Filippo Mancia

Ligand sensitivity in dimeric associations of the serotonin 5HT2c receptor

G‐protein‐coupled receptors (GPCRs) respond to external stimuli by activating heterotrimeric G proteins inside the cell. There is increasing evidence that many GPCRs exist as dimers or higher oligomers, but the biochemical nature of such dimers and what roles they have, if any, in signal transduction remains unclear. We conducted a comprehensive study of dimerization of the 5HT2c serotonin receptor using disulphide‐trapping experiments. We found a dimer interface between transmembrane (TM) helices IV and V that is markedly sensitive to the state of receptor activation. This dimer seems to be quasisymmetrical in interfacial geometry and asymmetrical in its association with its cognate Gα protein. We also found a second interface at TM I helices, which is insensitive to the state of activation.

Structures from anomalous diffraction of native biological macromolecules

Finessing Crystal Analysis Protein crystallography has revolutionized our understanding of a whole variety of biological processes (see the Perspective by Evans). In crystallography, the measure of agreement between the data and the calculated model is not on the same scale as the measure of data quality, making it challenging to choose an optimal high resolution limit beyond which the data should be discarded. Now, Karplus and Diederichs (p. 1030) introduce a statistical model that assesses agreement of model and data accuracy on the same scale. Determining the structures of biological macromolecules by x-ray crystallography requires solving the phase problem. The two techniques that dominate phase evaluation (multi- and single-wavelength anomalous diffraction) rely on element-specific scattering from incorporated heavy atoms. Liu et al. (p. 1033) present procedures for routine structure determination of native proteins with no heavy atom incorporation. The technique, which relies on combining data from multiple crystals, was used to determine the structures of four native proteins, including a 1200-residue complex. Don’t get MAD or be SAD; try lower energy. Crystal structure analyses for biological macromolecules without known structural relatives entail solving the crystallographic phase problem. Typical de novo phase evaluations depend on incorporating heavier atoms than those found natively; most commonly, multi- or single-wavelength anomalous diffraction (MAD or SAD) experiments exploit selenomethionyl proteins. Here, we realize routine structure determination using intrinsic anomalous scattering from native macromolecules. We devised robust procedures for enhancing the signal-to-noise ratio in the slight anomalous scattering from generic native structures by combining data measured from multiple crystals at lower-than-usual x-ray energy. Using this multicrystal SAD method (5 to 13 equivalent crystals), we determined structures at modest resolution (2.8 to 2.3 angstroms) for native proteins varying in size (127 to 1148 unique residues) and number of sulfur sites (3 to 28). With no requirement for heavy-atom incorporation, such experiments provide an attractive alternative to selenomethionyl SAD experiments.

The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins

The New York Consortium on Membrane Protein Structure (NYCOMPS) was formed to accelerate the acquisition of structural information on membrane proteins by applying a structural genomics approach. NYCOMPS comprises a bioinformatics group, a centralized facility operating a high-throughput cloning and screening pipeline, a set of associated wet labs that perform high-level protein production and structure determination by x-ray crystallography and NMR, and a set of investigators focused on methods development. In the first three years of operation, the NYCOMPS pipeline has so far produced and screened 7,250 expression constructs for 8,045 target proteins. Approximately 600 of these verified targets were scaled up to levels required for structural studies, so far yielding 24 membrane protein crystals. Here we describe the overall structure of NYCOMPS and provide details on the high-throughput pipeline.

Production and characterization of monoclonal antibodies sensitive to conformation in the 5HT2c serotonin receptor

mAbs that are sensitive to protein conformation can be helpful in studies of protein structure and function; in particular, mAb fragments are useful reagents in membrane protein crystallization. We immunized mice with the rat 5HT2c serotonin receptor and derived clonal hybridoma cells, which we tested for specific antigen reactivity by using the complementarity of purified protein from bacteria and receptor-embedded mammalian cell membranes. Nine mAbs met our criteria for specificity, affinity, and sensitivity to conformational features. Epitopes were mapped in various additional tests. Five of the nine mAbs have cytoplasmic epitopes, and two of these are sensitive to the ligand state of the receptor. These properties should be useful both for structural analysis and in probes of function.

High-throughput expression and purification of membrane proteins.

High-throughput (HT) methodologies have had a tremendous impact on structural biology of soluble proteins. High-resolution structure determination relies on the ability of the macromolecule to form ordered crystals that diffract X-rays. While crystallization remains somewhat empirical, for a given protein, success is proportional to the number of conditions screened and to the number of variants trialed. HT techniques have greatly increased the number of targets that can be trialed and the rate at which these can be produced. In terms of number of structures solved, membrane proteins appear to be lagging many years behind their soluble counterparts. Likewise, HT methodologies for production and characterization of these hydrophobic macromolecules are only now emerging. Presented here is an HT platform designed exclusively for membrane proteins that has processed over 5000 targets.

Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation

A bacterial defense mechanism Polymyxins are antibiotics that disrupt the bacterial cell membrane and are used to treat multidrug-resistant infections. A bacterial enzyme called ArnT can mediate resistance to polymyxins by transferring a sugar group from a lipid carrier to lipid A, a component of the bacterial outer membrane. Petrou et al. described structures of ArnT alone and in complex with a lipid carrier and identified a cavity where lipid A probably binds. Insights into the enzyme mechanism could be exploited to design drugs that combat polymyxin resistance. Science, this issue p. 608 Structural studies elucidate the mechanism of a reaction that contributes to antibiotic resistance in Gram-negative bacteria. Polymyxins are antibiotics used in the last line of defense to combat multidrug-resistant infections by Gram-negative bacteria. Polymyxin resistance arises through charge modification of the bacterial outer membrane with the attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose to lipid A, a reaction catalyzed by the integral membrane lipid-to-lipid glycosyltransferase 4-amino-4-deoxy-l-arabinose transferase (ArnT). Here, we report crystal structures of ArnT from Cupriavidus metallidurans, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.8 and 3.2 angstrom resolution, respectively. The structures show cavities for both lipidic substrates, which converge at the active site. A structural rearrangement occurs on undecaprenyl phosphate binding, which stabilizes the active site and likely allows lipid A binding. Functional mutagenesis experiments based on these structures suggest a mechanistic model for ArnT family enzymes.

Native Serotonin 5-HT2C Receptors are Expressed as Homodimers on the Apical Surface of Choroid Plexus Epithelial Cells.

G protein–coupled receptors (GPCRs) are a prominent class of plasma membrane proteins that regulate physiologic responses to a wide variety of stimuli and therapeutic agents. Although GPCR oligomerization has been studied extensively in recombinant cells, it remains uncertain whether native receptors expressed in their natural cellular environment are monomers, dimers, or oligomers. The goal of this study was to determine the monomer/oligomer status of a native GPCR endogenously expressed in its natural cellular environment. Native 5-HT2C receptors in choroid plexus epithelial cells were evaluated using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH). An anti–5-HT2C fragment antigen binding protein was used to label native 5-HT2C receptors. A known monomeric receptor (CD-86) served as a control for decoding the oligomer status of native 5-HT2C receptors by molecular brightness analysis. FCS with PCH revealed molecular brightness values for native 5-HT2C receptors equivalent to the molecular brightness of a homodimer. 5-HT2C receptors displayed a diffusion coefficient of 5 × 10−9 cm2/s and were expressed at 32 receptors/μm2 on the apical surface of choroid plexus epithelial cells. The functional significance and signaling capabilities of the homodimer were investigated in human embryonic kidney 293 cells using agonists that bind in a wash-resistant manner to one or both protomers of the homodimer. Whereas agonist binding to one protomer resulted in G protein activation, maximal stimulation required occupancy of both protomers. This study is the first to demonstrate the homodimeric structure of 5-HT2C receptors endogenously expressed in their native cellular environment, and identifies the homodimer as a functional signaling unit.

Structure of the STRA6 receptor for retinol uptake.

A window into the cell for vitamin A Vitamin A is an essential nutrient for mammals, and its metabolites affect diverse biological processes. It is carried in the bloodstream as retinol by retinol binding protein (RBP); a protein called STRA6 is implicated in facilitating retinol translocation across the cell membrane. Chen et al. determined the structure of zebrafish STRA6 to a resolution of 3.9 Å by electron microscopy. A lipophilic cleft is a likely binding site for RBP, and an opening in the cleft may allow retinol to diffuse into the membrane. Unexpectedly, the structure also includes bound calcium-modulated protein, but its function remains unclear. Science, this issue p. 887 The structure of a STRA6-calmodulin complex gives insight into how retinol (vitamin A) enters cells. INTRODUCTION Vitamin A is an essential nutrient for all mammals, being vital for vision and for transcription of a wide array of genes. Retinol (vitamin A alcohol) is the predominant circulating retinoid. In the fasting state, retinol from liver stores is mobilized bound to retinol-binding protein (RBP), which transports this highly hydrophobic molecule in the bloodstream. How retinol is released from RBP and internalized by target cells has been the subject of intense debate. The RBP receptor, STRA6, was cloned in 2007. STRA6 was predicted to be a 75-kDa multipass transmembrane (TM) protein without sequence similarity to any known transporter, channel, or receptor. STRA6 is expressed widely, with particular abundance in the eye and placenta. Mutations in the human STRA6 gene have been linked to Matthew-Wood syndrome, which presents with ocular abnormalities and developmental defects. RATIONALE Despite a wealth of biochemical work aimed at investigating how STRA6 mediates internalization of retinol from RBP, progress at the molecular level has been hindered by the absence of structural information. Purified STRA6 from zebrafish was a detergent-stable dimer in an unexpected association with calmodulin (CaM), forming a 180-kDa complex. RESULTS Using cryo-electron microscopy, we determined the structure of zebrafish STRA6 in complex with CaM to 3.9 Å resolution. The protein is assembled as an intricate dimer with a topology that includes 18 TM helices (nine per protomer) and two long horizontal intramembrane (IM) helices interacting at the dimer core. Each STRA6 protomer comprises an N-terminal domain (NTD) of the first five TM helices, connected by a linker containing the first CaM-binding peptide to a central domain at the dimer interface that includes TMs 6 to 9 and the IM helices, and a cytoplasmic C-terminal segment that interacts with CaM through two additional helices. Each protomer is compactly associated with one molecule of CaM, adopting an unconventional arrangement in which it is bound to three helical regions of STRA6. We characterized the STRA6-CaM interaction biophysically by isothermal titration calorimetry, showing that the affinity of CaM for one STRA6 peptide alone is subnanomolar, and structurally by x-ray crystallography. We also demonstrated that the STRA6-CaM association is physiological by performing immunoprecipitation experiments on native zebrafish tissue. Both the extra- and intracellular surfaces of the NTD feature conserved polar pockets. The outer NTD pocket spans half the bilayer. The central domain of STRA6 defines a large ~20,000 Å3 cleft on the extracellular side, which encompasses the space between previously characterized binding sites for RBP, ~25 Å above the membrane surface, and the IM helices located down at the mid-bilayer level. This outer cleft is hydrophobic, contains two ordered putative cholesterols, and is exposed to the membrane through two symmetry-related lateral windows defined by TMs 8 and 9 and the IM helices. CONCLUSIONS The structure of STRA6 suggests a mechanism for retinol release from RBP into the hydrophobic environment of the outer cleft and direct diffusion into the membrane through the lateral window. Our work also sets the basis for future experiments aimed at investigating how the system is regulated, whether STRA6 also has a role in signaling, and the functional relevance of its association with CaM. The structure of STRA6 in complex with CaM. The STRA6 dimer, drawn as a ribbon representation with one protomer in dark red and the other in black, is associated with two molecules of calmodulin, drawn in gray and gold. The internal volume of the outer cleft is represented as a semitransparent blue surface. Calcium ions are represented as green spheres. Vitamin A homeostasis is critical to normal cellular function. Retinol-binding protein (RBP) is the sole specific carrier in the bloodstream for hydrophobic retinol, the main form in which vitamin A is transported. The integral membrane receptor STRA6 mediates cellular uptake of vitamin A by recognizing RBP-retinol to trigger release and internalization of retinol. We present the structure of zebrafish STRA6 determined to 3.9-angstrom resolution by single-particle cryo-electron microscopy. STRA6 has one intramembrane and nine transmembrane helices in an intricate dimeric assembly. Unexpectedly, calmodulin is bound tightly to STRA6 in a noncanonical arrangement. Residues involved with RBP binding map to an archlike structure that covers a deep lipophilic cleft. This cleft is open to the membrane, suggesting a possible mode for internalization of retinol through direct diffusion into the lipid bilayer.

Identification of an Archaeal Presenilin-Like Intramembrane Protease

Background The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ) implicated in Alzheimers disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs). The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea. Methodology and Principal Findings We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the β-amyloid precursor protein (APP) without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aβ40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1. Conclusions and Significance Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.

Multi-crystal native SAD analysis at 6 keV.

Anomalous diffraction signals from typical native macromolecules are very weak, frustrating their use in de novo structure determination. Here, native SAD procedures are described to enhance signal to noise in anomalous diffraction by using multiple crystals in combination with synchrotron X-rays at 6 keV. Increased anomalous signals were obtained at 6 keV compared with 7 keV X-ray energy, which was used for previous native SAD analyses. A feasibility test of multi-crystal-based native SAD phasing was performed at 3.2 Å resolution for a known tyrosine protein kinase domain, and real-life applications were made to two novel membrane proteins at about 3.0 Å resolution. The three applications collectively serve to validate the robust feasibility of native SAD phasing at lower energy.