David N. Langelaan

Structural insight into G-protein coupled receptor binding by apelin.

Apelin peptides are the cognate ligands for the G-protein coupled receptor APJ, with functions in the cardiovascular and central nervous systems, in glucose metabolism and as a human immunodeficiency virus (HIV-1) coreceptor. Apelin is found in 13-36 residue forms in vivo. The structures of five isoforms of apelin at physiological versus low (5-6 degrees C) temperature are compared here using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, demonstrating increased structure at low temperature. Far-ultraviolet (UV) CD spectra are predominantly random coil for apelin isoforms, but are convoluted by unusual bands from the C-terminal phenylalanine side chain. These bands, assigned using F13A-apelin-13, are accentuated at 5 degrees C and imply conformational restriction. At 35 degrees C, the R6-L9 region of apelin-17 is well structured, consistent with previous mutagenesis results showing necessity of this segment for apelin-APJ binding and activation. At 5 degrees C, R6-L9 retains its structuring while the functionally critical C-terminal G13-F17 region also becomes highly structured. Type IV beta-turns and some polyproline-II structure alongside F17 side chain motional reduction correlate well with CD spectral properties. Cis-trans peptide bond isomerization at P14 and P16 produces two sequentially assignable conformers (both trans:both cis approximately 4:1) alongside less populated conformers. Chemical shift assignment of apelin-12, -13 and pyroglutamate-apelin-13 implies highly similar structuring and the same isomerization at the C-terminus. Based on the apelin-17 structure, a two-step binding and activation mechanism is hypothesized.

Improved Helix and Kink Characterization in Membrane Proteins Allows Evaluation of Kink Sequence Predictors

Although the α-helical secondary structure of proteins is well-defined, the exact causes and structures of helical kinks are not. This is especially important for transmembrane (TM) helices of integral membrane proteins, many of which contain kinks providing functional diversity despite predominantly helical structure. We have developed a Monte Carlo method based algorithm, MC-HELAN, to determine helical axes alongside positions and angles of helical kinks. Analysis of all nonredundant high-resolution α-helical membrane protein structures (842 TM helices from 205 polypeptide chains) revealed kinks in 64% of TM helices, demonstrating that a significantly greater proportion of TM helices are kinked than those indicated by previous analyses. The residue proline is over-represented by a factor >5 if it is two or three residues C-terminal to a bend. Prolines also cause kinks with larger kink angles than other residues. However, only 33% of TM kinks are in proximity to a proline. Machine learning techniques were used to test for sequence-based predictors of kinks. Although kinks are somewhat predicted by sequence, kink formation appears to be driven predominantly by other factors. This study provides an improved view of the prevalence and architecture of kinks in helical membrane proteins and highlights the fundamental inaccuracy of the typical topological depiction of helical membrane proteins as series of ideal helices.

Headgroup-dependent membrane catalysis of apelin-receptor interactions is likely.

Apelin is the peptidic ligand for the G-protein-coupled receptor APJ. The apelin−APJ system is important in cardiovascular regulation, fluid homeostasis, and angiogenesis, among other roles. In this study, we investigate interactions between apelin and membrane-mimetic micelles of the detergents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG). Far-ultraviolet circular dichroism spectropolarimetry and diffusion-ordered spectroscopy indicate that apelin peptides bind to micelles of the anionic detergents SDS and LPPG much more favorably than to zwitterionic DPC micelles. Nuclear magnetic resonance spectroscopy allowed full characterization of the interactions of apelin-17 with SDS micelles. Titration with paramagnetic agents and structural determination of apelin-17 in SDS indicate that R6−K12 is highly structured, with R6−L9 directly interacting with headgroups of the micelle. Type I β-turns are initiated between R6 and L9, and a well-defined type IV β-turn is initiated at S10. Furthermore, binding of apelin-17 to SDS micelles causes structuring of M15-F17, with no evidence for direct binding of this region to the micelles. These results are placed into the context of the membrane catalysis hypothesis for peptide−receptor binding, and a hypothetical mechanism of APJ binding and activation by apelin is advanced.

Structural features of the apelin receptor N-terminal tail and first transmembrane segment implicated in ligand binding and receptor trafficking.

G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins with rich functional diversity. Signaling through the apelin receptor (AR or APJ) influences the cardiovascular system, central nervous system and glucose regulation. Pathophysiological involvement of apelin has been shown in atherosclerosis, cancer, human immunodeficiency virus-1 (HIV-1) infection and obesity. Here, we present the high-resolution nuclear magnetic resonance (NMR) spectroscopy-based structure of the N-terminus and first transmembrane (TM) segment of AR (residues 1-55, AR55) in dodecylphosphocholine micelles. AR55 consists of two disrupted helices, spanning residues D14-K25 and A29-R55(1.59). Molecular dynamics (MD) simulations of AR built from a hybrid of experimental NMR and homology model-based restraints allowed validation of the AR55 structure in the context of the full-length receptor in a hydrated bilayer. AR55 structural features were functionally probed using mutagenesis in full-length AR through monitoring of apelin-induced extracellular signal-regulated kinase (ERK) phosphorylation in transiently transfected human embryonic kidney (HEK) 293A cells. Residues E20 and D23 form an extracellular anionic face and interact with lipid headgroups during MD simulations in the absence of ligand, producing an ideal binding site for a cationic apelin ligand proximal to the membrane-water interface, lending credence to membrane-catalyzed apelin-AR binding. In the TM region of AR55, N46(1.50) is central to a disruption in helical character. G42(1.46), G45(1.49) and N46(1.50), which are all involved in the TM helical disruption, are essential for proper trafficking of AR. In summary, we introduce a new correlative NMR spectroscopy and computational biochemistry methodology and demonstrate its utility in providing some of the first high-resolution structural information for a peptide-activated GPCR TM domain.

Membrane catalysis of peptide-receptor binding

The membrane catalysis hypothesis states that a peptide ligand activates its target receptor after an initial interaction with the surrounding membrane. Upon membrane binding and interaction, the ligand is structured such that receptor binding and activation is encouraged. As evidence for this hypothesis, there are numerous studies concerning the conformation that peptides adopt in membrane mimetic environments. This mini-review analyzes the features of ligand peptides with an available high-resolution membrane-induced structure and a characterized membrane-binding region. At the peptide-membrane interface, both amphipathic helices and turn structures are commonly formed in peptide ligands and both hydrophobic and electrostatic interactions can be responsible for membrane binding. Apelin is the ligand to the G-protein coupled receptor (GPCR) named APJ, with various important physiological effects, which we have recently characterized both in solution and bound to anionic micelles. The structural changes that apelin undergoes when binding to micelles provide strong evidence for membrane catalysis of apelin-APJ interactions.

Structure of a 1.5-MDa adhesin that binds its Antarctic bacterium to diatoms and ice

Structure of a bacterial adhesin reveals its role in forming a mixed-species symbiotic community with diatoms on sea ice. Bacterial adhesins are modular cell-surface proteins that mediate adherence to other cells, surfaces, and ligands. The Antarctic bacterium Marinomonas primoryensis uses a 1.5-MDa adhesin comprising over 130 domains to position it on ice at the top of the water column for better access to oxygen and nutrients. We have reconstructed this 0.6-μm-long adhesin using a “dissect and build” structural biology approach and have established complementary roles for its five distinct regions. Domains in region I (RI) tether the adhesin to the type I secretion machinery in the periplasm of the bacterium and pass it through the outer membrane. RII comprises ~120 identical immunoglobulin-like β-sandwich domains that rigidify on binding Ca2+ to project the adhesion regions RIII and RIV into the medium. RIII contains ligand-binding domains that join diatoms and bacteria together in a mixed-species community on the underside of sea ice where incident light is maximal. RIV is the ice-binding domain, and the terminal RV domain contains several “repeats-in-toxin” motifs and a noncleavable signal sequence that target proteins for export via the type I secretion system. Similar structural architecture is present in the adhesins of many pathogenic bacteria and provides a guide to finding and blocking binding domains to weaken infectivity.

Biophysical characterization of G-protein coupled receptor-peptide ligand binding.

G-protein coupled receptors (GPCRs) are ubiquitous membrane proteins allowing intracellular responses to extracellular factors that range from photons of light to small molecules to proteins. Despite extensive exploitation of GPCRs as therapeutic targets, biophysical characterization of GPCR-ligand interactions remains challenging. In this minireview, we focus on techniques that have been successfully used for structural and biophysical characterization of peptide ligands binding to their cognate GPCRs. The techniques reviewed include solution-state nuclear magnetic resonance (NMR) spectroscopy, solid-state NMR, X-ray diffraction, fluorescence spectroscopy and single-molecule fluorescence methods, flow cytometry, surface plasmon resonance, isothermal titration calorimetry, and atomic force microscopy. The goal herein is to provide a cohesive starting point to allow selection of techniques appropriate to the elucidation of a given GPCR-peptide interaction.

Identification of the first transcriptional activator of an archaellum operon in a euryarchaeon

The archaellum is the swimming organelle of the third domain, the Archaea. In the euryarchaeon Methanococcus maripaludis, genes involved in archaella formation, including the three archaellins flaB1, flaB2 and flaB3, are mainly located in the fla operon. Previous studies have shown that transcription of fla genes and expression of Fla proteins are regulated under different growth conditions. In this study, we identify MMP1718 as the first transcriptional activator that directly regulates the fla operon in M. maripaludis. Mutants carrying an in‐frame deletion in mmp1718 did not express FlaB2 detected by western blotting. Quantitative reverse transcription PCR analysis of purified RNA from the Δmmp1718 mutant showed that transcription of flaB2 was negligible compared to wildtype cells. In addition, no archaella were observed on the cell surface of the Δmmp1718 mutant. FlaB2 expression and archaellation were restored when the Δmmp1718 mutant was complemented with mmp1718 in trans. Electrophoretic motility shift assay and isothermal titration calorimetry results demonstrated the specific binding of purified MMP1718 to DNA fragments upstream of the fla promoter. Four 6 bp consensus sequences were found immediately upstream of the fla promoter and are considered the putative MMP1718‐binding sites. Herein, we designate MMP1718 as EarA, the first euryarchaeal archaellum regulator.

Characterization of a Basidiomycota hydrophobin reveals the structural basis for a high-similarity Class I subdivision

Class I hydrophobins are functional amyloids secreted by fungi. They self-assemble into organized films at interfaces producing structures that include cellular adhesion points and hydrophobic coatings. Here, we present the first structure and solution properties of a unique Class I protein sequence of Basidiomycota origin: the Schizophyllum commune hydrophobin SC16 (hyd1). While the core β-barrel structure and disulphide bridging characteristic of the hydrophobin family are conserved, its surface properties and secondary structure elements are reminiscent of both Class I and II hydrophobins. Sequence analyses of hydrophobins from 215 fungal species suggest this structure is largely applicable to a high-identity Basidiomycota Class I subdivision (IB). To validate this prediction, structural analysis of a comparatively distinct Class IB sequence from a different fungal order, namely the Phanerochaete carnosa PcaHyd1, indicates secondary structure properties similar to that of SC16. Together, these results form an experimental basis for a high-identity Class I subdivision and contribute to our understanding of functional amyloid formation.

Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.