Network analysis reveals how lipids and other cofactors influence membrane protein allostery

AbstractMany membrane proteins are modulated by external stimuli, such as small molecule binding or change in pH, transmembrane voltage or temperature. This modulation typically occurs at sites that are structurally distant from the functional site. Revealing the communication, known as allostery, between these two sites is key to understanding the mechanistic details of these proteins. Residue interaction networks of isolated proteins are commonly used to this end. Membrane proteins, however, are embedded in a lipid bilayer which may contribute to allosteric communication. The fast diffusion of lipids hinders direct use of standard residue interaction networks. Here, we present an extension which includes cofactors such as lipids and small molecules in the network. The novel framework is applied to three membrane proteins: a voltage-gated ion channel (KCNQ1), a G-protein coupled receptor (GPCR - β2 adrenergic receptor) and a pH-gated ion channel (KcsA). Through systematic analysis of the obtained networks and their components, we demonstrate the importance of lipids for membrane protein allostery. Finally, we reveal how small molecules may stabilize different protein states by allosterically coupling and decoupling the protein from the membrane.

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Interplay between hydrophobicity and the positive-inside rule in determining membrane-protein topology

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1605888113 ◽

2016 ◽

Vol 113 (37) ◽

pp. 10340-10345 ◽

Cited By ~ 22

Author(s):

Assaf Elazar ◽

Jonathan Jacob Weinstein ◽

Jaime Prilusky ◽

Sarel Jacob Fleishman

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Structure Prediction ◽

In Vitro Selection ◽

Prediction Algorithm ◽

Systematic Analysis ◽

Protein Topology ◽

Functional Sites ◽

Membrane Protein Topology

The energetics of membrane-protein interactions determine protein topology and structure: hydrophobicity drives the insertion of helical segments into the membrane, and positive charges orient the protein with respect to the membrane plane according to the positive-inside rule. Until recently, however, quantifying these contributions met with difficulty, precluding systematic analysis of the energetic basis for membrane-protein topology. We recently developed the dsTβL method, which uses deep sequencing and in vitro selection of segments inserted into the bacterial plasma membrane to infer insertion-energy profiles for each amino acid residue across the membrane, and quantified the insertion contribution from hydrophobicity and the positive-inside rule. Here, we present a topology-prediction algorithm called TopGraph, which is based on a sequence search for minimum dsTβL insertion energy. Whereas the average insertion energy assigned by previous experimental scales was positive (unfavorable), the average assigned by TopGraph in a nonredundant set is −6.9 kcal/mol. By quantifying contributions from both hydrophobicity and the positive-inside rule we further find that in about half of large membrane proteins polar segments are inserted into the membrane to position more positive charges in the cytoplasm, suggesting an interplay between these two energy contributions. Because membrane-embedded polar residues are crucial for substrate binding and conformational change, the results implicate the positive-inside rule in determining the architectures of membrane-protein functional sites. This insight may aid structure prediction, engineering, and design of membrane proteins. TopGraph is available online (topgraph.weizmann.ac.il).

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TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

Biochemical Journal ◽

10.1042/bcj20190359 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3241-3260

Author(s):

Sindhu Wisesa ◽

Yasunori Yamamoto ◽

Toshiaki Sakisaka

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Membrane Protein ◽

Mammalian Cells ◽

Coiled Coil ◽

Transmembrane Domains ◽

Domain Family ◽

Coiled Coil Domain ◽

U2os Cells ◽

Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

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Simulation studies of the interactions between membrane proteins and detergents

Biochemical Society Transactions ◽

10.1042/bst0330910 ◽

2005 ◽

Vol 33 (5) ◽

pp. 910-912 ◽

Cited By ~ 6

Author(s):

P.J. Bond ◽

J. Cuthbertson ◽

M.S.P. Sansom

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Self Assembly ◽

Kinetic Mechanism ◽

Coarse Grained ◽

Simulation Studies ◽

High Resolution Data ◽

Biologically Relevant ◽

Structure And Dynamics ◽

Detergent Interactions

Interactions between membrane proteins and detergents are important in biophysical and structural studies and are also biologically relevant in the context of folding and transport. Despite a paucity of high-resolution data on protein–detergent interactions, novel methods and increased computational power enable simulations to provide a means of understanding such interactions in detail. Simulations have been used to compare the effect of lipid or detergent on the structure and dynamics of membrane proteins. Moreover, some of the longest and most complex simulations to date have been used to observe the spontaneous formation of membrane protein–detergent micelles. Common mechanistic steps in the micelle self-assembly process were identified for both α-helical and β-barrel membrane proteins, and a simple kinetic mechanism was proposed. Recently, simplified (i.e. coarse-grained) models have been utilized to follow long timescale transitions in membrane protein–detergent assemblies.

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Network Analysis Identifies Drug Targets and Small Molecules to Modulate Apoptosis Resistant Cancers

Cancers ◽

10.3390/cancers13040851 ◽

2021 ◽

Vol 13 (4) ◽

pp. 851

Author(s):

Samreen Fathima ◽

Swati Sinha ◽

Sainitin Donakonda

Keyword(s):

Cell Death ◽

Small Molecules ◽

Drug Targets ◽

Apoptotic Cell ◽

Colon Adenocarcinoma ◽

Interaction Networks ◽

Small Cell Lung ◽

Protein Protein Interaction ◽

Cell Death Genes ◽

Death Genes

Programed cell death or apoptosis fails to induce cell death in many recalcitrant cancers. Thus, there is an emerging need to activate the alternate cell death pathways in such cancers. In this study, we analyzed the apoptosis-resistant colon adenocarcinoma, glioblastoma multiforme, and small cell lung cancers transcriptome profiles. We extracted clusters of non-apoptotic cell death genes from each cancer to understand functional networks affected by these genes and their role in the induction of cell death when apoptosis fails. We identified transcription factors regulating cell death genes and protein–protein interaction networks to understand their role in regulating cell death mechanisms. Topological analysis of networks yielded FANCD2 (ferroptosis, negative regulator, down), NCOA4 (ferroptosis, up), IKBKB (alkaliptosis, down), and RHOA (entotic cell death, down) as potential drug targets in colon adenocarcinoma, glioblastoma multiforme, small cell lung cancer phenotypes respectively. We also assessed the miRNA association with the drug targets. We identified tumor growth-related interacting partners based on the pathway information of drug-target interaction networks. The protein–protein interaction binding site between the drug targets and their interacting proteins provided an opportunity to identify small molecules that can modulate the activity of functional cell death interactions in each cancer. Overall, our systematic screening of non-apoptotic cell death-related genes uncovered targets helpful for cancer therapy.

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Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase

BMC Biology ◽

10.1186/s12915-021-01048-7 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Cheng-Wen He ◽

Xue-Fei Cui ◽

Shao-Jie Ma ◽

Qin Xu ◽

Yan-Peng Ran ◽

...

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Stationary Phase ◽

Molecular Mechanisms ◽

Multivesicular Body ◽

Degradation Process ◽

Vacuolar Membrane ◽

Yeast Cells ◽

Membrane Structures ◽

Membrane Recruitment

Abstract Background The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways. Results Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation. Conclusions Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.

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Fast Diffusion of the Unassembled PetC1-GFP Protein in the Cyanobacterial Thylakoid Membrane

Life ◽

10.3390/life11010015 ◽

2020 ◽

Vol 11 (1) ◽

pp. 15

Author(s):

Radek Kaňa ◽

Gábor Steinbach ◽

Roman Sobotka ◽

György Vámosi ◽

Josef Komenda

Keyword(s):

Membrane Proteins ◽

Single Molecule ◽

Protein Interactions ◽

Thylakoid Membrane ◽

Protein Complexes ◽

Correlation Spectroscopy ◽

Membrane Microdomains ◽

Fast Diffusion ◽

Light Harvesting Complexes ◽

Term Survival

Biological membranes were originally described as a fluid mosaic with uniform distribution of proteins and lipids. Later, heterogeneous membrane areas were found in many membrane systems including cyanobacterial thylakoids. In fact, cyanobacterial pigment–protein complexes (photosystems, phycobilisomes) form a heterogeneous mosaic of thylakoid membrane microdomains (MDs) restricting protein mobility. The trafficking of membrane proteins is one of the key factors for long-term survival under stress conditions, for instance during exposure to photoinhibitory light conditions. However, the mobility of unbound ‘free’ proteins in thylakoid membrane is poorly characterized. In this work, we assessed the maximal diffusional ability of a small, unbound thylakoid membrane protein by semi-single molecule FCS (fluorescence correlation spectroscopy) method in the cyanobacterium Synechocystis sp. PCC6803. We utilized a GFP-tagged variant of the cytochrome b6f subunit PetC1 (PetC1-GFP), which was not assembled in the b6f complex due to the presence of the tag. Subsequent FCS measurements have identified a very fast diffusion of the PetC1-GFP protein in the thylakoid membrane (D = 0.14 − 2.95 µm2s−1). This means that the mobility of PetC1-GFP was comparable with that of free lipids and was 50–500 times higher in comparison to the mobility of proteins (e.g., IsiA, LHCII—light-harvesting complexes of PSII) naturally associated with larger thylakoid membrane complexes like photosystems. Our results thus demonstrate the ability of free thylakoid-membrane proteins to move very fast, revealing the crucial role of protein–protein interactions in the mobility restrictions for large thylakoid protein complexes.

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Membrane Protein Stabilization Strategies for Structural and Functional Studies

Membranes ◽

10.3390/membranes11020155 ◽

2021 ◽

Vol 11 (2) ◽

pp. 155

Author(s):

Ekaitz Errasti-Murugarren ◽

Paola Bartoccioni ◽

Manuel Palacín

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Lipid Membrane ◽

Protein Stabilization ◽

New Drugs ◽

Cell Physiology ◽

Protein Preparation ◽

Functional Studies ◽

Membrane Protein Stability ◽

Druggable Targets

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

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A microfluidic strategy for the detection of membrane protein interactions

Lab on a Chip ◽

10.1039/d0lc00205d ◽

2020 ◽

Vol 20 (17) ◽

pp. 3230-3238

Author(s):

Yuewen Zhang ◽

Therese W. Herling ◽

Stefan Kreida ◽

Quentin A. E. Peter ◽

Tadas Kartanas ◽

...

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Protein Interactions ◽

Native Solution ◽

Exchange Of Information ◽

Extracellular Environment

Membrane proteins are gatekeepers for exchange of information and matter between the intracellular and extracellular environment. This paper opens up a route to probe membrane protein interactions under native solution conditions using microfluidics.

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Three-dimensional crystallization of membrane proteins

Quarterly Reviews of Biophysics ◽

10.1017/s0033583500004625 ◽

1988 ◽

Vol 21 (4) ◽

pp. 429-477 ◽

Cited By ~ 156

Author(s):

W. Kühlbrandt

Keyword(s):

High Resolution ◽

Membrane Proteins ◽

Membrane Protein ◽

Protein Complexes ◽

Three Dimensional ◽

Biological Macromolecules ◽

X Ray ◽

X Ray Crystallography ◽

Membrane Protein Complexes ◽

Essential Prerequisite

As recently as 10 years ago, the prospect of solving the structure of any membrane protein by X-ray crystallography seemed remote. Since then, the threedimensional (3-D) structures of two membrane protein complexes, the bacterial photosynthetic reaction centres of Rhodopseudomonas viridis (Deisenhofer et al. 1984, 1985) and of Rhodobacter sphaeroides (Allen et al. 1986, 1987 a, 6; Chang et al. 1986) have been determined at high resolution. This astonishing progress would not have been possible without the pioneering work of Michel and Garavito who first succeeded in growing 3-D crystals of the membrane proteins bacteriorhodopsin (Michel & Oesterhelt, 1980) and matrix porin (Garavito & Rosenbusch, 1980). X-ray crystallography is still the only routine method for determining the 3-D structures of biological macromolecules at high resolution and well-ordered 3-D crystals of sufficient size are the essential prerequisite.

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NCU-G1 is a highly glycosylated integral membrane protein of the lysosome

Biochemical Journal ◽

10.1042/bj20090567 ◽

2009 ◽

Vol 422 (1) ◽

pp. 83-90 ◽

Cited By ~ 17

Author(s):

Oliver Schieweck ◽

Markus Damme ◽

Bernd Schröder ◽

Andrej Hasilik ◽

Bernhard Schmidt ◽

...

Keyword(s):

Amino Acid ◽

Membrane Proteins ◽

Membrane Protein ◽

Mouse Liver ◽

Lysosomal Membrane ◽

Molecular Forms ◽

Type I ◽

Calculated Molecular Mass ◽

Sorting Motif ◽

Lysosomal Membrane Proteins

Until recently, a modest number of approx. 40 lysosomal membrane proteins had been identified and even fewer were characterized in their function. In a proteomic study, using lysosomal membranes from human placenta we identified several candidate lysosomal membrane proteins and proved the lysosomal localization of two of them. In the present study, we demonstrate the lysosomal localization of the mouse orthologue of the human C1orf85 protein, which has been termed kidney-predominant protein NCU-G1 (GenBank® accession number: AB027141). NCU-G1 encodes a 404 amino acid protein with a calculated molecular mass of 39 kDa. The bioinformatics analysis of its amino acid sequence suggests it is a type I transmembrane protein containing a single tyrosine-based consensus lysosomal sorting motif at position 400 within the 12-residue C-terminal tail. Its lysosomal localization was confirmed using immunofluorescence with a C-terminally His-tagged NCU-G1 and the lysosomal marker LAMP-1 (lysosome-associated membrane protein-1) as a reference, and by subcellular fractionation of mouse liver after a tyloxapol-induced density shift of the lysosomal fraction using an anti-NCU-G1 antiserum. In transiently transfected HT1080 and HeLa cells, the His-tagged NCU-G1 was detected in two molecular forms with apparent protein sizes of 70 and 80 kDa, and in mouse liver the endogenous wild-type NCU-G1 was detected as a 75 kDa protein. The remarkable difference between the apparent and the calculated molecular masses of NCU-G1 was shown, by digesting the protein with N-glycosidase F, to be due to an extensive glycosylation. The lysosomal localization was impaired by mutational replacement of an alanine residue for the tyrosine residue within the putative sorting motif.

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