A disordered acidic domain in GPIHBP1 harboring a sulfated tyrosine regulates lipoprotein lipase.

The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL's catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1's IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1-LPL interactions and the ability of GPIHBP1 to protect LPL against ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1-LPL encounter via electrostatic steering, increasing the association rate constant (kon) for LPL binding by >250-fold. Third, we show that LPL accumulates near capillary endothelial cells even in the absence of GPIHBP1. In wild-type mice, we expect that the accumulation of LPL in close proximity to capillaries would increase interactions with GPIHBP1. Fourth, we found that GPIHBP1's IDR is not a key factor in the pathogenicity of chylomicronemia in patients with the GPIHBP1 autoimmune syndrome. Finally, based on biophysical studies, we propose that the negatively charged IDR of GPIHBP1 traverses a vast space, facilitating capture of LPL by capillary endothelial cells and simultaneously contributing to GPIHBP1's ability to preserve LPL structure and activity.

Structure and Dynamics of the Central Lipid Pool and Proteins of the Bacterial Holo-Translocon.

The bacterial Sec translocon, SecYEG, associates with accessory proteins YidC and the SecDF-YajC subcomplex to form the bacterial holo-translocon (HTL). The HTL is a dynamic and flexible protein transport machine capable of coordinating protein secretion across the membrane and efficient lateral insertion of nascent membrane proteins. It has been hypothesized that a central lipid core facilitates the controlled passage of membrane proteins into the bilayer, ensuring the efficient formation of their native state. By performing small-angle neutron scattering on protein solubilized in "match-out" deuterated detergent, we have been able to interrogate a "naked" HTL complex, with the scattering contribution of the surrounding detergent micelle rendered invisible. Such an approach has allowed the confirmation of a lipid core within the HTL, which accommodates between 8 and 29 lipids. Coarse-grained molecular dynamics simulations of the HTL also demonstrate a dynamic, central pool of lipids. An opening at this lipid-rich region between YidC and the SecY lateral gate may provide an exit gateway for newly synthesized, correctly oriented, membrane protein helices, or even small bundles of helices, to emerge from the HTL.

Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex.

The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex under physiological pH, and to be capable of templating the three peptide sequences to constitute a small coiled-coil motif displaying remarkable α-helicity. The formed trimeric complex was characterized by ultraviolet thermal denaturation, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12 nm) in solution was revealed to be independent of concentration. The topological folding of the peptide moiety differed strongly from those of the individual POC strands and the unconjugated peptide, exclusively adopting the designed triple helical structure.

GUB06-046, a novel secretin/glucagon-like peptide 1 co-agonist, decreases food intake, improves glycemic control, and preserves beta cell mass in diabetic mice.

Bariatric surgery is currently the most effective treatment of obesity, which has spurred an interest in developing pharmaceutical mimetics. It is thought that the marked body weight-lowering effects of bariatric surgery involve stimulated secretion of appetite-regulating gut hormones, including glucagon-like peptide 1. We here report that intestinal expression of secretin is markedly upregulated in a rat model of Roux-en-Y gastric bypass, suggesting an additional role of secretin in the beneficial metabolic effects of Roux-en-Y gastric bypass. We therefore developed novel secretin-based peptide co-agonists and identified a lead compound, GUB06-046, that exhibited potent agonism of both the secretin receptor and glucagon-like peptide 1 receptor. Semi-acute administration of GUB06-046 to lean mice significantly decreased cumulative food intake and improved glucose tolerance. Chronic administration of GUB06-046 to diabetic db/db mice for 8 weeks improved glycemic control, as indicated by a 39% decrease in fasting blood glucose and 1.6% reduction of plasma HbA1c levels. Stereological analysis of db/db mice pancreata revealed a 78% increase in beta-cell mass after GUB06-046 treatment, with no impact on exocrine pancreas mass or pancreatic duct epithelial mass. The data demonstrate beneficial effects of GUB06-046 on appetite regulation, glucose homeostasis, and beta-cell mass in db/db mice, without proliferative effects on the exocrine pancreas and the pancreatic duct epithelium. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Dimeric peptides with three different linkers self-assemble with phospholipids to form peptide nanodiscs that stabilize membrane proteins.

Three dimers of the amphipathic α-helical peptide 18A have been synthesized with different interhelical linkers inserted between the two copies of 18A. The dimeric peptides were denoted 'beltides' where Beltide-1 refers to the 18A-dimer without a linker, Beltide-2 is the 18A-dimer with proline (Pro) as a linker and Beltide-3 is the 18A-dimer linked by two glycines (Gly-Gly). The self-assembly of the beltides with the phospholipid DMPC was studied with and without the incorporated membrane protein bacteriorhodopsin (bR) through a combination of coarse-grained MD simulations, size-exclusion chromatography (SEC), circular dichroism (CD) spectroscopy, small-angle scattering (SAS), static light scattering (SLS) and UV-Vis spectroscopy. For all three beltides, MD and combined small-angle X-ray and -neutron scattering were consistent with a disc structure composed by a phospholipid bilayer surrounded by a belt of peptides and with a total disc diameter of approximately 10 nm. CD confirmed that all three beltides were α-helical in the free form and with DMPC. However, as shown by SEC the different interhelical linkers clearly led to different properties of the beltides. Beltide-3, with the Gly-Gly linker, was very adaptable such that peptide nanodiscs could be formed for a broad range of different peptide to lipid stoichiometries and therefore also possible disc-sizes. On the other hand, both Beltide-2 with the Pro linker and Beltide-1 without a linker were less adaptable and would only form discs of certain peptide to lipid stoichiometries. SLS revealed that the structural stability of the formed peptide nanodiscs was also highly affected by the linkers and it was found that Beltide-1 gave more stable discs than the other two beltides. With respect to membrane protein stabilization, each of the three beltides in combination with DMPC stabilizes the seven-helix transmembrane protein bacteriorhodopsin significantly better than the detergent octyl glucoside, but no significant difference was observed between the three beltides. We conclude that adaptability, size, and structural stability can be tuned by changing the interhelical linker while maintaining the properties of the discs with respect to membrane protein stabilization.

Small-angle X-ray scattering of the cholesterol incorporation into human ApoA1-POPC discoidal particles.

Structural and functional aspects of high-density lipoproteins have been studied for over half a century. Due to the plasticity of this highly complex system, new aspects continue to be discovered. Here, we present a structural study of the human Apolipoprotein A1 (ApoA1) and investigate the role of its N-terminal domain, the so-called globular domain of ApoA1, in discoidal complexes with phospholipids and increasing amounts of cholesterol. Using a combination of solution-based small-angle x-ray scattering (SAXS) and molecular constrained data modeling, we show that the ApoA1-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-based particles are disk shaped with an elliptical cross section and composed by a central lipid bilayer surrounded by two stabilizing ApoA1 proteins. This structure is very similar to the particles formed in the so-called nanodisc system, which is based on N-terminal truncated ApoA1 protein. Although it is commonly agreed that the nanodisc is plain disk shaped, several more advanced structures have been proposed for the full-length ApoA1 in combination with POPC and cholesterol. This prompted us to make a detailed comparative study of the ApoA1 and nanodisc systems upon cholesterol uptake. Based on the presented SAXS analysis it is found that the N-terminal domains of ApoA1-POPC-cholesterol particles are not globular but instead an integrated part of the protein belt stabilizing the particles. Upon incorporation of increasing amounts of cholesterol, the presence of the N-terminal domain allows the bilayer thickness to increase while maintaining an overall flat bilayer structure. This is contrasted by the energetically more strained and less favorable lens shape required to fit the SAXS data from the N-terminal truncated nanodisc system upon cholesterol incorporation. This suggests that the N-terminal domain of ApoA1 actively participates in the stabilization of the ApoA1-POPC-cholesterol discoidal particle and allows for a more optimal lipid packing upon cholesterol uptake.

An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase.

LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

Small-angle scattering gives direct structural information about a membrane protein inside a lipid environment.

Monomeric bacteriorhodopsin (bR) reconstituted into POPC/POPG-containing nanodiscs was investigated by combined small-angle neutron and X-ray scattering. A novel hybrid approach to small-angle scattering data analysis was developed. In combination, these provided direct structural insight into membrane-protein localization in the nanodisc and into the protein-lipid interactions. It was found that bR is laterally decentred in the plane of the disc and is slightly tilted in the phospholipid bilayer. The thickness of the bilayer is reduced in response to the incorporation of bR. The observed tilt of bR is in good accordance with previously performed theoretical predictions and computer simulations based on the bR crystal structure. The result is a significant and essential step on the way to developing a general small-angle scattering-based method for determining the low-resolution structures of membrane proteins in physiologically relevant environments.

Self-assembling peptides form nanodiscs that stabilize membrane proteins.

New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self-assemble in combination with phospholipids to form discoidal shaped particles that can stabilize membrane proteins. In the present study, we have investigated an ApoA1 mimetic peptide with respect to its solution structure when in complex with phospholipids. This was achieved using a powerful combination of small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) supported by coarse-grained molecular dynamics simulations. The detailed structure of the discs was determined in unprecedented detail and it was found that they adopt a discoidal structure very similar to the ApoA1 based nanodiscs. We furthermore show that, like the ApoA1 and derived nanodiscs, these peptide discs can accommodate and stabilize a membrane protein. Finally, we exploit their dynamic properties and show that the 18A discs may be used for transferring membrane proteins and associated phospholipids directly and gently into phospholipid nanodiscs.

Anisotropic protein-protein interactions in dilute and concentrated solutions.

Interactions between biomolecules are ubiquitous in nature and crucial to many applications including vaccine development; environmentally friendly textile detergents; and food formulation. Using small angle X-ray scattering and structure-based molecular simulations, we explore protein-protein interactions in dilute to semi-concentrated protein solutions. We address the pertinent question, whether interaction models developed at infinite dilution can be extrapolated to concentrated regimes? Our analysis is based on measured and simulated osmotic second virial coefficients and solution structure factors at varying protein concentration and for different variants of the protein Thermomyces Lanuginosus Lipase (TLL). We show that in order to span the dilute and semi-concentrated regime, any model must carefully capture the balance between spatial and orientational correlations as the protein concentration is elevated. This requires consideration of the protein surface morphology, including possible patch interactions. Experimental data for TLL is most accurately described when assuming a patchy interaction, leading to dimer formation. Our analysis supports that the dimeric proteins predominantly exist in their open conformation where the active site is exposed, thereby maximising hydrophobic attractions that promote inter-protein alignment.

Aggregative adherence fimbriae form compact structures as seen by SAXS.

Bacterial colonization is mediated by fimbriae, which are thin hair-like appendages dispersed from the bacterial surface. The aggregative adherence fimbriae from enteroaggregative E. coli are secreted through the outer membrane and consist of polymerized minor and major pilin subunits. Currently, the understanding of the structural morphology and the role of the minor pilin subunit in the polymerized fimbriae are limited. In this study we use small-angle X-ray scattering to reveal the structural morphology of purified fimbriae in solution. We show that the aggregative fimbriae are compact arrangements of subunit proteins Agg5A + Agg3B which are assembled pairwise on a flexible string rather than extended in relatively straight filaments. Absence of the minor subunit leads to less compact fimbriae, but did not affect the length. The study provides novel insights into the structural morphology and assembly of the aggregative adherence fimbriae. Our study suggests that the minor subunit is not located at the tip of the fimbriae as previously speculated but has a higher importance for the assembled fimbriae by affecting the global structure.

Crystal structure of the TLDc domain of oxidation resistance protein 2 from zebrafish.

The oxidation resistance proteins (OXR) help to protect eukaryotes from reactive oxygen species. The sole C-terminal domain of the OXR, named TLDc is sufficient to perform this function. However, the mechanism by which oxidation resistance occurs is poorly understood. We present here the crystal structure of the TLDc domain of the oxidation resistance protein 2 from zebrafish. The structure was determined by X-ray crystallography to atomic resolution (0.97Å) and adopts an overall globular shape. Two antiparallel ß-sheets form a central ß-sandwich, surrounded by two helices and two one-turn helices. The fold shares low structural similarity to known structures.

Lipid-bound ApoE3 self-assemble into elliptical disc-shaped particles.

Apolipoproteins are vital to lipid metabolism and cholesterol transport in the human body. Here we present a structural study of the lipid-bound particles formed by ApoE3 in a full-length and a truncated version. The particles are formed with, respectively, POPC and DMPC and investigated by small-angle X-ray scattering and negative stain electron microscopy. We find that lipid-bound ApoE3 particles are elliptical, disc-shaped particles composed of a central lipid bilayer encircled by two amphipathic ApoE3 proteins. We went on to investigate a truncated form of ApoE3 containing only residue 80 to 255 (ApoE380-255), which is the central helical repeat segment of ApoE3. The lipid-bound ApoE380-255 particles are found to have the same morphology as the particles with full-length ApoE3. However, they are larger, and form more heterogeneous discoidal structures with four proteins per particle. This behavior is in contrast to ApoA1 where the highly similar helical repeat domain determines the size and stoichiometry of the formed particles both in the case of full-length and truncated ApoA1. Our data hence points towards different mechanisms for lipid bilayer structural modulation by ApoA1 and ApoE3 due to different roles of the non-repeat segments.

Controlling the fractal dimension in self-assembly of terpyridine modified insulin by Fe2+ and Eu3+ to direct in vivo effects.

Metal ion-induced self-assembly (SA) of proteins into higher-order structures can provide new, dynamic nano-assemblies. Here, the synthesis and characterization of a human insulin (HI) analog modified at LysB29 with the tridentate chelator 2,2':6',2''-terpyridine (Tpy) is described. SA of this new insulin analog (LysB29Tpy-HI) in the presence of the metal ions Fe2+ and Eu3+ at different concentrations was studied in solution by fluorescence luminescence and CD spectroscopy, dynamic light scattering, and small-angle X-ray scattering, while surface assembly was probed by AFM. Unique oligomerization was observed in solution, as Fe2+ yielded small magenta-colored discrete non-native assemblies, while Eu3+ caused the formation of large fractal assemblies. Binding of both metal ions to Tpy was demonstrated spectroscopically, and emission lifetime experiments revealed a distinct Eu3+ coordination geometry that included two water molecules. SAXS suggested that LysB29Tpy-HI with Fe2+ oligomerized to a discrete, roughly octameric species, while LysB29Tpy-HI with Eu3+ gave very large assemblies that could be modelled as fractals. The fractal dimensionality increased with the Eu3+ concentration. We propose that this is a consequence of Eu3+ binding to both Tpy and to free carboxylic acid groups on the insulin surface. LysB29Tpy-HI maintained insulin receptor affinity, and showed extended blood glucose lowering and plasma concentration after subcutaneous injection in rats. The combination of metal ion directed SA and native SA provides control of nano-scale fractal dimensionality and points towards use in therapeutics.

Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument.

Coupling of size-exclusion chromatography with biological solution small-angle X-ray scattering (SEC-SAXS) on dedicated synchrotron beamlines enables structural analysis of challenging samples such as labile proteins and low-affinity complexes. For this reason, the approach has gained increased popularity during the past decade. Transportation of perishable samples to synchrotrons might, however, compromise the experiments, and the limited availability of synchrotron beamtime renders iterative sample optimization tedious and lengthy. Here, the successful setup of laboratory-based SEC-SAXS is described in a proof-of-concept study. It is demonstrated that sufficient quality data can be obtained on a laboratory instrument with small sample consumption, comparable to typical synchrotron SEC-SAXS demands. UV/vis measurements directly on the SAXS exposure cell ensure accurate concentration determination, crucial for direct molecular weight determination from the scattering data. The absence of radiation damage implies that the sample can be fractionated and subjected to complementary analysis available at the home institution after SEC-SAXS. Laboratory-based SEC-SAXS opens the field for analysis of biological samples at the home institution, thus increasing productivity of biostructural research. It may further ensure that synchrotron beamtime is used primarily for the most suitable and optimized samples.

Small-angle neutron scattering studies on the AMPA receptor GluA2 in the resting, AMPA-bound and GYKI-53655-bound states.

The AMPA receptor GluA2 belongs to the family of ionotropic glutamate receptors, which are responsible for most of the fast excitatory neuronal signalling in the central nervous system. These receptors are important for memory and learning, but have also been associated with brain diseases such as Alzheimer's disease and epilepsy. Today, one drug is on the market for the treatment of epilepsy targeting AMPA receptors, i.e. a negative allosteric modulator of these receptors. Recently, crystal structures and cryo-electron microscopy (cryo-EM) structures of full-length GluA2 in the resting (apo), activated and desensitized states have been reported. Here, solution structures of full-length GluA2 are reported using small-angle neutron scattering (SANS) with a novel, fully matched-out detergent. The GluA2 solution structure was investigated in the resting state as well as in the presence of AMPA and of the negative allosteric modulator GYKI-53655. In solution and at neutral pH, the SANS data clearly indicate that GluA2 is in a compact form in the resting state. The solution structure resembles the crystal structure of GluA2 in the resting state, with an estimated maximum distance (D max) of 179 ± 11 Šand a radius of gyration (R g) of 61.9 ± 0.4 Å. An ab initio model of GluA2 in solution generated using DAMMIF clearly showed the individual domains, i.e. the extracellular N-terminal domains and ligand-binding domains as well as the transmembrane domain. Solution structures revealed that GluA2 remained in a compact form in the presence of AMPA or GYKI-53655. At acidic pH only, GluA2 in the presence of AMPA adopted a more open conformation of the extracellular part (estimated D max of 189 ± 5 Šand R g of 65.2 ± 0.5 Å), resembling the most open, desensitized class 3 cryo-EM structure of GluA2 in the presence of quisqualate. In conclusion, this methodological study may serve as an example for future SANS studies on membrane proteins.

Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering.

A novel and generally applicable method for determining structures of membrane proteins in solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic neutron scattering length difference between hydrogen and deuterium. Individual hydrogen/deuterium levels of the detergent head and tail groups were achieved such that the formed micelles became effectively invisible in heavy water (D2 O) when investigated by neutrons. This way, only the signal from the membrane protein remained in the SANS data. We demonstrate that the method is not only generally applicable on five very different membrane proteins but also reveals subtle structural details about the sarco/endoplasmatic reticulum Ca2+ ATPase (SERCA). In all, the synthesis of isotope-substituted detergents makes solution structure determination of membrane proteins by SANS and subsequent data analysis available to nonspecialists.

Peptide-oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic.

Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide-oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design.

Aquaporin-Based Biomimetic Polymeric Membranes: Approaches and Challenges.

In recent years, aquaporin biomimetic membranes (ABMs) for water separation have gained considerable interest. Although the first ABMs are commercially available, there are still many challenges associated with further ABM development. Here, we discuss the interplay of the main components of ABMs: aquaporin proteins (AQPs), block copolymers for AQP reconstitution, and polymer-based supporting structures. First, we briefly cover challenges and review recent developments in understanding the interplay between AQP and block copolymers. Second, we review some experimental characterization methods for investigating AQP incorporation including freeze-fracture transmission electron microscopy, fluorescence correlation spectroscopy, stopped-flow light scattering, and small-angle X-ray scattering. Third, we focus on recent efforts in embedding reconstituted AQPs in membrane designs that are based on conventional thin film interfacial polymerization techniques. Finally, we describe some new developments in interfacial polymerization using polyhedral oligomeric silsesquioxane cages for increasing the physical and chemical durability of thin film composite membranes.

PET/CT Based In Vivo Evaluation of 64Cu Labelled Nanodiscs in Tumor Bearing Mice.

64Cu radiolabelled nanodiscs based on the 11 α-helix MSP1E3D1 protein and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine lipids were, for the first time, followed in vivo by positron emission tomography for evaluating the biodistribution of nanodiscs. A cancer tumor bearing mouse model was used for the investigations, and it was found that the approximately 13 nm nanodiscs, due to their size, permeate deeply into cancer tissue. This makes them promising candidates for both drug delivery purposes and as advanced imaging agents. For the radiolabelling, a simple approach for 64Cu radiolabelling of proteins via a chelating agent, DOTA, was developed. The reaction was performed at sufficiently mild conditions to be compatible with labelling of the protein part of a lipid-protein particle while fully conserving the particle structure including the amphipathic protein fold.