrane and result in the formation of >300 nm diameter holes.^2g,3

Historically, genetics of the coliphages T4, λ, and 21 have been extensively investigated, and therefore our current understanding of holin function and regulation is largely derived from the lysis effector S105 (or S²¹68) holin and the antiholin S107 (or S²¹71).^2d,2g,4 Based on more recent functional characterization of members from eight holin superfamilies comprising several bacteriophages,⁵ we now know that the number of transmembrane α-helical segments (TMSs) can vary from 1-4; of this, the 3-TMS is widely prevalent.^1b,6

Despite conceptual and experimental advances in our understanding of holin function, very few studies have translated these findings to the mechanism of holin regulation in mycobacteriophages (Mφ). Currently, >4000 documented Mφ species exist, of which >600 have been sequenced.⁷ 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 two transmembrane segments, typically observed in class II holins.⁸ However, an antiholin sequence is conspicuously missing in the Mφ D29 genome, raising concerns on how this phage achieves holin regulation.

A previous finding from our laboratory demonstrated that the first transmembrane domain (TM1) of D29 Mφ holin could undergo a conformational switch from a helical form to an extended structure, and a centrally located Pro-Gly segment was important for such interconversion.⁹ This opened further questions on the biophysical nature of such a conformational conversion, the functional implications during holin assembly and whether such interconversion did indeed possess any regulational role within the mycobacterial cell. In this study, we demonstrate that the D29 Mφ TM1 undergoes a helix <-> β-hairpin conversion that is abolished in Pro -> Ala/^DPro/Aib mutation. We also show that proline internally regulates assembly of TM1 in the membrane, and could potentially function as the ‘missing’ antiholin in D29 Mφ.

RESULTS AND DISCUSSION

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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 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.^9-10 Final peptide was 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. On-resin 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 λ_max­ and unlabeled : labeled peptide ratios for all reactions were maintained at ~1.0:0.5. Details are in the electronic supplementary information (ESI).

Peptide folding and circular dichroism experiments. Desired quantity of peptide in the powder form was dissolved in 100 mM LDAO (lauryldimethylamine oxide) or 100 mM DPC (n-dodeyclphosphocholine) micelles prepared in 50 mM sodium phosphate pH 7.2, and were subjected to repeated cycles of heating and vortexing to promote peptide folding.⁹ All biophysical experiments were carried out using 0.022-0.024 mM samples, unless otherwise specified. 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.^9,11 Thermal denaturation and recovery measurements were carried out between 5-95 °C and 95-5 °C, respectively at a ramp rate of 1 °C/min. Details are provided in the ESI.

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-molecular 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 were normalized against Trp emission intensities and acceptor intensity at λ_max­ were plotted (Alexa Fluor® 350 λ_max = 442 nm and dansyl chloride λ_max = 500 nm).

Anisotropy values were acquired using λ_ex-max = 442 nm and λ_em = 345 nm for both the labeled and unlabeled peptides. Lifetime measurements were carried out using time correlated single photon counting. Trp excitation was achieved at 292 nm using a pulsed LED and fluorescence decays were monitored at the λ_em-max for the respective samples (345 nm in LDAO; 347 nm in DPC; 355 nm in buffer). All data were fitted to a triple exponential decay to derive lifetimes (τ_i) and their respective amplitudes (α_i). The average lifetime was given as <τ> = Στ_i.α_i.¹²Details are provided in the ESI.

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 … mM sodium phosphate pH 7.2 containing 0.5 M KCl. 0.022-0.024 mM peptide was added to the cis chamber and electric 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 using the formula: [observed current in pA] / 10 mV = conductance in nS. Details are described in the ESI.

ASSOCIATED CONTENT

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ABBREVIATIONS

CCR2, CC chemokine receptor 2; CCL2, CC chemokine ligand 2; CCR5, CC chemokine receptor 5; TLC, thin layer chromatography.

REFERENCES

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