Continuous Wave Electron Paramagnetic Resonance Spectroscopy Reveals the Structural Topology and Dynamic Properties of Active Pinholin S2168 in a Lipid Bilayer

Abstract

Pinholin S²¹68 is an essential part of the phage Φ21 lytic protein system to release the virus progeny at the end of the infection cycle. It is known as the simplest natural timing system for its precise control of hole formation in the inner cytoplasmic membrane. Pinholin S²¹68 is a 68 amino acid integral membrane protein consisting of two transmembrane domains (TMDs) called TMD1 and TMD2. Despite its biological importance, structural and dynamic information of the S²¹68 protein in a membrane environment is not well understood. Systematic site-directed spin labeling and continuous wave electron paramagnetic resonance (CW-EPR) spectroscopic studies of pinholin S²¹68 in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) proteoliposomes are used to reveal the structural topology and dynamic properties in a native-like environment. CW-EPR spectral line-shape analysis of the R1 side chain for 39 residue positions of S²¹68 indicates that the TMDs have more restricted mobility when compared to the N- and C-termini. CW-EPR power saturation data indicate that TMD1 partially externalizes from the lipid bilayer and interacts with the membrane surface, whereas TMD2 remains buried in the lipid bilayer in the active conformation of pinholin S²¹68. A tentative structural topology model of pinholin S²¹68 is also suggested based on EPR spectroscopic data reported in this study.

Figure 1.

(A) Primary sequence of S²¹68, boxes indicate TMD1 and TMD2. The amino acid positions studied by EPR spectroscopy are shown in blue. (B) Predicted topology of S²¹68 is adapted from the literature.^,,, TMD1 completely externalizes from the lipid bilayer and remains in the periplasm or partially externalizes and stays on the surface of the lipid bilayer, where TMD2 remains in the lipid bilayer. (C) R1 side chain shown in the dotted box which is attached to the protein through the disulfide bond of a Cys residue.

Figure 2.

CD spectrum of pinholin S²¹68 G40R1 in DMPC proteoliposomes was collected at pH 7 and 298 K. Mean residue ellipticity is plotted against incident radiation wavelength.

Figure 3.

CW-EPR spectra of R1 side chains attached at indicated positions by replacing the native amino acid with Cys. All spectra were normalized to the highest spectral intensity. (A) EPR spectra from the N-terminal and TMD1 of pinholin S²¹68. (B) EPR spectra from the loop to the C-terminal of pinholin S²¹68 including TMD2. CW-EPR spectra composed of multiple components were marked with (*).

Figure 4.

EPR mobility analysis of the R1 side chain of S²¹68, calculated from the inverse width of the central resonance line (δ⁻¹). The larger δ⁻¹ value indicates increasing motion.

Figure 5.

Scaled mobility as a function of the residue position for pinholin S²¹68.

Figure 6.

Rotational correlation time (τ) of SL pinholin S²¹68 in DMPC proteoliposomes as a function of the residue position.

Figure 7.

Representative CW-EPR power saturation curves of S²¹68 in DMPC proteoliposomes. (A) A12R1 and (B) Q26R1 are in TMD1 and (C) L50R1 is in TMD2. Red triangle represents NiEDDA, green circle represents oxygen, and blue square represents nitrogen spectra with their fitted line from eq 3. The amplitudes of first derivative m_I = 0 peak were plotted against the square root of the incident microwave power. (D) Color-coded primary sequence of S²¹68, where green residues are buried in the lipid bilayer and red residues are solvent-exposed based on CW-EPR power saturation data. Black residues are not studied by the CW-EPR power saturation experiment.

Figure 8.

Membrane depth parameter (Φ) as a function of S²¹68 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 topology of active pinholin S²¹68 after partial externalization of TMD1 from the lipid bilayer. The red amino acids represent solvent-exposed and the green amino acids represent the lipid buried residues based on CW-EPR power saturation data. Black letters are for those residues which were not studied via EPR power saturation.