Summary CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human Class A G protein-coupled receptors (GPCRs). CCR2 is expressed on monocytes, immature dendritic cells and T cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL21. CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see ClinicalTrials.gov) in search of therapies that target the CCR2:chemokine axis. To aid drug discovery efforts5, we solved a structure of CCR2 in a ternary complex with an orthosteric (BMS-6816) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in Class A GPCRs to date; this site spatially overlaps the G protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive GPCR structures solved to date. Like other protein:protein interactions, receptor:chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome drug design obstacles.

The membrane protein complex between the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) and phospholamban (PLN) controls Ca 2+ transport in cardiomyocytes, thereby modulating cardiac contractility. β-Adrenergic-stimulated phosphorylation of PLN at Ser-16 enhances SERCA activity via an unknown mechanism. Using solid-state nuclear magnetic resonance spectroscopy, we mapped the physical interactions between SERCA and both unphosphorylated and phosphorylated PLN in membrane bilayers. We found that the allosteric regulation of SERCA depends on the conformational equilibrium of PLN, whose cytoplasmic regulatory domain interconverts between three different states: a ground T state (helical and membrane associated), an excited R state (unfolded and membrane detached), and a B state (extended and enzymebound), which is noninhibitory. Phosphorylation at Ser-16 of PLN shifts the populations toward the B state, increasing SERCA activity. We conclude that PLN's conformational equilibrium is central to maintain SERCA's apparent Ca 2+ affinity within a physiological window. This model represents a paradigm shift in our understanding of SERCA regulation by posttranslational phosphorylation and suggests strategies for designing innovative therapeutic approaches to enhance cardiac muscle contractility. -ATPase (SERCA)/phospholamban (PLN) complex regulates Ca 2+ translocation into the sarcoplasmic reticulum (SR) of cardiomyocytes and constitutes the main mechanism of cardiac relaxation (diastole) (1-3). SERCA is a P-type ATPase that translocates two Ca 2+ ions per ATP molecule hydrolyzed in exchange for three H + ( Fig. 1) (4, 5). PLN binds and allosterically inhibits SERCA function, decreasing its apparent affinity for Ca 2+ ions (3, 6). On β-adrenergic stimulation, cAMP-dependent protein kinase A phosphorylates PLN at Ser-16, reversing the inhibition and augmenting cardiac output (3). Disruptions in this regulatory mechanism degenerate into Ca 2+ mishandling and heart failure (3). Several X-ray structures of SERCA have been determined along its enzymatic coordinates, providing atomic details on the structural transitions in the absence of PLN (4, 5). The first image of the SERCA/PLN complex resulted from cryo-EM studies (7), but the low-resolution data prevent an atomic view of PLN structure and architecture within the complex. In addition, mutagenesis and cross-linking data were used to model the complex, suggesting that the inhibitory transmembrane (TM) region of PLN is positioned into a binding groove far from the putative Ca 2+ entry, as well as the ATP binding site, and located between TM helices M2, M4, M6, and M9 of SERCA. The location of PLN's TM domain agrees with a recent crystal structure of the SERCA/PLN complex (8) and is remarkably similar to the one recently identified for a PLN homolog, sarcolipin, in complex with SERCA (9, 10) (Fig. 1).In the SERCA/PLN model, which was further refined using NMR constraints (11), the loop bridging the TM and cytoplasmic domain of PLN adopts an unfolded configuration, stretching tow...

The integral membrane protein complex between phospholamban (PLN) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) regulates cardiac contractility. In the unphosphorylated form, PLN binds SERCA and inhibits Ca2+ flux. Upon phosphorylation of PLN at Ser16, the inhibitory effect is reversed. Although structural details on both proteins are emerging from X-ray crystallography, cryo-electron microscopy, and NMR studies, the molecular mechanisms of their interactions and regulatory process are still lacking. It has been speculated that SERCA regulation depends on PLN structural transitions (order to disorder, i.e., folding/unfolding). Here, we investigated PLN conformational changes upon chemical unfolding by a combination of electron paramagnetic resonance and NMR spectroscopies, revealing that the conformational transitions involve mostly the cytoplasmic regions, with two concomitant phenomena: (1) membrane binding and folding of the amphipathic domain Ia and (2) folding/unfolding of the juxtamembrane domain Ib of PLN. Analysis of phosphorylated and unphosphorylated PLN with two phosphomimetic mutants of PLN (S16E and S16D) shows that the population of an unfolded state in domains Ia and Ib (T′ state) is linearly correlated to the extent of SERCA inhibition measured by activity assays. Inhibition of SERCA is carried out by the folded ground state (T state) of the protein (PLN), while the relief of inhibition involves promotion of PLN to excited conformational states (Ser16 phosphorylated PLN). We propose that PLN population shifts (folding/unfolding) are a key regulatory mechanism for SERCA.

Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor.