Structure and Function of Lysosomal Phospholipase A2 and Lecithin:Cholesterol Acyltransferase.

Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of lipid metabolizing enzymes responsible for lung surfactant catabolism and reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause familial LCAT deficiency (FLD). Herein are described multiple high resolution crystal structures of human LPLA2 and a low resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/β hydrolase core of LPLA2 form domains with unique folds that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The wide opening of the LPLA2 active site faces the membrane for an easy access to glycerophospholipids and lipophilic alcohols, its preferred substrates. Based on these structures, substrate modeling, and the position of disease-causing mutations in LCAT, we propose that orientation of the bound phospholipid in the active site underlies the specificity of LPLA2 for fatty acids in the sn-2 vs. sn-1 position, and that preference for length of the acyl chains is dictated by two hydrophobic grooves leading away from the catalytic triad of the enzyme. We proposed a 2-step mechanism for LPLA2 membrane binding, consisting of a transient LPLA2 interaction with the membrane via its surface hydrophobic residues. High-affinity membrane interaction occurs through the bound substrate and the catalytic intermediate. Based on LPLA2 structure we built an LCAT homology model and validated it by solving the 8.7 Å structure of LCAT. Then we used this model to map known genetic LCAT mutations leading to either fish eye disease (FED) or FLD. We determined that most FLD-causing mutations result in either structural defects or catalytic impairments. On the other hand, FED mutations mostly cluster on the surface of the hydrolase domain and might define a surface for LCAT interactions with apolipoprotein A (ApoA)-I, a key component of HDL. The LCAT structure thus paves the way for rational development of new therapeutics to treat FLD, atherosclerosis, and acute coronary syndrome.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/110334/1/alisagl_1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110334/2/alisagl_2.pd

Dynamics of GLP-1R peptide agonist engagement are correlated with kinetics of G protein activation

The glucagon-like peptide-1 receptor (GLP-1R) has broad physiological roles and is a validated target for treatment of metabolic disorders. Despite recent advances in GLP-1R structure elucidation, detailed mechanistic understanding of how different peptides generate profound differences in G protein-mediated signalling is still lacking. Here we combine cryo-electron microscopy, molecular dynamics simulations, receptor mutagenesis and pharmacological assays, to interrogate the mechanism and consequences of GLP-1R binding to four peptide agonists; glucagon-like peptide-1, oxyntomodulin, exendin-4 and exendin-P5. These data reveal that distinctions in peptide N-terminal interactions and dynamics with the GLP-1R transmembrane domain are reciprocally associated with differences in the allosteric coupling to G proteins. In particular, transient interactions with residues at the base of the binding cavity correlate with enhanced kinetics for G protein activation, providing a rationale for differences in G protein-mediated signalling efficacy from distinct agonists

Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor

Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that plays a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and the type 1 transmembrane domain protein, receptor activity modifying protein (RAMP) 1. Herein, we report the 3.3 Å structure of the human CGRP receptor in complex with CGRP and the Gs40 protein heterotrimer determined by Volta phase plate cryo-electron microscopy. The RAMP transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilises CLR extracellular loop 2. RAMP1 makes only limited direct interaction with CGRP, consistent with allosteric modulation of CLR as its key function. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly the location of the CLR extracellular domain. The work provides novel insight into the control of G-protein-coupled receptor function

The Molecular Control of Calcitonin Receptor Signaling

The calcitonin receptor (CTR) is a class B G protein-coupled receptor (GPCR) that responds to the peptide hormone calcitonin (CT). CTs are clinically approved for the treatment of bone diseases. We previously reported a 4.1 Å structure of the activated CTR bound to salmon CT (sCT) and heterotrimeric Gs protein by cryo-electron microscopy (Liang, Y.-L., et al. Phase-plate cryo- EM structure of a class B GPCR-G protein complex. Nature 2017, 546, 118–123). In the current study, we have reprocessed the electron micrographs to yield a 3.3 Å map of the complex. This has allowed us to model extracellular loops (ECLs) 2 and 3, and the peptide N-terminus that previously could not be resolved. We have also performed alanine scanning mutagenesis of ECL1 and the upper segment of transmembrane helix 1 (TM1) and its extension into the receptor extracellular domain (TM1 stalk), with effects on peptide binding and function assessed by cAMP accumulation and ERK1/2 phosphorylation. These data were combined with previously published alanine scanning mutagenesis of ECL2 and ECL3 and the new structural information to provide a comprehensive 3D map of the molecular surface of the CTR that controls binding and signaling of distinct CT and related peptides. The work highlights distinctions in how different, related, class B receptors may be activated. The new mutational data on the TM1 stalk and ECL1 have also provided critical insights into the divergent control of cAMP versus pERK signaling and, collectively with previous mutagenesis data, offer evidence that the conformations linked to these different signaling pathways are, in many ways, mutually exclusive. This study furthers our understanding of the complex nature of signaling elicited by GPCRs and, in particular, that of the therapeutically important class B subfamily

Inhibitory Investigations of Acyl-CoA Derivatives against Human Lipoxygenase Isozymes.

Lipid metabolism is a complex process crucial for energy production resulting in high levels of acyl-coenzyme A (acyl-CoA) molecules in the cell. Acyl-CoAs have also been implicated in inflammation, which could be possibly linked to lipoxygenase (LOX) biochemistry by the observation that an acyl-CoA was bound to human platelet 12-lipoxygenase via cryo-EM. Given that LOX isozymes play a pivotal role in inflammation, a more thorough investigation of the inhibitory effects of acyl-CoAs on lipoxygenase isozymes was judged to be warranted. Subsequently, it was determined that C18 acyl-CoA derivatives were the most potent against h12-LOX, human reticulocyte 15-LOX-1 (h15-LOX-1), and human endothelial 15-LOX-2 (h15-LOX-2), while C16 acyl-CoAs were more potent against human 5-LOX. Specifically, oleoyl-CoA (18:1) was most potent against h12-LOX (IC50 = 32 μM) and h15-LOX-2 (IC50 = 0.62 μM), stearoyl-CoA against h15-LOX-1 (IC50 = 4.2 μM), and palmitoleoyl-CoA against h5-LOX (IC50 = 2.0 μM). The inhibition of h15-LOX-2 by oleoyl-CoA was further determined to be allosteric inhibition with a Ki of 82 +/- 70 nM, an α of 3.2 +/- 1, a β of 0.30 +/- 0.07, and a β/α = 0.09. Interestingly, linoleoyl-CoA (18:2) was a weak inhibitor against h5-LOX, h12-LOX, and h15-LOX-1 but a rapid substrate for h15-LOX-1, with comparable kinetic rates to free linoleic acid (kcat = 7.5 +/- 0.4 s-1, kcat/KM = 0.62 +/- 0.1 µM-1s-1). Additionally, it was determined that methylated fatty acids were not substrates but rather weak inhibitors. These findings imply a greater role for acyl-CoAs in the regulation of LOX activity in the cell, either through inhibition of novel oxylipin species or as a novel source of oxylipin-CoAs

Membrane Orientation and Binding Determinants of G Protein-Coupled Receptor Kinase 5 as Assessed by Combined Vibrational Spectroscopic Studies

<div><p>G-protein coupled receptors (GPCRs) are integral membrane proteins involved in a wide variety of biological processes in eukaryotic cells, and are targeted by a large fraction of marketed drugs. GPCR kinases (GRKs) play important roles in feedback regulation of GPCRs, such as of β-adrenergic receptors in the heart, where GRK2 and GRK5 are the major isoforms expressed. Membrane targeting is essential for GRK function in cells. Whereas GRK2 is recruited to the membrane by heterotrimeric Gβγ subunits, the mechanism of membrane binding by GRK5 is not fully understood. It has been proposed that GRK5 is constitutively associated with membranes through elements located at its N-terminus, its C-terminus, or both. The membrane orientation of GRK5 is also a matter of speculation. In this work, we combined sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) to help determine the membrane orientation of GRK5 and a C-terminally truncated mutant (GRK5_1-531) on membrane lipid bilayers. It was found that GRK5 and GRK5_1-531 adopt a similar orientation on model cell membranes in the presence of PIP₂ that is similar to that predicted for GRK2 in prior studies. Mutation of the N-terminal membrane binding site of GRK5 did not eliminate membrane binding, but prevented observation of this discrete orientation. The C-terminus of GRK5 does not have substantial impact on either membrane binding or orientation in this model system. Thus, the C-terminus of GRK5 may drive membrane binding in cells via interactions with other proteins at the plasma membrane or bind in an unstructured manner to negatively charged membranes.</p> </div

Modeled membrane orientations of GRK5_1-531.

<p>Possible membrane orientations of GRK5_1-531 on POPG lipid bilayers as determined from SFG and ATR-FTIR experimental measurements by using the 2ACX crystal structure: (A) Twist=40°, Tilt=10°, (B) Twist=300°, Tilt=26°. The plane of the membrane relative to the protein is shown as a blue rectangle.</p

SFG signal of GRK5 and GRK5_1-531.

<p>No discernible SFG amide I signals were observed for 336 nM (A) GRK5 and (B) GRK5_1-531 interacting with a 9:1 POPC:POPG lipid bilayer. SFG polarized amide I signals of 336 nM (C) GRK5 and (D) GRK5_1-531 interacting with a 1:1 POPC:POPG lipid bilayer. SFG polarized amide I signals of 336 nM (E) GRK5 and (F) GRK5_1-531 interacting with a POPG lipid bilayer. SFG polarized amide I signal of 336 nM (G) GRK5_1-531 and (F) GRK5_NT interacting with a 1:1 POPC:PIP₂ lipid bilayer. The circles and squares are experimental data. The solid lines indicate the fitting results.</p

GRK5 in the reference orientation.

<p>The GRK5 (PDB entry: 3NYN) and definition of twist (ψ), tilt (θ) and azimuthal (ϕ) angles which rotate the protein from the molecular (x´, y´, z´) to the macroscopic (X, Y, Z) coordinate system. The regulator of G protein signaling homology (RH) domain is colored grey, the C-terminal region containing the amphipathic helix is colored red, the kinase domain yellow, and the αN helix green (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082072#B18" target="_blank">18</a>). The side chains of residues proposed to be involved in the N-terminal phospholipid binding site (K26A, K28A, K29A, K31A, K35A) are shown as purple spheres. An approximate membrane plane (defined to be consistent with Ref. 18), is shown as blue rectangle, and lies parallel to the X-Y plane. The GRK5 is depicted in the reference orientation (ψ=0°, θ=0°, ϕ=0°) used as a starting point for data analysis. In our calculation, the molecule is rotated using an Eulerian rotation scheme according to three angles: first twist (ψ) then tilt (θ) and finally azimuthal (ϕ).</p

Modeled membrane orientations of full length GRK5.

<p>Possible membrane orientations of GRK5 on POPG lipid bilayers as determined from SFG and ATR-FTIR experimental measurements using the 3NYN crystal structure: (A) twist=190°, tilt=35°, (B) twist=245°, tilt=50°. Possible membrane orientations of GRK5 as determined from SFG and ATR-FTIR experimental measurements by using the 2ACX crystal structure: (C) twist=70°, tilt=2°, (D) twist=340°, tilt=10°. The plane of the membrane relative to the protein is shown as a blue rectangle.</p