Structure Determination Of Membrane Proteins Research Articles

NDT-C11 as a Viable Novel Detergent for Single Particle Cryo-EM.

Single particle cryo electron microscopy (cryo-EM) is now the major method for the determination of integral membrane protein structure. For the success of a given project the type of membrane mimetic used for extraction from the native cell membrane, purification to homogeneity and finally cryo-grid vitrification is crucial. Although small molecule amphiphiles - detergents - are the most widely used membrane mimetic, specific tailoring of detergent structure for single particle cryo-EM is rare and the demand for effective detergents not satisfied. Here, we compare the popular detergent lauryl maltose-neopentyl glycol (LMNG) with the novel detergent neopentyl glycol-derived triglucoside-C11 (NDT-C11) in its behavior as free detergent and when bound to two types of multisubunit membrane protein complexes - cyanobacterial photosystem I (PSI) and mammalian F-ATP synthase. We conclude that NDT-C11 has high potential to become a very useful detergent for single particle cryo-EM of integral membrane proteins.

Limitations in membrane protein structure determination by lipid nanodiscs

Lipid nanodiscs are popular mimetics of biological membranes for determining membrane protein structures. However, a recent study revealed that the choice of nanodisc scaffold directly influenced the structure of an ion channel. This finding prompts us to be cautious and calls for improved membrane mimetics for structure determination.

Magic-angle spinning NMR structure of Opa60 in lipid bilayers

Here we report the structure of Opa60 in lipid bilayers using proton-detected magic-angle spinning nuclear magnetic resonance (MAS NMR). Preparations including near-native oligosaccharide lipids reveal a consistent picture of a stable transmembrane beta barrel with a minor increase in the structured region as compared with the previously reported detergent structure. The large variable loops known to interact with host proteins could not be detected, confirming their dynamic nature even in a lipid bilayer environment. The structure provides a starting point for investigation of the functional role of Opa60 in gonococcal infection, which is understood to involve interaction with host proteins. At the same time, it demonstrates the recent advances in proton-detected methodology for membrane protein structure determination at atomic resolution by MAS NMR.

Rational Approach to Improve Detergent Efficacy for Membrane Protein Stabilization.

Membrane protein structures are essential for the molecular understanding of diverse cellular processes and drug discovery. Detergents are not only widely used to extract membrane proteins from membranes but also utilized to preserve native protein structures in aqueous solution. However, micelles formed by conventional detergents are suboptimal for membrane protein stabilization, necessitating the development of novel amphiphilic molecules with enhanced protein stabilization efficacy. In this study, we prepared two sets of tandem malonate-derived glucoside (TMG) variants, both of which were designed to increase the alkyl chain density in micelle interiors. The alkyl chain density was modulated either by reducing the spacer length (TMG-Ms) or by introducing an additional alkyl chain between the two alkyl chains of the original TMGs (TMG-Ps). When evaluated with a few membrane proteins including a G protein-coupled receptor, TMG-P10,8 was found to be substantially more efficient at extracting membrane proteins and also effective at preserving protein integrity in the long term compared to the previously described TMG-A13. This result reveals that inserting an additional alkyl chain between the two existing alkyl chains is an effective way to optimize detergent properties for membrane protein study. This new biochemical tool and the design principle described have the potential to facilitate membrane protein structure determination.

Challenges of Protein-Protein Docking of the Membrane Proteins.

Despite the recent advances in the determination of high-resolution membrane protein (MP) structures, the structural and functional characterization of MPs remains extremely challenging, mainly due to the hydrophobic nature, low abundance, poor expression, purification, and crystallization difficulties associated with MPs. Whereby the major challenges/hurdles for MP structure determination are associated with the expression, purification, and crystallization procedures. Although there have beensignificant advances in the experimental determination of MP structures, only a limited number of MP structures (approximately less than 1% of all) are available in the Protein Data Bank (PDB). Therefore, the structures of a large number of MPs still remain unresolved, which leads to the availability of widely unplumbed structural and functional information related to MPs. As a result, recent developments in the drug discovery realm and the significant biological contemplation have led to the development of several novel, low-cost, and time-efficient computational methods that overcome the limitations of experimental approaches, supplement experiments, and provide alternatives for the characterization of MPs. Whereby the fine tuning and optimizations of these computational approaches remains an ongoing endeavor.Computational methods offer a potential way for the elucidation of structural features andthe augmentation of currently available MP information. However, the use of computational modeling can be extremely challenging for MPs mainly due to insufficient knowledge of (or gaps in) atomic structures of MPs. Despite the availability of numerous in silico methods for 3D structure determination the applicability of these methods to MPs remains relatively low since all methods are not well-suited or adequate for MPs. However, sophisticated methods for MP structure predictions are constantly being developed and updated to integrate the modifications required for MPs. Currently, different computational methods for (1) MP structure prediction, (2) stability analysis of MPs through molecular dynamics simulations, (3) modeling of MP complexes through docking, (4) prediction of interactions between MPs, and (5) MP interactions with its soluble partner are extensively used. Towards this end, MP docking is widely used. It is notable that the MP docking methods yet few in number might show greater potential in terms of filling the knowledge gap. In this chapter, MP docking methods and associated challenges have been reviewed to improve the applicability, accuracy, and the ability to model macromolecular complexes.

LipIDens: simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. We demonstrate this by application of our open-source LipIDens code to ten diverse membrane protein structures which exhibit lipid-like densities.

Solid-state NMR structure determination of a membrane protein in E. coli cellular inner membrane.

Structure determination of membrane proteins in native cellular membranes is critical to precisely reveal their structures in physiological conditions. However, it remains challenging for solid-state nuclear magnetic resonance (ssNMR) due to the low sensitivity and high complexity of ssNMR spectra of cellular membranes. Here, we present the structure determination of aquaporin Z (AqpZ) by ssNMR in Escherichia coli inner membranes. To enhance the signal sensitivity of AqpZ, we optimized protein overexpression and removed outer membrane components. To suppress the interference of background proteins, we used a "dual-media" expression approach and antibiotic treatment. Using 1017 distance restraints obtained from two-dimensional 13C-13C spectra based on the complete chemical shift assignments, the 1.7-Å ssNMR structure of AqpZ is determined in E. coli inner membranes. This cellular ssNMR structure determination paves the way for analyzing the atomic structural details for membrane proteins in native cellular membranes.

Se-MAG Is a Convenient Additive for Experimental Phasing and Structure Determination of Membrane Proteins Crystallised by the Lipid Cubic Phase (In Meso) Method

Both intensity and phase information are needed for structure determination by macromolecular X-ray crystallography. The diffraction experiment provides intensities. Phases must be accessed indirectly by molecular replacement, or by experimental phasing. A popular method for crystallising membrane proteins employs a lipid cubic mesophase (the in meso method). Monoolein is the most popular lipid for in meso crystallisation. Invariably, the lipid co-crystallises with the protein recapitulating the biomembrane from whence it came. We reasoned that such a lipid bearing a heavy atom could be used for experimental phasing. In this study, we replaced half the monoolein in the mesophase with a seleno-labelled analogue (Se-MAG), which has a selenium atom in the fatty acyl chain of the lipid. The lipid mixture formed the cubic mesophase and grew crystals by the in meso method of the alginate transporter, AlgE, and the lipoprotein N-acyltransferase, Lnt. Se-MAGs co-crystallised with both proteins and were used to obtain phases for high-resolution structure determination by the selenium single-wavelength anomalous diffraction method. The use of such a mixed lipid system may prove to be a general strategy for the experimental phasing part of crystallographic structure determination of membrane proteins that crystallise via the in meso method.

`Cryo-EM': electron cryomicroscopy, cryo electron microscopy or something else?

Structural biology continues to benefit from an expanding toolkit, which is helping to gain unprecedented insight into the assembly and organization of multi-protein machineries, enzyme mechanisms and ligand/inhibitor binding. During the last ten years, cryoEM has become widely available and has provided a major boost to structure determination of membrane proteins and large multi-protein complexes. Many of the structures have now been made available at resolutions around 2 Å, where fundamental questions regarding enzyme mechanisms can be addressed. Over the years, the abbreviation cryoEM has been understood to stand for different things. We wish the wider community to engage and clarify the definition of cryoEM so that the expanding literature involving cryoEM is unified.

The Structure-property Relationships of GPCR-targeted Drugs Approved between 2011 and 2021.

G-protein-coupled receptors (GPCRs) are the largest family of membrane receptors and the most intensively studied drug targets. Given the physiological importance of signal transduction by GPCRs and the recent progress in the structure determination of membrane proteins, the development of GPCR antagonists and agonists is expected to continue to be a major area of medicinal chemistry research. The structure-property relationship illustrates how the modification of the chemical structure influences the absorption, distribution, metabolism, excretion, and other related properties of drug compounds. Understanding the structure-property relationships of clinically approved GPCR-targeted drugs and their analogues could provide useful information on the lead-to-candidate optimization strategies. Among more than 50 GPCR antagonists and agonists approved in the last decade, the structure-property relationships of 17 drugs are compiled from medicinal chemistry literature, in which detailed pharmacokinetic and toxicological properties are disclosed not only for the final drug candidate but also for key analogues generated during the lead optimization campaign. The structure-property relationships hereby summarized demonstrate how in vitro and in vivo properties of the membrane protein-targeted ligands could be effectively optimized, in many cases, without requiring a significant change in the molecular size. This information is expected to provide valuable insights to expedite new GPCR-targeted drug development.

Development of a novel lipid for the in meso crystallisation and structure determination of membrane proteins and the impact of 2-MAG on mesophase behaviour

Development of a novel lipid for the <i>in meso</i> crystallisation and structure determination of membrane proteins and the impact of 2-MAG on mesophase behaviour

Lipid nanodiscs as a template for high-resolution cryo-EM structures of peripheral membrane proteins

Peripheral membrane proteins are ubiquitous throughout cell biology and are required for a variety of cellular processes such as signal transduction, membrane trafficking, and autophagy. Transient binding to the membrane has a profound impact on protein function, serving to induce conformational changes and alter biochemical and biophysical parameters by increasing the local concentration of factors and restricting diffusion to two dimensions. Despite the centrality of the membrane in serving as a template for cell biology, there are few reported high-resolution structures of peripheral membrane proteins bound to the membrane. We analyzed the utility of lipid nanodiscs to serve as a template for cryo-EM analysis of peripheral membrane proteins. We tested a variety of nanodiscs and we report a 3.3 Å structure of the AP2 clathrin adaptor complex bound to a 17-nm nanodisc, with sufficient resolution to visualize a bound lipid head group. Our data demonstrate that lipid nanodiscs are amenable to high-resolution structure determination of peripheral membrane proteins and provide a framework for extending this analysis to other systems.

Membrane protein isolation and structure determination in cell-derived membrane vesicles

Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of proteins in membrane vesicles derived directly from cells. Structures of the ion channel Slo1 from total cell membranes and from cell plasma membranes were determined at 3.8Å and 2.7Å resolution, respectively. The plasma membrane environment stabilizes Slo1, revealing an alteration of global helical packing, polar lipid, and cholesterol interactions that stabilize previously unresolved regions of the channel and an additional ion binding site in the Ca2+ regulatory domain. The two methods presented enable structural analysis of both internal and plasma membrane proteins without disrupting weakly interacting proteins, lipids, and cofactors that are essential to biological function.

Halobacterium salinarum: Life with more than a grain of salt.

Halobacterium salinarum is a halophilic (salt-loving) archaeon that grows in salt concentrations near or at saturation. Although isolated from salted fish a century ago, it was the 1971 discovery of bacteriorhodopsin, the light-driven proton pump, that raised interest in Hbt. salinarum across a range of disciplines, including biophysics, chemistry, molecular evolution and biotechnology. Hbt. salinarum have since contributed to numerous discoveries, such as advances in membrane protein structure determination and the first example of a non-eukaryal glycoprotein. Work on Hbt. salinarum, one of the species used to define Archaea, has also elucidated molecular workings in the third domain. Finally, Hbt. salinarum presents creative solutions to the challenges of life in high salt.

Intrinsic fiducials for small membrane protein structure determination.

Solute carrier proteins (SLC) are small membrane transport proteins that play a critical role in many physiological processes. Despite their critical importance, our understanding of how these proteins function is limited due to the difficulty of structure determination. This is particularly true for major facilitator superfamily (MFS) proteins where there are often limited protein features to drive computational alignment of particles. While tools like cryo-EM have greatly enhanced the ability to determine membrane protein structures, fiducial markers are needed to help promote particle alignment. Fragment antigen-binding antibodies (Fab) or nanobodies (Nb) are the most commonly used examples, but generating these is not a trivial task. Often this is the largest barrier for membrane protein structure determination, taking a significant amount of time and resources. In the end, the resulting fiducial may be too flexible to enable high resolution structure determination, requiring the whole process to be done again. A feature intrinsic to the protein sequence would eliminate this barrier as it would require no additional generation time, and can be purified concurrently with the transporter of interest. Here we investigate the use of an intrinsic fiducial consisting of GFP and anti-GFP Nb labeled at the each of the protein's termini to solve a novel structure of an MFS transporter. We have generated three fully functional constructs, each with varying termini lengths, and demonstrated successive improvements in fiducial rigidity and cryo-EM reconstructions. We believe this method will not only enable us to solve a previously unknown structure, but also prove to be an accessible means for membrane protein structure determination.

Subtomogram averaging of protein structures in isolated HEK293 cell plasma membranes

The plasma membrane and its associated proteins together are key to the function and disfunction of many vital cell processes such as adhesion, sensing/signaling, endocytosis/exocytosis. To better understand their function and treat their disfunction with precision, we need high-resolution structures of membrane-associated molecular components in their native context. Cryo-electron tomography (cryoET) is a rapidly evolving structural determination technique capable of resolving proteins and protein complexes to ångström-level resolution. However, high-resolution reconstruction of membrane-associated proteins and organelles in situ has been met with obstacles including the necessity for ultrathin (<200 nm thick) sample preparation. Here, we isolate plasma membranes using a technique called “unroofing” immediately prior to sample vitrification. This produces 50-200-nm-thick vitreous samples containing the plasma membrane, the actin cortex, and the myriad of other protein complexes associated with the membrane. To verify this technique as a tool for membrane protein structure determination, we are performing a quantitative assessment of the preserved resolution using subtomogram averaging. We are currently working to utilize and modify existing subtomogram averaging tools to identify, segment, process, and refine objects of interest within reconstructed tomograms. We compare our reconstructions with known protein structures. This ongoing work is part of an effort aimed to develop a correlative pipeline for visualizing the proteome of the eukaryotic plasma membrane with efficiency and precision.

Fabrication of membrane proteins in the form of native cell membrane nanoparticles using novel membrane active polymers.

Membrane proteins are a widespread class of bio-macromolecules responsible for numerous vital biological processes and serve as therapeutic targets for a vast array of contemporary medications. For membrane protein isolation and purification, detergents have historically been used. Despite this, detergents frequently result in protein instability. Consequently, their application was limited. Recent detergent-free approaches have been invented. Among these, styrene-maleic acid lipid particle (SMALP), diisobutylene-maleic acid lipid particle (DIBMALP), and native cell membrane nanoparticle (NCMN) systems are the most prevalent. The NCMN system intends to create a library of membrane-active polymers suitable for high-resolution structure determination of membrane protein. Design, synthesis, characterization, and comparative application evaluations of three novel classes of NCMN polymers, NCMNP13-x, NCMNP21-x, and NCMNP21b-x, are presented in this article. Although each NCMN polymer can solubilize distinct model membrane proteins and retain native lipids in NCMN particles, only the NCMNP21b-x family produces lipid-protein particles with ideal buffer compatibility and high homogeneity suitable for single-particle cryo-EM analysis. NCMNP21b-x polymers that generate high-quality NCMN particles are particularly desirable for membrane protein structural biology.

Melamine-cored glucosides for membrane protein solubilization and stabilization: importance of water-mediated intermolecular hydrogen bonding in detergent performance.

Membrane proteins play essential roles in a number of biological processes, and their structures are important in elucidating such processes at the molecular level and also for rational drug design and development. Membrane protein structure determination is notoriously challenging compared to that of soluble proteins, due largely to the inherent instability of their structures in non-lipid environments. Micelles formed by conventional detergents have been widely used for membrane protein manipulation, but they are suboptimal for long-term stability of membrane proteins, making downstream characterization difficult. Hence, there is an unmet need for the development of new amphipathic agents with enhanced efficacy for membrane protein stabilization. In this study, we designed and synthesized a set of glucoside amphiphiles with a melamine core, denoted melamine-cored glucosides (MGs). When evaluated with four membrane proteins (two transporters and two G protein-coupled receptors), MG-C11 conferred notably enhanced stability compared to the commonly used detergents, DDM and LMNG. These promising findings are mainly attributed to a unique feature of the MGs, i.e., the ability to form dynamic water-mediated hydrogen-bond networks between detergent molecules, as supported by molecular dynamics simulations. Thus, MG-C11 is the first example of a non-peptide amphiphile capable of forming intermolecular hydrogen bonds within a protein-detergent complex environment. Detergent micelles formed via a hydrogen-bond network could represent the next generation of highly effective membrane-mimetic systems useful for membrane protein structural studies.

Structure determination of inactive-state GPCRs with a universal nanobody.

Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. Despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that the same single-chain camelid antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained structures of neurotensin 1 receptor bound to antagonist SR48692, μ-opioid receptor bound to alvimopan, apo somatostatin receptor 2 and histamine receptor 2 bound to famotidine. We expect this rapid, straightforward approach to facilitate the broad exploration of GPCR inactive states without the need for extensive engineering and crystallization.

Characterization of heterogeneity in nanodisc samples using Feret signatures

Nanodiscs have become a popular tool in structure determination of membrane proteins using cryogenic electron microscopy and single particle analysis. However, the structure determination of small membrane proteins remains challenging. When the embedded protein is in the same size range as the nanodisc, the nanodisc can significantly contribute to the alignment and classification during the structure determination process. In those cases, it is crucial to minimize the heterogeneity in the nanodisc preparations to assure maximum accuracy in the classification and alignment steps of single particle analysis. Here, we introduce a new in-silico method for the characterization of nanodisc samples that is based on analyzing the Feret diameter distribution of their particle projection as imaged in the electron microscope. We validated the method with comprehensive simulation studies and show that Feret signatures can detect subtle differences in nanodisc morphologies and composition that might otherwise go unnoticed. We used the method to identify a specific biochemical nanodisc preparation with low size variations, allowing us to obtain a structure of the 23-kDa single-span membrane protein Bcl-xL while embedded in a nanodisc. Feret signature analysis can steer experimental data collection strategies, allowing more efficient use of high-end data collection hardware, as well as image analysis investments in studies where nanodiscs significantly contribute to the total volume of the full molecular species.