Structural Dynamics and Topology of the Inactive Form of S²¹ Holin in a Lipid Bilayer Using Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Abstract

The bacteriophage infection cycle plays a crucial role in recycling the world's biomass. Bacteriophages devise various cell lysis systems to strictly control the length of the infection cycle for an efficient phage life cycle. Phages evolved with lysis protein systems, which can control and fine-tune the length of this infection cycle depending on the host and growing environment. Among these lysis proteins, holin controls the first and ratelimiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time and concentration hence known as the simplest molecular clock. Pinholin S 21 is the holin from phage Φ21, which defines the cell lysis time through a predefined ratio of active pinholin and antipinholin (inactive form of pinholin). Active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously we reported the structural dynamics and topology of active pinholin S 21 68. Currently, there is no detailed structural study of the antipinholin using biophysical techniques. In this study, the structural dynamics and topology of antipinholin S 21 68 IRS in DMPC proteoliposomes is investigated using electron paramagnetic resonance (EPR) spectroscopic techniques. Continuous-wave (CW) EPR line shape analysis experiments of 35 different R1 side chains of S 21 68 IRS indicated restricted mobility of the transmembrane domains (TMDs), which were predicted to be inside the lipid bilayer when compared to the N-and C-termini R1 side chains. In addition, the R1 accessibility test performed on 24 residues using the CW-EPR power saturation experiment indicated that TMD1 and TMD2 of S 21 68 IRS were incorporated into the lipid bilayer where N-and C-termini were located outside of the lipid bilayer. Based on this study, a tentative model of S 21 68 IRS is proposed where both TMDs remain incorporated into the lipid bilayer and N-and C-termini are located outside of the lipid bilayer. This work will pave the way for the further studies of other holins using biophysical techniques and will give structural insights into these biological clocks in molecular detail.

Probing Structural Dynamics of Membrane Proteins Using Electron Paramagnetic Resonance Spectroscopic Techniques

“…However, both TMDs remain incorporated in the lipid bilayer for the inactive S 21 68 IRS form. This study was consistent with earlier CW-EPR spectral lineshape analysis and power saturation data obtained on pinholin S 21 [139,140].…”

“…Structural dynamic properties and topology of several biologically important protein systems have been investigated using SDSL EPR spectroscopic techniques. Some of the important systems include Escherichia coli ferric citrate transporter FecA, GM2 activator protein, pentameric ligand-gated ion channels (pLGICs), ABC cassette transporter MsbA, cytochrome C oxidase subunit IV (COX IV), the prokaryotic potassium channel KcsA, KCNE1, KCNQ1-voltage sensing domain (VSD), lactose permease protein, integrin β 1a , functional amyloid Obr2A, C99 domain of the amyloid precursor protein, bacteriorhodopsin, mechanosensitive channel of small conductance (MscS), KvAP voltage-sensing domain, phospholamban (PLB), and bacteriophase pinholin S 21 [78,81,87,119,124,[126][127][128][129][130][131][132][133][134][135][136][137][138][139][140][141].…”

“…A recent example of using SDSL CW-EPR spectroscopy is the study of the structural dynamics of the inactive form of pinholin S 21 [139]. Holins work as porters during the infected cell lysis process.…”

“…S 21 68 IRS delays the formation of the active dimer which is a prerequisite for the pinhole formation [143,144]. Ahammad et al extensively utilized CW-EPR spectra collected for spin-labeled antipinholin S 21 68 IRS to investigate the structural dynamics of the inactive form of pinholin S 21 68 IRS in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers [139]. The CW-EPR spectral line shape analysis of the R1 side-chain for 35 residue positions of S 21 68 IRS suggested that both transmembrane domains (TMDs) have more restricted mobility inside the lipid bilayers when compared to the N-and C-termini R1 side-chains.…”

“…Figure 3 shows the relative mobility of R1 side-chain (δ −1 ) as a function of residue positions of the primary sequence of S 21 68 IRS , membrane depth parameter (Φ) as a function of S 21 68 IRS residue positions in DMPC proteoliposomes at room temperature, and the proposed structural topology model of inactive pinholin S 21 68 IRS incorporated into lipid bilayers. The depth parameter (φ) was determined by analyzing the power saturation data obtained under three equilibrium sample conditions: samples were equilibrated with (1) lipid soluble paramagnetic relaxant (21% oxygen), (2) nitrogen gas as a control, and (3) water-soluble paramagnetic relaxant (2 mM NiEDDA) with a continuous purge of nitrogen gas using the Equation (1) [139].…”