Conformational Differences Are Observed for the Active and Inactive Forms of Pinholin S²¹ Using DEER Spectroscopy

Abstract

Bacteriophages have evolved with an efficient host cell lysis mechanism to terminate the infection cycle and release the new progeny virions at the optimum time, allowing adaptation with the changing host and environment. Among the lytic proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time known as "holin triggering". Pinholin S 21 is a prototype holin of phage Φ21 which makes many nanoscale holes and destroys the proton motive force, which in turn activates the signal anchor release (SAR) endolysin system to degrade the peptidoglycan layer of the host cell and destruction of the outer membrane by the spanin complex. Like many others, phage Φ21 has two holin proteins: active pinholin and antipinholin. The antipinholin form differs only by three extra amino acids at the N-terminus; however, it has a different structural topology and conformation with respect to the membrane. Predefined combinations of active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously, the dynamics and topology of active pinholin and antipinholin were investigated (Ahammad et al. JPCB 2019, 2020) using continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. However, detailed structural studies and direct comparison of these two forms of pinholin S 21 are absent in the literature. In this study, the structural topology and conformations of active pinholin (S 21 68) and inactive antipinholin (S 21 68 IRS ) in DMPC (1,2-dimyristoyl-snglycero-3-phosphocholine) proteoliposomes were investigated using the four-pulse double electron−electron resonance (DEER) EPR spectroscopic technique to measure distances between transmembrane domains 1 and 2 (TMD1 and TMD2). Five sets of interlabel distances were measured via DEER spectroscopy for both the active and inactive forms of pinholin S 21 . Structural models of the active pinholin and inactive antipinholin forms in DMPC proteoliposomes were obtained using the experimental DEER distances coupled with the simulated annealing software package Xplor-NIH. TMD2 of S 21 68 remains in the lipid bilayer, and TMD1 is partially externalized from the bilayer with some residues located on the surface. However, both TMDs remain incorporated in the lipid bilayer for the inactive S 21 68 IRS form. This study demonstrates, for the first time, clear structural topology and conformational differences between the two forms of pinholin S 21 . This work will pave the way for further studies of other holin systems using the DEER spectroscopic technique and will give structural insight into these biological clocks in molecular detail.

The Functional Nanopore Screen: A Versatile High-throughput Assay to Study and Engineer Protein Nanopores inEscherichia coli

“…The capacity of the FuN Screen is demonstrated by dissecting the molecular features that underlie the insertion, activation and stability of S 21 68 nanopores. In this regard, early genetic and recent biophysical studies hypothesized that TMD2 is necessary and sufficient to form functional nanopores while the N-terminal affects the efficiency of activation [44][45][46][47][48] . Truncation experiments by means of the FuN Screen complemented by electrophysiological characterisation of chemically synthesized peptides confirm that TMD2 is necessary and sufficient to form nanopores in the inner membrane of E. coli albeit very transient ones.…”

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

“…Nitroxide spin labeling based SDSL DEER spectroscopy is a widely used biophysical technique for studying secondary, tertiary and quaternary structures, and conformational dynamics of a numerous membrane proteins [6,11,32,41,83,98,[104][105][106][107][108][109][110]. However, other spin labels including functionalized chelators of paramagnetic lanthanides (Gd III ), carbonbased radicals ((trityl), and metals such as copper (Cu II ) have been recently applied for DEER experiments for studying membrane proteins [111][112][113][114][115][116].…”

“…The use of an arbitrary waveform generator (AWG) to EPR has further improved data quality in DEER pulsed EPR experiments [121]. DEER distance restraints in association with the computational methods of molecular dynamics simulations have also been widely used to refine the structural properties of membrane proteins [32,78,110,[168][169][170].…”

“…A recent example of using nitroxide based SDSL DEER spectroscopy is the study of the conformational changes of the active and inactive forms of Pinholin S 21 [110] . Pinholin S21 is a class-II holin, encoded by the S21 gene of phage Φ21.…”

“…The antipinholin form differs from pinholin only by three extra amino acids at the N-terminus. Ahmmad et al applied the four pulse DEER technique to measure distances between transmembrane domains 1 and 2 (TMD1 and TMD2) to investigate the structural topology and conformations of active pinholin (S 21 68) and inactive antipinholin (S 21 68 IRS ) in DMPC (1,2dimyristoyl-snglycero-3-phosphocholine) proteoliposomes [110]. The researchers utilized five sets of interlabel DEER distances obtained for both the active and inactive forms of pinholin S 21 as experimental DEER distance restraints coupled with the simulated annealing software package Xplor-NIH to predict structural models of the active pinholin and inactive antipinholin.…”