Mimicking the Cell: Bio-Inspired Functions of Supramolecular Assemblies

TAR-DNA-binding protein-43 (TDP-43) C-terminus encodes a prion-like domain widely presented in RNA-binding proteins, which functions to form dynamic oligomers and also, amazingly, hosts most amyotrophic lateral sclerosis (ALS)-causing mutations. Here, as facilitated by our previous discovery, by circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopy, we have successfully determined conformations, dynamics, and self-associations of the full-length prion-like domains of the wild type and three ALS-causing mutants (A315E, Q331K, and M337V) in both aqueous solutions and membrane environments. The study decodes the following: (1) The TDP-43 prion-like domain is intrinsically disordered only with some nascent secondary structures in aqueous solutions, but owns the capacity to assemble into dynamic oligomers rich in β-sheet structures. By contrast, despite having highly similar conformations, three mutants gained the ability to form amyloid oligomers. The wild type and three mutants all formed amyloid fibrils after incubation as imaged by electron microscopy. (2) The interaction with nucleic acid enhances the self-assembly for the wild type but triggers quick aggregation for three mutants. (3) A membrane-interacting subdomain has been identified over residues Met311-Gln343 indispensable for TDP-43 neurotoxicity, which transforms into a well-folded Ω-loop-helix structure in membrane environments. Furthermore, despite having very similar membrane-embedded conformations, three mutants will undergo further self-association in the membrane environment. Our study implies that the TDP-43 prion-like domain appears to have an energy landscape, which allows the assembly of the wild-type sequence into dynamic oligomers only under very limited condition sets, and ALS-causing point mutations are sufficient to remodel it to more favor the amyloid formation or irreversible aggregation, thus supporting the emerging view that the pathologic aggregation may occur via the exaggeration of functionally important assemblies. Furthermore, the coupled capacity of TDP-43 in aggregation and membrane interaction may critically account for its high neurotoxicity, and therefore its decoupling may represent a promising therapeutic strategy to treat TDP-43 causing neurodegenerative diseases.

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ALS-Causing Mutations Significantly Perturb the Self-Assembly and Interaction with Nucleic Acid of the Intrinsically Disordered Prion-Like Domain of TDP-43

We developed a method that allows release of intact membrane protein complexes from amphipols, bicelles and nanodiscs in the gas phase for observation by mass spectrometry (MS). Current methods involve release of membrane protein complexes from detergent micelles, which reveals subunit composition and lipid binding. We demonstrated that oligomeric complexes or proteins requiring defined lipid environments are stabilized to a greater extent in the absence of detergent.

/pdf/detergent-free-mass-spectrometry-of-membrane-protein-3kycr7uuxz.pdf

Detergent-free mass spectrometry of membrane protein complexes

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2014.06.007

Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes

Crystallogenesis of Membrane Proteins Mediated by Polymer-Bounded Lipid Nanodiscs.

1.1. Why Study Membranes and Membrane Proteins?
Biological membranes and membrane proteins, responsible for numerous exciting biological processes, present one of the paramount challenges in biophysics today. Membranes are present in great number and variety in all organisms. They form the boundary between the inside and outside for any bacterium or cell, and they delimit the host of organelles that make up their inner subunits. Each biological membrane is made up of dozens of different types of lipids and sterols, and any particular type of membrane has a characteristic content of these different constituents. As a very basic example, we mention that prokaryotic membranes contain a notable component of negatively charged lipids but almost no cholesterol, while eukaryotic membranes are mostly zwitterionic but have a significant amount of cholesterol. Since the driving biophysical principles of membrane formation are very simple—they lie in the amphipathic properties of any lipid molecule—a single lipid type is sufficient to form membrane-like bilayers in an aqueous environment. Such model membranes are used extensively to study biophysical properties that are representative for most membrane systems. A particularly interesting effect is observed when detergent molecules are added to lipid bilayer samples: the detergents solubilize the bilayers, and in certain regimes so-called bilayered mixed micelles or “bicelles” are formed. In the simplest case, they can be described as microscopic disks where a bilayer patch is encircled by a “rim” of detergent molecules. Bicelles represent a new instance of lipid morphology and are extensively applicable to structural studies of lipid membranes and protein structure.1

Membranes delimit any cell and all of its compartments. They form natural borders for metabolic substances and signaling molecules. Membrane proteins are the porters and gatekeepers that make sure that only proper molecules or signals make it across the membrane. Since membrane proteins perform numerous key functions in cell metabolism and signaling, they contribute over 30% of the genes in typical eukaryotic genomes,2 and they form the targets for over 50% of drugs in use today.3 The number of elucidated structures of membrane proteins has grown exponentially after the first structure was published in 1985, thus equaling the rate at which structure determination of soluble proteins emerged early on.4 Still, the number of available high-resolution structures of membrane proteins is limited. There are Internet sites that keep track of newly published structures of membrane proteins. The crystallography-oriented Web site of Dr. Stephen White [http://blanco.biomol.uci.edu/mpstruc] has recently been joined by another site maintained by Dr. Dror Warschawski that is dedicated to structures of membrane proteins elucidated by nuclear magnetic resonance (NMR) spectroscopy [www.drorlist.com/nmr/MPNMR.html]. Another equally important site of Dr. Hartmut Michel [www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html] with an emphasis on crystallization conditions is no longer updated, but states that access is still enabled.

In this review article, we aim to give a general overview of lipid bicelles as employed in the study of protein structure. Recent advances in the field of protein structural biology that have been made possible by exploiting the unique properties of lipid bicelles, in both solution and solid-state NMR spectroscopy, will be discussed. During the last five years, review contributions have presented bicelles either within the far more general context of reconstitution media for solution NMR studies (see section 1.4) or have focused on macroscopically aligned bicelles as used for solid-state NMR studies.5,6 One very recent contribution has tackled the formidable task of reviewing all membrane mimetics employed in both solution and solid-state NMR studies.7 As mentioned above, we will limit the contents of this review article to applications of lipid bicelles, but will cover both the isotropic and the aligned bicelles as used in NMR studies. Some parts of this article can be viewed as an update on the review articles of Opella and Marassi,8 Marcotte and Auger,9 and Prosser et al.10 In addition, some of our own recent research involving bicelles is presented in detail.

The magic of bicelles lights up membrane protein structure.

Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge. While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes. In this study, we report temperature resistant bicelles that can magnetically-align for a broad range of temperatures and demonstrate their advantages in the structural studies of full-length microsomal cytochrome-P450 and cytochrome-b5 by solid-state NMR spectroscopy. Our results reveal that the N-terminal region of rabbit cytochromeP4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal.

Probing the Transmembrane Structure and Topology of Microsomal Cytochrome-P450 by Solid-State NMR on Temperature-Resistant Bicelles

9123 Background: RET rearrangements and RET kinase domain mutations represent targetable genomic alterations in various cancer types. However, the frequency and characteristics of RET amplification in cancer and its potential role as a targetable oncogenic driver are not well-characterized. Methods: In a pan-cancer cohort of patients at the Dana-Farber Cancer Institute (DFCI) whose tumors underwent next-generation sequencing (NGS), we evaluated the frequency of wild-type (wt) RET amplification (defined as ≥6 copies) in the absence of RET rearrangements or activating mutations. We validated our findings using merged data from the TCGA, GENIE, and China Pan-Cancer datasets. Results: The frequency of wt RET amplification across all cancers was 0.08% (28/34,463) in the DFCI cohort and 0.16% (145/91,466) in the validation cohort. In NSCLC, the frequency of RET amplification was 0.10% (5/4773) in the DFCI cohort and 0.13% (15/11,622) in the validation cohort. Other cancer types with RET amplification included hepatobiliary cancer, prostate cancer, and breast cancer, among others. For 22 RET-amplified cases with available copy number data , the median RET gene copy number was 7 (range 6-36). Among 20 RET-amplified NSCLCs, 10 had no other known driver mutations, and 10 had concurrent driver mutations (including MET exon 14 skipping, MET amplification, KRAS G12C/S/D, EGFR L858R, EGFR exon 20 insertion, and EGFR exon 19 deletion). In cases with available smoking status, 5 out of 6 patients with RET-amplified NSCLC had a history of tobacco use. A 69-year-old man with unresectable stage III NSCLC had disease recurrence after chemoradiation and durvalumab. Both the initial and recurrent biopsy samples showed focal high-level RET amplification (22 and 28 copies, respectively); genomic and RNA sequencing showed no other driver mutations or RET rearrangements. He experienced partial response to the selective RET inhibitor selpercatinib with decrease of both intracranial and systemic tumor burden. Conclusions: Amplification of wt RET represents a novel, targetable, rare molecular subset of NSCLC and other cancers.

Melissa Gildenberg

Papers

The magic of bicelles lights up membrane protein structure.

Probing the Transmembrane Structure and Topology of Microsomal Cytochrome-P450 by Solid-State NMR on Temperature-Resistant Bicelles

Amplification of wild-type RET and clinical response to selpercatinib for non–small-cell lung cancer (NSCLC).

Fork-remodeling helicase Rad5 preferentially reverses replication forks with gaps in the leading strand.