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 rate-limiting 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 S²¹ 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 S²¹68. Currently, there is no detailed structural study of the antipinholin using biophysical techniques. In this study, the structural dynamics and topology of antipinholin S²¹68_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 S²¹68_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 S²¹68_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 S²¹68_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.

Figure 1.

Primary sequences of (A) wild type antipinholin S²¹71, (B) wild type active pinholin S²¹68, and (C) inactive analog of pinholin S²¹ (S²¹68_IRS). The amino acid positions studied by EPR spectroscopy are shown in blue. The inactive tag is color-coded red and indicating its incorporation site by the arrow. The helical region of TMD1 and TMD2 are indicated by blue and green boxes, respectively. (D) MTSL attached to Cys side chain of the protein via a disulfide bond (also known as R1). (E) Predicted topology of S²¹68_IRS where both TMDs remain incorporated in the lipid bilayer and TMD1 has not been externalized from the lipid bilayer.^,,,

Figure 2.

DLS spectrum for S²¹68_IRS F49R1 incorporated into the DMPC proteoliposomes. Signal intensity is plotted as a log function of the particle diameter.

Figure 3.

(A) Circular dichroism spectra for inactive pinholin S²¹68_IRS without spin-label (black) and S²¹68_IRS F24R1, with spin-label (blue). Spectra were signal-averaged for three scans. Mean residue molar ellipticity (MRE) is plotted against the incident radiation wavelength.

Figure 4.

Representative CW-EPR spectra with R1 situated at the indicated positions. Here, blue EPR spectra represent TMD1 residues, green represents TMD2, and black indicates loop and terminal regions. All spectra were normalized to the highest spectral intensity. CW-EPR spectra composed of multiple components were marked with (*).

Figure 5.

Relative mobility of R1 (δ⁻¹) as a function of residue positions of the primary sequence of S²¹68_IRS. A higher value of (δ⁻¹) indicates the higher mobility of the nitroxide spin-label at that corresponding position. Here, blue closed circles represent TMD1 residues, green represents TMD2, and black indicates loop and terminal regions.

Figure 6.

Rotational correlational time (τ) as a function of residue positions of the primary sequence of S²¹68_IRS. The same color code is used as in Figure 5.

Figure 7.

Representative CW-EPR power saturation curves of S²¹68_IRS in DMPC proteoliposomes. (A) A20R1 and (B) A67R1. The red triangle represents NiEDDA, green circle represents oxygen, and blue square represents nitrogen spectra with their fitted line from eq 2. The amplitudes of the first derivative m_I = 0 peak were plotted against the square root (SQRT) of the incident microwave (MW) power. (C) Color-coded primary sequence of S²¹68_IRS, where green residues are buried in the lipid bilayer and red residues are solvent-exposed based on the CW-EPR power saturation data.

Figure 8.

Calculated depth parameter (Φ) as a function of S²¹68_IRS residue positions in DMPC proteoliposomes. Positive (Φ) values (green) indicate that the R1 side chains are embedded inside the lipid bilayer and negative (Φ) values (red) indicate that the R1 side chains are solvent-exposed.

Figure 9.

Proposed structural topology of inactive pinholin S²¹68_IRS incorporated into a lipid bilayer. The red amino acids represent solvent-exposed and the green amino acids represent the lipid buried residues based on the CW-EPR power saturation data. Black letters were not studied by the EPR power saturation experiment. Red filled circles are the RYIRS tag used for the inactive conformation of pinholin S²¹.