Christopher B. Roth

Flexibility in the ABC transporter MsbA: Alternating access with a twist.

ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a wide variety of substrates across cellular membranes and are conserved from bacteria to humans. Here we compare four x-ray structures of the bacterial ABC lipid flippase, MsbA, trapped in different conformations, two nucleotide-bound structures and two in the absence of nucleotide. Comparison of the nucleotide-free conformations of MsbA reveals a flexible hinge formed by extracellular loops 2 and 3. This hinge allows the nucleotide-binding domains to disassociate while the ATP-binding half sites remain facing each other. The binding of the nucleotide causes a packing rearrangement of the transmembrane helices and changes the accessibility of the transporter from cytoplasmic (inward) facing to extracellular (outward) facing. The inward and outward openings are mediated by two different sets of transmembrane helix interactions. Altogether, the conformational changes between these structures suggest that large ranges of motion may be required for substrate transport.

Structural basis for allosteric regulation of GPCRs by sodium ions.

GPCR Close-Up Structures of G protein–coupled receptors (GPCRs) determined in the past few years, have provided insight into the function of this important family of membrane proteins. Liu et al. (p. 232) used a protein-engineering strategy to produce a stabilized version of the human A2Aadenosine receptor (A2AAR). The high-resolution structure reveals the position of about 60 internal waters, which suggests an almost continuous channel in the GPCR and can explain the allosteric effects of Na+ on ligand binding and how cholesterol may contribute to GPCR stabilization. A protein-engineering strategy yields a closer look at the receptor-bound water, sodium, and lipid molecules. Pharmacological responses of G protein–coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A2A adenosine receptor by replacing its third intracellular loop with apocytochrome b562RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp2.50. Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.

Crystal structure of a lipid G protein-coupled receptor.

A Lipid-Sensing GPCR Sphingosine 1-phosphate (S1P) is a sphingolipid that binds to the G protein–coupled receptor subtype 1 (S1P1) to activate signaling pathways involved in regulation of the vascular and immune systems. Hanson et al. (p. 851) determined the crystal structure of S1PR in complex with an antagonist sphingolipid mimic. Ligand access to the receptor from the extracellular milieu is occluded, and a gap between helices I and VII may provide ligand access from within the membrane. The structural information, together with mutagenesis and structure activity relationship data, provides insight into the molecular recognition events that modulate signaling. A channel in a lipid-dependent G protein–coupled receptor allows a ligand to access its binding site from within the plasma membrane. The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein–coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P1-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P1, resulting in the modulation of immune and stromal cell responses.

Analysis of Full and Partial Agonists Binding to β2-Adrenergic Receptor Suggests a Role of Transmembrane Helix V in Agonist-Specific Conformational Changes

The 2.4 Å crystal structure of the β2‐adrenergic receptor (β2AR) in complex with the high‐affinity inverse agonist (−)‐carazolol provides a detailed structural framework for the analysis of ligand recognition by adrenergic receptors. Insights into agonist binding and the corresponding conformational changes triggering G‐protein coupled receptor (GPCR) activation mechanism are of special interest. Here we show that while the carazolol pocket captured in the β2AR crystal structure accommodates (−)‐isoproterenol and other agonists without steric clashes, a finite movement of the flexible extracellular part of TM‐V helix (TM‐Ve) obtained by receptor optimization in the presence of docked ligand can further improve the calculated binding affinities for agonist compounds. Tilting of TM‐Ve towards the receptor axis provides a more complete description of polar receptor–ligand interactions for full and partial agonists, by enabling optimal engagement of agonists with two experimentally identified anchor sites, formed by Asp113/Asn312 and Ser203/Ser204/Ser207 side chains. Further, receptor models incorporating a flexible TM‐V backbone allow reliable prediction of binding affinities for a set of diverse ligands, suggesting potential utility of this approach to design of effective and subtype‐specific agonists for adrenergic receptors. Systematic differences in capacity of partial, full and inverse agonists to induce TM‐V helix tilt in the β2AR model suggest potential role of TM‐V as a conformational “rheostat” involved in the whole spectrum of β2AR responses to small molecule signals. Copyright

Stabilization of the Human β2-Adrenergic Receptor TM4–TM3–TM5 Helix Interface by Mutagenesis of Glu1223.41, A Critical Residue in GPCR Structure

G protein-coupled receptor (GPCR) instability represents one of the most profound obstacles in the structural study of GPCRs that bind diffusible ligands. The introduction of targeted mutations at nonconserved residues that lie proximal to helix interfaces has the potential to enhance the fold stability of the receptor helix bundle while maintaining wild-type receptor function. To test this hypothesis, we studied the effect of amino acid substitutions at Glu122(3.41) in the well-studied beta(2)-adrenergic receptor (beta(2)AR), which was predicted from sequence conservation to lie at a position equivalent to a tryptophan residue in rhodopsin at the 3,4,5 helix interface among transmembrane (TM) domains 3, 4, and 5. Replacement of Glu122(3.41) with bulky hydrophobic residues, such as tryptophan, tyrosine, and phenylalanine, increases the yield of functionally folded beta(2)AR by as much as 5-fold. Receptor stability in detergent solution was studied by isothermal denaturation, and it was found that the E122W and E122Y mutations enhanced the beta(2)AR thermal half-life by 9.3- and 6.7-fold, respectively, at 37 degrees C. The beta(1)AR was also stabilized by the introduction of tryptophan at Glu147(3.41), and the effect on protein behavior was similar to the rescue of the unstable wild-type receptor by the antagonist propranolol. Molecular modeling of the E122W and E122Y mutants revealed that the tryptophan ring edge and tyrosine hydroxyl are positioned proximal to the helical break in TM5 introduced by the conserved Pro211(5.50) and may stabilize the helix by interacting favorably with the unpaired carbonyl oxygen of Val206(5.45). Conformational flexibility of TM5 is likely to be a general property of class A GPCRs; therefore, engineering of the TM4-TM3-TM5 interface at the 3.41 position may provide a general strategy for the stabilization of other receptors.

CHAPTER 19 – Signal Transduction and Integral Membrane Proteins

The classic paradigms for signal transduction across the cell membrane are the membrane-bound receptors. Crystal structures of a bacterial ion channels and transporters have been elucidated, and the next frontiers of membrane protein structural biology will likely focus on smaller mammalian targets. Cells need to adapt their behavior continuously in response to a barrage of external stimuli. Integral membrane proteins are the best positioned to interact with outside stimuli and are, therefore, critical components of signal transduction. Ion channels, transporters, and receptors are integral membrane proteins that can mediate signaling across the cellular membrane. The availability of detailed structural information on these proteins has been limited by technical challenges unique to the high-resolution structure determination of membrane proteins by X-ray crystallography. In recent years, however, the X-ray structures of a few of these important proteins have been solved, providing insight into the molecular structural basis of signal transduction across the cell membrane.

CHAPTER 7 – X-RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA*

ABC exporters transport a diverse array of substrates including peptides, toxins, lipids, and hydrophobic drug molecules from the cytoplasmic side of the cell membrane to either the outer membrane leaflet or the outside of the cell. Many of these transporters are believed to be “flippases”, transporting or “flipping” drugs and/or lipids from the inner to the outer membrane leaflet. Some of the best-studied MDR-ABC transporters are the human P-glycoprotein (Pgp) and other related drug transporters. The lipid flippase, MsbA, from Escherichia coli (Eco-MsbA) is one of the closest bacterial ABC transporters to human Pgp. MsbA transports lipid A, a major component of the bacterial outer cell membrane, and is essential for cell viability. Loss of MsbA expression in the cell membrane or a disruption of transport by mutation results in a lethal accumulation of lipid A in the cytoplasmic leaflet. The recent X-ray structure of the lipid flippase MsbA from E. coli at 4.5 A in resolution establishes the overall structural architecture of an ABC transporter and suggests a model for the structural basis of the flipping mechanism that moves hydrophobic substrates from the inner to the outer membrane leaflet of the cell membrane.