PML and PML-like exonucleases restrict retrotransposons in jawed vertebrates.

We have uncovered a role for the promyelocytic leukemia (PML) gene and novel PML-like DEDDh exonucleases in the maintenance of genome stability through the restriction of LINE-1 (L1) retrotransposition in jawed vertebrates. Although the mammalian PML protein forms nuclear bodies, we found that the spotted gar PML ortholog and related proteins in fish function as cytoplasmic DEDDh exonucleases. In contrast, PML proteins from amniote species localized both to the cytoplasm and formed nuclear bodies. We also identified the PML-like exon 9 (Plex9) genes in teleost fishes that encode exonucleases. Plex9 proteins resemble TREX1 but are unique from the TREX family and share homology to gar PML. We also characterized the molecular evolution of TREX1 and the first non-mammalian TREX1 homologs in axolotl. In an example of convergent evolution and akin to TREX1, gar PML and zebrafish Plex9 proteins suppressed L1 retrotransposition and could complement TREX1 knockout in mammalian cells. Following export to the cytoplasm, the human PML-I isoform also restricted L1 through its conserved C-terminus by enhancing ORF1p degradation through the ubiquitin-proteasome system. Thus, PML first emerged as a cytoplasmic suppressor of retroelements, and this function is retained in amniotes despite its new role in the assembly of nuclear bodies.

Characterization of the structure and self-assembly of two distinct class IB hydrophobins.

Hydrophobins are small proteins secreted by fungi that accumulate at interfaces, modify surface hydrophobicity, and self-assemble into large amyloid-like structures. These unusual properties make hydrophobins an attractive target for commercial applications as emulsifiers and surface modifying agents. Hydrophobins have diverse sequences and tertiary structures, complicating attempts to characterize how they function. Here we describe the atomic resolution structure of the unusual hydrophobin SLH4 (86 aa, 8.4 kDa) and compare its function to another hydrophobin, SC16 (99 aa, 10.2 kDa). Despite containing only one charged residue, SLH4 has a similar structure to SC16 yet has strikingly different rodlet morphology, propensity to self-assemble, and preferred assembly conditions. Secondary structure analysis of both SC16 and SLH4 suggest that during rodlet formation residues in the first intercysteine loop undergo conformational changes. This work outlines a representative structure for class IB hydrophobins and illustrates how hydrophobin surface properties govern self-assembly, which provides context to rationally select hydrophobins for applications as surface modifiers. KEY POINTS: • The atomic-resolution structure of the hydrophobin SLH4 was determined using nuclear magnetic resonance spectroscopy. • The structure of SLH4 outlines a representative structure for class IB hydrophobins. • The assembly characteristics of SLH4 and SC16 are distinct, outlining how surface properties of hydrophobins influence their function.

Structural insights into TAZ2 domain-mediated CBP/p300 recruitment by transactivation domain 1 of the lymphopoietic transcription factor E2A.

The E-protein transcription factors guide immune cell differentiation, with E12 and E47 (hereafter called E2A) being essential for B-cell specification and maturation. E2A and the oncogenic chimera E2A-PBX1 contain three transactivation domains (ADs), with AD1 and AD2 having redundant, independent, and cooperative functions in a cell-dependent manner. AD1 and AD2 both mediate their functions by binding to the KIX domain of the histone acetyltransferase paralogues CREB-binding protein (CBP) and E1A-binding protein P300 (p300). This interaction is necessary for B-cell maturation and oncogenesis by E2A-PBX1 and occurs through conserved ΦXXΦΦ motifs (with Φ denoting a hydrophobic amino acid) in AD1 and AD2. However, disruption of this interaction via mutation of the KIX domain in CBP/p300 does not completely abrogate binding of E2A and E2A-PBX1. Here, we determined that E2A-AD1 and E2A-AD2 also interact with the TAZ2 domain of CBP/p300. Characterization of the TAZ2:E2A-AD1(1-37) complex indicated that E2A-AD1 adopts an α-helical structure and uses its ΦXXΦΦ motif to bind TAZ2. Whereas this region overlapped with the KIX recognition region, key KIX-interacting E2A-AD1 residues were exposed, suggesting that E2A-AD1 could simultaneously bind both the KIX and TAZ2 domains. However, we did not detect a ternary complex involving E2A-AD1, KIX, and TAZ2 and found that E2A containing both intact AD1 and AD2 is required to bind to CBP/p300. Our findings highlight the structural plasticity and promiscuity of E2A-AD1 and suggest that E2A binds both the TAZ2 and KIX domains of CBP/p300 through AD1 and AD2.

Expression, purification, and refolding of diverse class IB hydrophobins.

Hydrophobins are low molecular weight proteins secreted by fungi that are extremely surface-active and able to self-assemble into larger structures. Due to their unusual biochemical properties, hydrophobins are an attractive target for commercial applications such as drug emulsification and surface modification. When produced in E. coli, hydrophobins are often not soluble and need to be refolded. In this work we use SHuffle T7 Express E. coli coupled with glutathione redox buffers to produce and refold four distinct class IB hydrophobins that originate from Phanerochaete carnosa (PC1), Wallemia ichthyophaga (WI1), Serpula lacrymans (SL1), and Schizophyllum commune (SC16). Proper refolding and function of these purified hydrophobins was confirmed using nuclear magnetic resonance spectroscopy and thioflavin T assays. These results indicate that class IB hydrophobins can be consistently produced and purified from E. coli, aiding future structural and biochemical studies that require highly pure hydrophobins.

Periodicity in Structure, Bonding, and Reactivity for p-Block Complexes of a Geometry Constraining Triamide Ligand.

The use of pincer ligands to access non-VSEPR geometries at main-group centers is an emerging strategy for eliciting new stoichiometric and catalytic reactivity. As part of this effort, several different tridentate trianionic substituents have to date been employed at a range of different central elements, providing a patchwork dataset that precludes rigorous structure-function correlation. An analysis of periodic trends in structure (solid, solution, and computation), bonding, and reactivity based on systematic variation of the central element (P, As, Sb, or Bi) with retention of a single tridentate triamide substituent is reported herein. In this homologous series, the central element can adopt either a bent or planar geometry. The tendency to adopt planar geometries increases descending the group with the phosphorus triamide (1) and its arsenic congener (2) exhibiting bent conformations, and the antimony (3) and bismuth (4) analogues exhibiting a predominantly planar structure in solution. This trend has been rationalized using an energy decomposition analysis. A rare phase-dependent dynamic covalent dimerization was observed for 3 and the associated thermodynamic parameters were established quantitatively. Planar geometries were found to engender lower LUMO energies and smaller band gaps than bent ones, resulting in different reactivity patterns. These results provide a benchmark dataset to guide further research in this rapidly emerging area.

Properties of a family 56 carbohydrate-binding module and its role in the recognition and hydrolysis of ß-1,3-glucan.

BH0236 from Bacillus halodurans is a multimodular ß-1,3-glucanase comprising an N-terminal family 81 glycoside hydrolase catalytic module, an internal family 6 carbohydrate-binding module (CBM) that binds the nonreducing end of ß-1,3-glucan chains, and an uncharacterized C-terminal module classified into CBM family 56. Here, we determined that this latter CBM, BhCBM56, bound the soluble ß-1,3-glucan laminarin with a dissociation constant (Kd ) of ∼26 µm and displayed higher affinity for insoluble ß-1,3-glucans with Kd values of ∼2-10 µm but lacked affinity for ß-1,3-glucooligosaccharides. The X-ray crystal structure of BhCBM56 and NMR-derived chemical shift mapping of the binding site revealed a ß-sandwich fold, with the face of one ß-sheet possessing the ß-1,3-glucan-binding surface. On the basis of the functional and structural properties of BhCBM56, we propose that it binds a quaternary polysaccharide structure, most likely the triple helix adopted by polymerized ß-1,3-glucans. Consistent with the BhCBM56 and BhCBM6/56 binding profiles, deletion of the CBM56 from BH0236 decreased activity of the enzyme on the insoluble ß-1,3-glucan curdlan but not on soluble laminarin; additional deletion of the CBM6 also did not affect laminarin degradation but further decreased curdlan hydrolysis. The pseudo-atomic solution structure of BH0236 determined by small-angle X-ray scattering revealed structural insights into the nature of avid binding by the BhCBM6/56 pair and how the orientation of the active site in the catalytic module factors into recognition and degradation of ß-1,3-glucans. Our findings reinforce the notion that catalytic modules and their cognate CBMs have complementary specificities, including targeting of polysaccharide quaternary structure.

Structure of the Single-lobe Myosin Light Chain C in Complex with the Light Chain-binding Domains of Myosin-1C Provides Insights into Divergent IQ Motif Recognition.

Myosin light chains are key regulators of class 1 myosins and typically comprise two domains, with calmodulin being the archetypal example. They bind IQ motifs within the myosin neck region and amplify conformational changes in the motor domain. A single lobe light chain, myosin light chain C (MlcC), was recently identified and shown to specifically bind to two sequentially divergent IQ motifs of the Dictyostelium myosin-1C. To provide a molecular basis of this interaction, the structures of apo-MlcC and a 2:1 MlcC·myosin-1C neck complex were determined. The two non-functional EF-hand motifs of MlcC pack together to form a globular four-helix bundle that opens up to expose a central hydrophobic groove, which interacts with the N-terminal portion of the divergent IQ1 and IQ2 motifs. The N- and C-terminal regions of MlcC make critical contacts that contribute to its specific interactions with the myosin-1C divergent IQ motifs, which are contacts that deviate from the traditional mode of calmodulin-IQ recognition.

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.

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.

Functional redundancy between the transcriptional activation domains of E2A is mediated by binding to the KIX domain of CBP/p300.

The E-protein transcription factors play essential roles in lymphopoiesis, with E12 and E47 (hereafter called E2A) being particularly important in B cell specification and maturation. The E2A gene is also involved in a chromosomal translocation that results in the leukemogenic oncoprotein E2A-PBX1. The two activation domains of E2A, AD1 and AD2, display redundant, independent, and cooperative functions in a cell-dependent manner. AD1 of E2A functions by binding the transcriptional co-activator CBP/p300; this interaction is required in oncogenesis and occurs between the conserved ϕ-x-x-ϕ-ϕ motif in AD1 and the KIX domain of CBP/p300. However, co-activator recruitment by AD2 has not been characterized. Here, we demonstrate that the first of two conserved ϕ-x-x-ϕ-ϕ motifs within AD2 of E2A interacts at the same binding site on KIX as AD1. Mutagenesis uncovered a correspondence between the KIX-binding affinity of AD2 and transcriptional activation. Although AD2 is dispensable for oncogenesis, experimentally increasing the affinity of AD2 for KIX uncovered a latent potential to mediate immortalization of primary hematopoietic progenitors by E2A-PBX1. Our findings suggest that redundancy between the two E2A activation domains with respect to transcriptional activation and oncogenic function is mediated by binding to the same surface of the KIX domain of CBP/p300.

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.

Qualitative and Quantitative Characterization of Protein-Carbohydrate Interactions by NMR Spectroscopy.

Solution-state nuclear magnetic resonance (NMR) spectroscopy can be used to monitor protein-carbohydrate interactions. Two-dimensional 1H-15N heteronuclear single quantum coherence (HSQC)-based techniques described in this chapter can be used quickly and effectively to screen a set of possible carbohydrate-binding partners, to quantify the dissociation constant (Kd) of any identified interactions, and to the map the carbohydrate-binding site on the structure of a protein. Here, we describe the titration of a family 32 carbohydrate-binding module from Clostridium perfringens (CpCBM32) with the monosaccharide N-acetylgalactosamine (GalNAc), in which we calculate the apparent dissociation of the interaction and map the GalNAc binding site onto the structure of CpCBM32. This approach can be applied to other CBM- and protein-ligand systems.

The C-terminal transactivation domain of MITF interacts promiscuously with co-activator CBP/p300.

The microphthalmia-associated transcription factor (MITF) is one of four closely related members of the MiT/TFE family (TFEB, TFE3, TFEC) that regulate a wide range of cellular processes. MITF is a key regulator of melanocyte-associated genes, and essential to proper development of the melanocyte cell lineage. Abnormal MITF activity can contribute to the onset of several diseases including melanoma, where MITF is an amplified oncogene. To enhance transcription, MITF recruits the co-activator CREB-binding protein (CBP) and its homolog p300 to gene promoters, however the molecular determinants of their interaction are not yet fully understood. Here, we characterize the interactions between the C-terminal MITF transactivation domain and CBP/p300. Using NMR spectroscopy, protein pulldown assays, and isothermal titration calorimetry we determine the C-terminal region of MITF is intrinsically disordered and binds with high-affinity to both TAZ1 and TAZ2 of CBP/p300. Mutagenesis studies revealed two conserved motifs within MITF that are necessary for TAZ2 binding and critical for MITF-dependent transcription of a reporter gene. Finally, we observe the transactivation potential of the MITF C-terminal region is reliant on the N-terminal transactivation domain for function. Taken together, our study helps elucidate the molecular details of how MITF interacts with CBP/p300 through multiple redundant interactions that lend insight into MITF function in melanocytes and melanoma.

ß-Catenin interacts with the TAZ1 and TAZ2 domains of CBP/p300 to activate gene transcription.

The transcriptional co-regulator ß-catenin is a critical member of the canonical Wnt signaling pathway, which plays an important role in regulating cell fate. Deregulation of the Wnt/ß-catenin pathway is characteristic in the development of major types of cancer, where accumulation of ß-catenin promotes cancer cell proliferation and renewal. ß-catenin gene expression is facilitated through recruitment of co-activators such as histone acetyltransferases CBP/p300; however, the mechanism of their interaction is not fully understood. Here we investigate the interaction between the C-terminal transactivation domain of ß-catenin and CBP/p300. Using a combination of pulldown assays, isothermal titration calorimetry, and nuclear resonance spectroscopy we determine the disordered C-terminal region of ß-catenin binds promiscuously to the TAZ1 and TAZ2 domains of CBP/p300. We then map the interaction site of the C-terminal ß-catenin transactivation domain onto TAZ1 and TAZ2 using chemical-shift perturbation studies. Luciferase-based gene reporter assays indicate Asp750-Leu781 is critical to ß-catenin gene activation, and mutagenesis revealed that acidic and hydrophobic residues within this region are necessary to maintain TAZ1 binding. These results outline a mechanism of Wnt/ß-catenin gene regulation that underlies cell development and provides a framework to develop methods to block ß-catenin dependent signaling.

Structural basis of CBP/p300 recruitment by the microphthalmia-associated transcription factor.

The microphthalmia-associated transcription factor (MITF) is a master regulator of the melanocyte cell lineage. Aberrant MITF activity can lead to multiple malignancies including skin cancer, where it modulates the progression and invasiveness of melanoma. MITF-regulated gene expression requires recruitment of the transcriptional co-regulator CBP/p300, but details of this process are not fully defined. In this study, we investigate the structural and functional interaction between the MITF N-terminal transactivation domain (MITFTAD) and CBP/p300. Using pulldown assays and nuclear magnetic resonance spectroscopy we determined that MITFTAD is intrinsically disordered and binds to the TAZ1 and TAZ2 domains of CBP/p300 with moderate affinity. The solution-state structure of the MITFTAD:TAZ2 complex reveals that MITF interacts with a hydrophobic surface of TAZ2, while remaining somewhat dynamic. Peptide array and mutagenesis experiments determined that an acidic motif is integral to the MITFTAD:TAZ2 interaction and is necessary for transcriptional activity of MITF. Peptides that bind to the same surface of TAZ2 as MITFTAD, such as the adenoviral protein E1A, are capable of displacing MITF from TAZ2 and inhibiting transactivation. These findings provide insight into co-activator recruitment by MITF that are fundamental to our understanding of MITF targeted gene regulation and melanoma biology.

The N-terminal tail of the hydrophobin SC16 is not required for rodlet formation.

Hydrophobins are small proteins that are secreted by fungi, accumulate at interfaces, modify surface hydrophobicity, and self-assemble into large amyloid-like structures. These unusual properties make hydrophobins an attractive target for commercial applications as green emulsifiers and surface modifying agents. Hydrophobins have diverse sequences and tertiary structures, and depending on the hydrophobin, different regions of their structure have been proposed to be required for self-assembly. To provide insight into the assembly process, we determined the first crystal structure of a class I hydrophobin, SC16. Based on the crystal structure, we identified a putative intermolecular contact that may be important for rodlet assembly and was formed in part by the N-terminal tail of SC16. Surprisingly, removal of the N-terminal tail did not influence the self-assembly kinetics of SC16 or the morphology of its rodlets. These results suggest that other regions of this hydrophobin class are required for rodlet formation and indicate that the N-terminal tail of SC16 is amenable to modification so that functionalized hydrophobin assemblies can be created.

Bactericidal Activity of Carvacrol against Streptococcus pyogenes Involves Alteration of Membrane Fluidity and Integrity through Interaction with Membrane Phospholipids.

BACKGROUND: Carvacrol, a mono-terpenoid phenol found in herbs, such as oregano and thyme, has excellent antibacterial properties against Streptococcus pyogenes. However, its mechanism of bactericidal activity on S. pyogenes has not been elucidated. OBJECTIVES: This study investigated the bactericidal mechanism of carvacrol using three strains of S. pyogenes. METHODS: Flow cytometry (FCM) experiments were conducted to determine carvacrol's membrane permeabilization and cytoplasmic membrane depolarization activities. Protoplasts of S. pyogenes were used to investigate carvacrol's effects on the membrane, followed by gel electrophoresis. The carvacrol-treated protoplasts were examined by transmission electron microscopy (TEM) to observe ultrastructural morphological changes. The fluidity of the cell membrane was measured by steady-state fluorescence anisotropy. Thin-layer chromatographic (TLC) profiling was conducted to study the affinity of carvacrol for membrane phospholipids. RESULTS: Increased membrane permeability and decreased membrane potential from FCM and electron microscopy observations revealed that carvacrol killed the bacteria primarily by disrupting membrane integrity, leading to whole-cell lysis. Ultra-structural morphological changes in the membrane induced by carvacrol over a short period were confirmed using the S. pyogenes protoplast and membrane isolate models in vitro. In addition, changes in the other biophysical properties of the bacterial membrane, including concentration- and time-dependent increased fluidity, were observed. TLC experiments showed that carvacrol preferentially interacts with membrane phosphatidylglycerol (P.G.), phosphatidylethanolamine (P.E.), and cardiolipins (CL). CONCLUSIONS: Carvacrol exhibited rapid bactericidal action against S. pyogenes by disrupting the bacterial membrane and increasing permeability, possibly due to affinity with specific membrane phospholipids, such as P.E., P.G., and CL. Therefore, the bactericidal concentration of carvacrol (250 µg/mL) could be used to develop safe and efficacious natural health products for managing streptococcal pharyngitis or therapeutic applications.

Microbial rhodoquinone biosynthesis proceeds via an atypical RquA-catalyzed amino transfer from S-adenosyl-L-methionine to ubiquinone.

Rhodoquinone (RQ) is a close analogue of ubiquinone (UQ) that confers diverse bacterial and eukaryotic taxa the ability to utilize fumarate as an electron acceptor in hypoxic conditions. The RquA protein, identified in a Rhodospirillum rubrum RQ-deficient mutant, has been shown to be required for RQ biosynthesis in bacteria. In this report, we demonstrate that RquA, homologous to SAM-dependent methyltransferases, is necessary and sufficient to catalyze RQ biosynthesis from UQ in vitro. Remarkably, we show that RquA uses SAM as the amino group donor in a substitution reaction that converts UQ to RQ. In contrast to known aminotransferases, RquA does not use pyridoxal 5'-phosphate (PLP) as a coenzyme, but requires the presence of Mn2+ as a cofactor. As these findings reveal, RquA provides an example of a non-canonical SAM-dependent enzyme that does not catalyze methyl transfer, instead it uses SAM in an atypical amino transfer mechanism.

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.

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.