RESUMEN
There have recently been advances in methods for detecting local secondary structures of membrane protein using electron paramagnetic resonance (EPR). A three pulsed electron spin echo envelope modulation (ESEEM) approach was used to determine the local helical secondary structure of the small hole forming membrane protein, S21 pinholin. This ESEEM approach uses a combination of site-directed spin labeling and 2H-labeled side chains. Pinholin S21 is responsible for the permeabilization of the inner cytosolic membrane of double stranded DNA bacteriophage host cells. In this study, we report on the overall global helical structure using circular dichroism (CD) spectroscopy for the active form and the negative-dominant inactive mutant form of S21 pinholin. The local helical secondary structure was confirmed for both transmembrane domains (TMDs) for the active and inactive S21 pinholin using the ESEEM spectroscopic technique. Comparison of the ESEEM normalized frequency domain intensity for each transmembrane domain gives an insight into the α-helical folding nature of these domains as opposed to a π or 310-helix which have been observed in other channel forming proteins.
Asunto(s)
Bacteriófagos/metabolismo , Espectroscopía de Resonancia por Spin del Electrón/métodos , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Proteínas Virales/química , Secuencia de Aminoácidos , Estructura Secundaria de ProteínaRESUMEN
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 S21 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 S21 are absent in the literature. In this study, the structural topology and conformations of active pinholin (S2168) and inactive antipinholin (S2168IRS) in DMPC (1,2-dimyristoyl-sn-glycero-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 S21. 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 S2168 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 S2168IRS form. This study demonstrates, for the first time, clear structural topology and conformational differences between the two forms of pinholin S21. 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.
Asunto(s)
Bacteriófagos , Proteínas Virales , Pared Celular , Espectroscopía de Resonancia por Spin del Electrón , Membrana Dobles de LípidosRESUMEN
Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes1, aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state.
Asunto(s)
Modelos Animales de Enfermedad , Células Precursoras de Granulocitos/patología , Mutación , Neutropenia/genética , Neutropenia/patología , Neutrófilos/patología , Animales , Candida albicans/inmunología , Candida albicans/patogenicidad , Linaje de la Célula , Proteínas de Unión al ADN/genética , Femenino , Humanos , Inmunidad Innata , Masculino , Ratones , Ratones Transgénicos , Neutropenia/congénito , Neutropenia/inmunología , Neutrófilos/inmunología , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Proteínas Virales/química , Bacteriófagos/metabolismo , Dimiristoilfosfatidilcolina/química , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/metabolismo , Marcadores de Spin , Proteínas Virales/metabolismoRESUMEN
The mechanism for the lysis pathway of double-stranded DNA bacteriophages involves a small hole-forming class of membrane proteins, the holins. This study focuses on a poorly characterized class of holins, the pinholin, of which the S21 protein of phage Ï21 is the prototype. Here we report the first in vitro synthesis of the wildtype form of the S21 pinholin, S2168, and negative-dominant mutant form, S21IRS, both prepared using solid phase peptide synthesis and studied using biophysical techniques. Both forms of the pinholin were labeled with a nitroxide spin label and successfully incorporated into both bicelles and multilamellar vesicles which are membrane mimetic systems. Circular dichroism revealed the two forms were both >80% alpha helical, in agreement with the predictions based on the literature. The molar ellipticity ratio [θ]222/[θ]208 for both forms of the pinholin was 1.4, suggesting a coiled-coil tertiary structure in the bilayer consistent with the proposed oligomerization step in models for the mechanism of hole formation. 31P solid-state NMR spectroscopic data on pinholin indicate a strong interaction of both forms of the pinholin with the membrane headgroups. The 31P NMR data has an axially symmetric line shape which is consistent with lamellar phase proteoliposomes lipid mimetics.