SUMMARY G protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the β2-adrenergic receptor (β2AR) using 19F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound β2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium towards a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate and active states. Complete transition to the active conformation requires subsequent interaction with a G-protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.

G protein-coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein α subunit Ras and helical domains-previously observed to separate widely upon receptor binding to expose the nucleotide-binding site-separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism.G protein-coupled receptors (GPCRs), which represent the largest class of drug targets, trigger cellular responses to external stimuli primarily by activating heterotrimeric G proteins: an activated GPCR, upon binding an inactive, GDP-bound G protein, dramatically accelerates GDP release, thus allowing GTP to bind spontaneously to the vacated nucleotide-binding site (1-2). This nucleotide exchange initiates G protein-mediated † To whom correspondence should be addressed. Ron.Dror@DEShawResearch.com, Telephone: (212) Fax: (212) 845-1981, David.Shaw@DEShawResearch.com, Telephone: (212) Fax: (212) Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript intracellular signaling. Despite breakthroughs in GPCR structure determination (3-5), key aspects of the molecular mechanism by which GPCRs accelerate GDP release remain unresolved.Heterotrimeric G proteins undergo a dramatic conformational change upon binding activated GPCRs (Fig. 1, A and B). Double electron-electron resonance (DEER) spectroscopy has demonstrated that the Ras and helical domains of the G protein α subunit (Gα), which tightly sandwich the nucleotide in all nucleotide-bound G protein crystal structures, separate by tens of angstroms upon GPCR binding and GDP release (6). A crystal structure of a GPCR-G protein complex (4), and accompanying deuterium exchange and electron microscopy data (7,8), confirmed this dramatic domain separation.These observations have raised several unresolved questions (4, 9). What is the role of domain separation in GDP release? Does a GPCR catalyze GDP release by forcing the domains to separate, or does the GPCR force out GDP, with the absence of GDP leading to subsequent domain separation? More generally, what is the structural mechanism by which a GPCR brings about GDP release?To address these questions, we performed atomic-level molecular dynamics (MD) simulations of heterotrimeric G proteins with and without bound GPCRs. We initiated simulations from crystal structures of nucleotide-bound G protein heterotrimers (in particular, G i (10) and a chimeric G t (11)), inc...

Molecular flexibility over a wide time range is of central importance to the function of many proteins, both soluble and membrane. Revealing the modes of flexibility, their amplitudes, and time scales under physiological conditions is the challenge for spectroscopic methods, one of which is site-directed spin labeling EPR (SDSL-EPR). Here we provide an overview of some recent technological advances in SDSL-EPR related to investigation of structure, structural heterogeneity, and dynamics of proteins. These include new classes of spin labels, advances in measurement of long range distances and distance distributions, methods for identifying backbone and conformational fluctuations, and new strategies for determining the kinetics of protein motion.

These papers demonstrate how 19 F NMR has been applied to study the role of dynamics in GPCR function and provide a broad context for understanding the advance coming out of our combined application of 19 F NMR and DEER spectroscopy to uncover conformational changes during b 2 AR activation.

Pulsed electron spin resonance (ESR) dipolar spectroscopy (PDS) in combination with site-directed spin labeling is unique in providing nanometer- range distances and distributions in biological systems. To date, most of the pulsed ESR techniques require frozen solutions at cryogenic temperatures to reduce the rapid electron spin relaxation rate and to prevent averaging of electron-electron dipolar interaction due to the rapid molecular tumbling. To enable measurements in liquid solution, we are exploring a triarylmethyl (TAM)-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. In this preliminary study, TAM radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or 65 and 76 in T4 lysozyme immobilized on Sepharose. Interspin distances determined using double quantum coherence (DQC) in solution are close to those expected from models, and the narrow distance distribution in each case indicates that the TAM-based spin label is relatively localized.

As a first-line vertebrate immune defense, the polymeric immunoglobulin receptor (pIgR) transports polymeric IgA and IgM across epithelia to mucosal secretions, where the cleaved ectodomain (secretory component; SC) becomes a component of secretory antibodies, or when unliganded, binds and excludes bacteria. Here we report the 2.6Å crystal structure of unliganded human SC (hSC) and comparisons with a 1.7Å structure of teleost fish SC (tSC), an early pIgR ancestor. The hSC structure comprises five immunoglobulin-like domains (D1-D5) arranged as a triangle, with an interface between ligand-binding domains D1 and D5. Electron paramagnetic resonance measurements confirmed the D1-D5 interface in solution and revealed that it breaks upon ligand binding. Together with binding studies of mutant and chimeric SCs, which revealed domain contributions to secretory antibody formation, these results provide detailed models for SC structure, address pIgR evolution, and demonstrate that SC uses multiple conformations to protect mammals from pathogens.DOI: http://dx.doi.org/10.7554/eLife.10640.001

Enzyme immobilization in metal–organic frameworks (MOFs) offers retained enzyme integrity and activity, enhanced stability, and reduced leaching. Trapping enzymes on MOF surfaces would allow for catalysis involving large substrates. In both cases, the catalytic efficiency and selectivity depend not only on enzyme integrity/concentration but also orientation. However, it has been a challenge to determine the orientation of enzymes that are supported on solid matrices, which is even more challenging for enzymes immobilized/trapped in MOFs due to the interferences of the MOF background signals. To address such challenge, we demonstrate in this work the utilization of site-directed spin labeling in combination with Electron Paramagnetic Resonance spectroscopy, which allows for the first time the characterization of the orientation of enzymes trapped on MOF surfaces. The obtained insights are fundamentally important for MOF-based enzyme immobilization design and understanding enzyme orientation once trapped in solid matrices or even cellular confinement conditions.

Membrane proteins are assembled through balanced interactions among protein, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements in membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping which couples unfolding of a doubly-biotinylated protein to binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to an intramembrane protease GlpG of Escherichia coli, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain the stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.