Structural Interconversion of Holin Transmembrane Domain I

structural Interconversion of Holin Transtissue layer Domain IStructural Interconversion of Holin Transmembrane Domain I is Dictated by a Single masterline A FRET-based Analysis and its Functional Importance in Pore Formation.Muralikrishna Lella, Soumya Kamilla, Vikas Jain,* and Radhakrishnan Mahalakshmi,*KEYWORDS (Word port BG_Keywords). If you are submitting your paper to a journal that requires keywords, provide signifi domiciliatet keywords to aid the reader in literature retrieval. rear Mycobacterial cell lysis during the lytic D29 bacteriophage infection is timed by perfect orchestration amongst/between components of the holin- deatholysin cassette. In Gram-negative bacteria, progressively accumulating holin in the interior membrane, retain in its inactive form by anti-holin, is triggered into active hole formation, termin utilize in the canonical host cell lysis. However, the molecular mechanism of regulation and fleshly basis of pore formation in the mycobacterial i nner 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 give of fluorescence resonance transfer measurements to demonstrate that the first transmembrane domain (TM1) of D29 holin undergoes a scroll -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, musical composition its rigid variants show accelerated lipid bilayer disruption. The biological implications of D29 holin structural fitting is presented as a holin self-regulatory mechanism and its implications are discussed in the context of data-driven peptide-based ther apeutics.The bleak host cell lysis step during bacteriophage infection is one of the around 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 manufacturing into holes that are large enough for endolysin release.1b,2 It is believed that ion leak through pinholes create 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,3Historically, genetics of the coliphages T4, , and 21 see been extensively investigated, and therefore our current understanding of holin position and regulation is generally arrive atd from the lysis effector S105 (or S2168) holin and the antiholin S107 (or S2171).2d,2g,4 Based on more recent functional word picture of members from eight holin superfamilies comprising several bacteriophages,5 we now know that the number of transmembrane -helical segments (TMSs) can straggle from 1-4 of this, the 3-TMS is widely prevalent.1b,6Despite conceptual and experimental advances in our understanding of holin function, actually few studies have translated these findings to the mechanism of holin regulation in mycobacteriophages (M). Currently, 4000 documented M species exist, of which 600 have been sequenced.7 Of particular interest is the lytic M D29, which is the predator for Mycobacterium tuberculosis, among different mycobacteria. M 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 M D29 genome, raising concerns on ho w this phage wins holin regulation.A previous finding from our laboratory demonstrated that the first transmembrane domain (TM1) of D29 M holin could undergo a conformational switch from a helical form to an across-the-board structure, and a centrally located Pro-Gly segment was important for such interconversion.9 This clear 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 M TM1 undergoes a helix -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 M.RESULTS AND DISCUSSIONASDASDMETHODSPeptide synthesis and labeling with fluorescent fixture probes. All peptides were synthesized employ Fmoc chemi stry 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 examination peptide was generated using the cleavage cocktail (TFA water phenol ethanedithiol thioanisole in the ratio 85552.52.5), 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 confirm by mass spectrometry. Labeling efficiency was calculated using designate peptide absorbance at fluorophore max and unlabeled labeled peptide ratios for all reactions were maintained at 1.00.5. enlarge are in the electronic supplementary information (ESI).Peptide folding and pear-shaped 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 estrus 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 space quartz cuvette at scan speeds of 100 nm/min. Data were corporate 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, on an individual basis at a ramp rate of 1 C/min. expatiate are provided in the ESI.Fluorescence and anisotropy measurements. Steady state Frster Resonance zip fastener Transfer (FRET) measu rements 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 11, 10.8, 10.6, 10.4, 10.2, 10. Data were normalized against Trp emission intensities and acceptor intensity at max were plotted (Alexa Fluor 350 max = 442 nm and dansyl chloride max = ergocalciferol nm).Anisotropy values were acquired using ex-max = 442 nm and em = 345 nm for both the labeled and unlabeled peptides. Lifetime measurements were carried out using time correlated whizz photon counting. Trp excitation was achieved at 292 nm using a pulsed LED and fluorescence decays were monitored at the 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 (i) and their respective amplitudes (i). The average lifetime was given as = i.i.12 expatiate are provided in the ESI.Pore formation measurement using planar lipid bilayers. nasty lipid membranes were generated using DiPhPC (diphytanoyl phosphatidylcholine) on a planar lipid bilayer workstation in which the membrane bilayer was painted across a 150 m aperture generated in the septum of a Delrin cup. A constant 10 mV voltage was employ 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 culmination 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(Word Style TE_Supporting_Information). Supporting Information. A brief sta tement in nonsentence format listing the contents of hearty supplied as Supporting Information should be included, ending with This material is uncommitted free of charge via the Internet at http//pubs.acs.org. For instructions on what should be included in the Supporting Information as well as how to prepare this material for publication, refer to the journals Instructions for Authors.ABBREVIATIONSCCR2, CC chemokine receptor 2 CCL2, CC chemokine ligand 2 CCR5, CC chemokine receptor 5 TLC, thin layer chromatography.REFERENCES(Word Style TF_References_Section). References are placed at the end of the manuscript. Authors are responsible for the accuracy and completeness of all references. Examples of the recommended formats for the various reference types can be found at http//pubs.acs.org/page/4authors/index.html. Detailed information on reference style can be found in The ACS Style Guide, available from Oxford Press.(1) (a) Smith, D. L. Young, R. J. 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