YidC is a membrane protein that facilitates the insertion of newly synthesized proteins into lipid membranes. Through YidC, proteins are inserted into the lipid bilayer via the SecYEG-dependent complex. Additionally, YidC functions as a chaperone in protein folding processes. Several studies have provided evidence of its independent insertion mechanism. However, the mechanistic details of the YidC SecY-independent protein insertion mechanism remain elusive at the molecular level. This study elucidates the insertion mechanism of YidC at an atomic level through a combination of equilibrium and non-equilibrium molecular dynamics (MD) simulations. Different docking models of YidC-Pf3 in the lipid bilayer were built in this study to better understand the insertion mechanism. To conduct a complete investigation of the conformational difference between the two docking models developed, we used classical molecular dynamics simulations supplemented with a non-equilibrium technique. Our findings indicate that the YidC transmembrane (TM) groove is essential for this high-affinity interaction and that the hydrophilic nature of the YidC groove plays an important role in protein transport across the cytoplasmic membrane bilayer to the periplasmic side. At different stages of the insertion process, conformational changes in YidC’s TM domain and membrane core have a mechanistic effect on the Pf3 coat protein. Furthermore, during the insertion phase, the hydration and dehydration of the YidC’s hydrophilic groove are critical. These results demonstrate that Pf3 coat protein interactions with the membrane and YidC vary in different conformational states during the insertion process. Finally, this extensive study directly confirms that YidC functions as an independent insertase.
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The allosteric mechanism leading to an open-groove lipid conductive state of the TMEM16F scramblase
TMEM16F is a Ca2+-activated phospholipid scramblase in the TMEM16 family of membrane proteins. Unlike other TMEM16s exhibiting a membrane-exposed hydrophilic groove that serves as a translocation pathway for lipids, the experimentally determined structures of TMEM16F shows the groove in a closed conformation even under conditions of maximal scramblase activity. It is currently unknown if/how TMEM16F groove can open for lipid scrambling. Here we describe the analysis of ~400 µs all-atom molecular dynamics (MD) simulations of the TMEM16F revealing an allosteric mechanism leading to an open-groove, lipid scrambling competent state of the protein. The groove opens into a continuous hydrophilic conduit that is highly similar in structure to that seen in other activated scramblases. The allosteric pathway connects this opening to an observed destabilization of the Ca2+ion bound at the distal site near the dimer interface, to the dynamics of specific protein regions that produces the open-groove state to scramble phospholipids.
more » « less- Award ID(s):
- 1741057
- PAR ID:
- 10480951
- Publisher / Repository:
- PubMed
- Date Published:
- Journal Name:
- Communications Biology
- Volume:
- 5
- Issue:
- 1
- ISSN:
- 2399-3642
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Lipid oxidation by reactive oxygen species (ROS) provide several different oxidation products that have been implicated in inflammatory responses. Ground state atomic oxygen [O(3P)] is produced by the photodeoxygenation of certain heterocyclic oxides and has a reactivity that is unique from other ROS. Due to the reactive nature of O(3P), the site of O(3P)‐generation is expected to influence the products in heterogenous solutions or environments. In this work, the oxidation of low‐density lipoprotein (LDL) by lipids with covalently bound O(3P)‐photoprecursors was compared to more hydrophilic O(3P)‐photoprecursors. Lipid oxidation products were quantified after Bligh‐Dyer extraction and pentafluorobenzyl bromide (PFB) derivatization by GC–MS. Unlike the more hydrophilic O(3P)‐photoprecursors, the oxidation of LDL during the irradiation of lipid‐(O3P)‐photoprecursor conjugates showed little quenching by the addition of the O(3P)‐scavenging sodium allyl sulfonate. This indicated that lipophilic O(3P)‐photoprecursors are expected to generate lipid oxidation products where other more hydrophilic O(3P)‐photoprecursors could be quenched by other reactive groups present in solution or the environment.
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ABSTRACT G‐protein‐coupled receptors (GPCRs) constitute one of the most prominent families of integral membrane receptor proteins that mediate most transmembrane signaling processes. Malfunction of these signal transduction processes is one of the underlying causes of many human pathologies (Parkinson's, Huntington's, heart diseases, etc), provoking that GPCRs are the largest family of druggable proteins. However, these receptors have been targeted traditionally by orthosteric ligands, which usually causes side effects due to the simultaneous targeting of homologous receptor subtypes. Allosteric modulation offers a promising alternative approach to circumvent this problematic and, thus, comprehending its details is a most important task. Here we use the Cannabinoid type‐1 receptor (CB1R) in trying to shed light on this issue, focusing on positive allosteric modulation. This is done by using the protein‐dipole Langevin‐dipole (PDLD) within the linear response approximation (LRA) framework (PDLD/S‐2000) along with our coarse‐grained (CG) model of membrane proteins to evaluate the dissociation constants (
K Bs) and cooperativity factors (αs) for a diverse series of CB1R positive allosteric modulators belonging to the 2‐phenylindole structural class, considering CP55940 as an agonist. The agreement with the experimental data evinces that significantly populated allosteric modulator:CB1R and allosteric modulator:CP55940:CB1R complexes have been identified and characterized successfully. Analyzing them, it has been determined that CB1R positive allosteric modulation lies in an outwards displacement of transmembrane α helix (TM) 4 extracellular end and in the regulation of the range of motion of a compound TM7 movement for binary and ternary complexes, respectively. In this respect, we achieved a better comprehension of the molecular architecture of CB1R positive allosteric site, identifying Lys1923.28and Gly1943.30as key residues regarding electrostatic interactions inside this cavity, and to rationalize (at both structural and molecular level) the exhibited stereoselectivity in relation to positive allosteric modulation activity by considered CB1R allosteric modulators. Additionally, putative/postulated allosteric binding sites have been screened successfully, identifying the real CB1R positive allosteric site, and most structure–activity relationship (SAR) studies of CB1R 2‐phenylindole allosteric modulators have been rationalized. All these findings point out towards the predictive value of the methodology used in the current work, which can be applied to other biophysical systems of interest. The results presented in this study contribute significantly to understand GPCRs allosteric modulation and, hopefully, will encourage a more thorough exploration of the topic. -
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SUMMARY Extracellular ATP (eATP) is known to act as a danger signal in both plants and animals. In plants, eATP is recognized by the plasma membrane (PM)‐localized receptor P2K1 (LecRK‐I.9). Among the first measurable responses to eATP addition is a rapid rise in cytoplasmic free calcium levels ([Ca2+]cyt), which requires P2K1. However, the specific transporter/channel proteins that mediate this rise in [Ca2+]cytare unknown. Through a forward genetic screen, we identified an Arabidopsis ethylmethanesulfonate (EMS) mutant impaired in the [Ca2+]cytresponse to eATP. Positional cloning revealed that the mutation resided in the
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Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔ
G ). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 μs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔG , we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR’s G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule–derived ΔΔG for mutant L223A (−2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔG for mutant V217A was 2.2-fold larger (−2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val217and a natively bound squalene lipid, highlighting the contribution of membrane protein–lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔG for a fully folded membrane protein embedded in its native bilayer.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s42003-022-03930-8
