Structural determinants of lipid specificity within Ups/PRELI lipid transfer proteins

Conserved lipid transfer proteins of the Ups/PRELI family regulate lipid accumulation in mitochondria by shuttling phospholipids in a lipid-specific manner across the intermembrane space. Here, we combine structural analysis, unbiased genetic approaches in yeast and molecular dynamics simulations to unravel determinants of lipid specificity within the conserved Ups/PRELI family. We present structures of human PRELID1–TRIAP1 and PRELID3b–TRIAP1 complexes, which exert lipid transfer activity for phosphatidic acid and phosphatidylserine, respectively. Reverse yeast genetic screens identify critical amino acid exchanges that broaden and swap their lipid specificities. We find that amino acids involved in head group recognition and the hydrophobicity of flexible loops regulate lipid entry into the binding cavity. Molecular dynamics simulations reveal different membrane orientations of PRELID1 and PRELID3b during the stepwise release of lipids. Our experiments thus define the structural determinants of lipid specificity and the dynamics of lipid interactions by Ups/PRELI proteins

The importance of the membrane for biophysical measurements

Within cell membranes numerous protein assemblies reside. Among their many functions, these assemblies regulate the movement of molecules between membranes, facilitate signaling into and out of cells, allow movement of cells by cell-matrix attachment, and regulate the electric potential of the membrane. With such critical roles, membrane protein complexes are of considerable interest for human health, yet they pose an enduring challenge for structural biologists because it is difficult to study these protein structures at atomic resolution in in situ environments. To advance structural and functional insights for these protein assemblies, membrane mimetics are typically employed to recapitulate some of the physical and chemical properties of the lipid bilayer membrane. However, extraction from native membranes can sometimes change the structure and lipid-binding properties of these complexes, leading to conflicting results and fueling a drive to study complexes directly from native membranes. Here we consider the co-development of membrane mimetics with technological breakthroughs in both cryo-electron microscopy (cryo-EM) and native mass spectrometry (nMS). Together, these developments are leading to a plethora of high-resolution protein structures, as well as new knowledge of their lipid interactions, from different membrane-like environments

Structure of native lens connexin 46/50 intercellular channels by cryo-EM

Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of different connexin isoforms produces channels with unique properties and enables communication across cell types. Here we used single-particle cryo-electron microscopy to investigate the structural basis of connexin co-assembly in native lens gap junction channels composed of connexin 46 and connexin 50 (Cx46/50). We provide the first comparative analysis to connexin 26 (Cx26), which—together with computational studies—elucidates key energetic features governing gap junction permselectivity. Cx46/50 adopts an open-state conformation that is distinct from the Cx26 crystal structure, yet it appears to be stabilized by a conserved set of hydrophobic anchoring residues. ‘Hot spots’ of genetic mutations linked to hereditary cataract formation map to the core structural–functional elements identified in Cx46/50, suggesting explanations for many of the disease-causing effects

Structure of native lens connexin 46/50 intercellular channels by cryo-EM

Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of different connexin isoforms produces channels with unique properties and enables communication across cell types. Here we used single-particle cryo-electron microscopy to investigate the structural basis of connexin co-assembly in native lens gap junction channels composed of connexin 46 and connexin 50 (Cx46/50). We provide the first comparative analysis to connexin 26 (Cx26), which—together with computational studies—elucidates key energetic features governing gap junction permselectivity. Cx46/50 adopts an open-state conformation that is distinct from the Cx26 crystal structure, yet it appears to be stabilized by a conserved set of hydrophobic anchoring residues. ‘Hot spots’ of genetic mutations linked to hereditary cataract formation map to the core structural–functional elements identified in Cx46/50, suggesting explanations for many of the disease-causing effects

Capturing a rhodopsin receptor signalling cascade across a native membrane

G protein-coupled receptors (GPCRs) are cell-surface receptors that respond to various stimuli to induce signalling pathways across cell membranes. Recent progress has yielded atomic structures of key intermediates1,2 and roles for lipids in signalling3,4. However, capturing signalling events of a wild-type receptor in real time, across a native membrane to its downstream effectors, has remained elusive. Here we probe the archetypal class A GPCR, rhodopsin, directly from fragments of native disc membranes using mass spectrometry. We monitor real-time photoconversion of dark-adapted rhodopsin to opsin, delineating retinal isomerization and hydrolysis steps, and further showing that the reaction is significantly slower in its native membrane than in detergent micelles. Considering the lipids ejected with rhodopsin, we demonstrate that opsin can be regenerated in membranes through photoisomerized retinal–lipid conjugates, and we provide evidence for increased association of rhodopsin with unsaturated long-chain phosphatidylcholine during signalling. Capturing the secondary steps of the signalling cascade, we monitor light activation of transducin (Gt) through loss of GDP to generate an intermediate apo-trimeric G protein, and observe Gαt•GTP subunits interacting with PDE6 to hydrolyse cyclic GMP. We also show how rhodopsin-targeting compounds either stimulate or dampen signalling through rhodopsin–opsin and transducin signalling pathways. Our results not only reveal the effect of native lipids on rhodopsin signalling and regeneration but also enable us to propose a paradigm for GPCR drug discovery in native membrane environments

The use of sonicated lipid vesicles for mass spectrometry of membrane protein complexes

Recent applications of mass spectrometry (MS) to study membrane protein complexes are yielding valuable insights into the binding of lipids and their structural and functional roles. To date, most native MS experiments with membrane proteins are based on detergent solubilization. Many insights into the structure and function of membrane proteins have been obtained using detergents; however, these can promote local lipid rearrangement and can cause fluctuations in the oligomeric state of protein complexes. To overcome these problems, we developed a method that does not use detergents or other chemicals. Here we report a detailed protocol that enables direct ejection of protein complexes from membranes for analysis by native MS. Briefly, lipid vesicles are prepared directly from membranes of different sources and subjected to sonication pulses. The resulting destabilized vesicles are concentrated, introduced into a mass spectrometer and ionized. The mass of the observed protein complexes is determined and this information, in conjunction with ‘omics’-based strategies, is used to determine subunit stoichiometry as well as cofactor and lipid binding. Within this protocol, we expand the applications of the method to include peripheral membrane proteins of the S-layer and amyloid protein export machineries overexpressed in membranes from which the most abundant components have been removed. The described experimental procedure takes approximately 3 d from preparation to MS. The time required for data analysis depends on the complexity of the protein assemblies embedded in the membrane under investigation

Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry

Membrane proteins reside in lipid bilayers and are typically extracted from this environment for study, which often compromises their integrity. In this work, we ejected intact assemblies from membranes, without chemical disruption, and used mass spectrometry to define their composition. From Escherichia coli outer membranes, we identified a chaperone-porin association and lipid interactions in the β-barrel assembly machinery. We observed efflux pumps bridging inner and outer membranes, and from inner membranes we identified a pentameric pore of TonB, as well as the protein-conducting channel SecYEG in association with F1FO adenosine triphosphate (ATP) synthase. Intact mitochondrial membranes from Bos taurus yielded respiratory complexes and fatty acid–bound dimers of the ADP (adenosine diphosphate)/ATP translocase (ANT-1). These results highlight the importance of native membrane environments for retaining small-molecule binding, subunit interactions, and associated chaperones of the membrane proteome

Characterizing exons and introns by regularity of nucleotide strings

BACKGROUND: Translation of nucleotides into a numeric form has been approached in many ways and has allowed researchers to investigate the properties of protein-coding sequences and noncoding sequences. Typically, more pronounced long-range correlations and increased regularity were found in intron-containing genes and in non-transcribed regulatory DNA sequences, compared to cDNA sequences or intron-less genes. The regularity is assessed by spectral tools defined on numerical translates. In most popular approaches of numerical translation the resulting spectra depend on the assignment of numerical values to nucleotides. Our contribution is to propose and illustrate a spectra which remains invariant to the translation rules used in traditional approaches. RESULTS: We outline a methodology for representing sequences of DNA nucleotides as numeric matrices in order to analytically investigate important structural characteristics of DNA. This representation allows us to compute the 2-dimensional wavelet transformation and assess regularity characteristics of the sequence via the slope of the wavelet spectra. In addition to computing a global slope measure for a sequence, we can apply our methodology for overlapping sections of nucleotides to obtain an “evolutionary slope.” To illustrate our methodology, we analyzed 376 gene sequences from the first chromosome of the honeybee. CONCLUSION: For the genes analyzed, we find that introns are significantly more regular (lead to more negative spectral slopes) than exons, which agrees with the results from the literature where regularity is measured on “DNA walks”. However, unlike DNA walks where the nucleotides are assigned numerical values depending on nucleotide characteristics (purine-pyrimidine, weak-strong hydrogen bonds, keto-amino, etc.) or other spatial assignments, the proposed spectral tool is invariant to the assignment of nucleotides. Thus, ambiguity in numerical translation of nucleotides is eliminated. REVIEWERS: This article was reviewed by Dr. Vladimir Kuznetsov, Professor Marek Kimmel and Dr. Natsuhiro Ichinose (nominated by Professor Masanori Arita). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13062-016-0108-7) contains supplementary material, which is available to authorized users