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92. Annual Meeting of the International Association of Applied Mathematics and Mechanics (GAMM), GAMM, Aachen, Germany, 15 Aug 2022 - 19 Aug 2022; Proceedings in applied mathematics and mechanics : PAMM 23(1), e202200069 (2023). doi:10.1002/pamm.202200069 special issue: "Special Issue: 92. Annual Meeting of the International Association of Applied Mathematics and Mechanics (GAMM) / Issue Edited by: Ch. Böhm, K. Mang, B. Markert, S. Reese, M. Schmidtchen, J. Waimann, M. Kaliske", Published by Wiley-VCH, Weinheim

SummaryG protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating the exchange of guanine nucleotide in the Gα subunit. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G protein complex. Using variability analysis to monitor the transitions of the stimulatory Gs protein in complex with the β2-adrenergic receptor (β2AR) at short sequential time points after GTP addition, we identified the conformational trajectory underlying G protein activation and functional dissociation from the receptor. Twenty transition structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of events driving G protein activation upon GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα Switch regions and the α5 helix that weaken the G protein-receptor interface. Molecular dynamics (MD) simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP upon closure of the alpha-helical domain (AHD) against the nucleotide-bound Ras-homology domain (RHD) correlates with irreversible α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signaling events.

Decision support systems can provide real-time process information and correlations, which in turn assists process experts in making decisions and thus further increase productivity. This also applies to well-established and already highly automated processes in continuous production employed in various industrial sectors. Continuous production refers to processes in which the produced material, either fluid or solid form, is continuously in motion and processed. As a result, the process can usually not be stopped. It is only possible to influence the running process. However, the highly nonlinear interactions between process parameters and product quality are not always known in their entirety which led to inferior product quality in terms of mechanical properties and surface quality. This requires accurate representations of the processes and the products in real-time, so-called digital shadows.Therefore, this contribution shows the necessary steps to provide a digital shadow based on numerical, physical models and process data and to couple the digital shadow with data analysis and machine learning to enable automatic decision support. This is exemplified at various stages throughout two different process chains with continuous processes: first, by using a thermoplastic production process called profile extrusion, and second, on the example of a metal processing process chain, from which three processes are described in more detail, namely, hot rolling, tempering, and fine blanking. Finally, the presented approaches and results are summarized.

The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R–Gs-protein complexes with two drugs recently approved by the FDA, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-α-MSH, a high-affinity linear variant of the endogenous agonist α-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor–Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs.

G protein-coupled receptors (GPCRs) play critical roles in the regulation of human physiology in response to a wide array of different extracellular stimuli and thus represent one of the largest groups of therapeutic drug targets. Recent advances in the structural characterization of GPCRs in different conformations and in complex with G proteins and arrestins have provided important insights into the mechanism and function of GPCRs. However, in order to truly understand the molecular basis of the functional versatility of GPCRs, the structural snapshots obtained by X-ray crystallography or cryo-EM need to be complimented with information about the conformational dynamics of receptors and their signaling complexes. In the last decade, a combination of biophysical approaches and computational studies has been utilized to examine the molecular motions of GPCRs and their transducer complexes and how they are regulated by ligands of different efficacy and bias. These studies revealed that GPCRs are highly dynamic allosteric proteins that can sample multiple conformational states. Ligands with distinct signaling profiles not only impact the conformational landscape of GPCRs but also of the receptor-engaged G proteins and arrestins. The conformational dynamics of GPCRs and their signaling complexes and the ligand-dependent bias sampling of distinct functional states are important underlying principles behind the complex signaling behavior of GPCRs.

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). The glucagon receptor (GCGR), a Family B GPCR responsible for maintenance of proper blood sugar levels, interacts with RAMP2, though the purpose and consequence of this interaction is poorly understood. Using a series of biochemical and cell-based assays, we show that RAMP2 interacts with and broadly inhibits GCGR-induced downstream signaling. Hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) demonstrates that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) as well as at a key region in the 6th transmembrane helix, while single-molecule fluorescence resonance energy transfer (smFRET) experiments show that this RAMP2-induced ECD disorder results in inhibition of active and intermediate states of the intracellular face of the receptor. Using cryo-electron microscopy (cryoEM), we determined the structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner, yet the ECD of GCGR is indeed largely disordered in the presence of RAMP2. This disorder is accompanied by rearrangements of several key areas of the receptor, resulting in the formation of a likely unproductive complex. Together, our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

Cell-free expression enables direct cotranslational insertion of G protein coupled receptors (GPCRs) and other membrane proteins into the defined membrane environments of nanodiscs. This technique avoids GPCR contacts with detergents and allows rapid identification of lipid effects on GPCR function as well as fast screening of receptor derivatives. Critical steps of conventional GPCR preparation from cellular membranes followed by detergent-based reconstitution into nanodisc membranes are thus eliminated. We report the efficient cotranslational insertion of full-length human β

G protein-coupled receptors (GPCRs) play critical roles in human physiology and are one of the prime targets for marketed drugs. While traditional drug discovery programs have focused on the development of ligands targeting the binding site of endogenous ligands (orthosteric site), allosteric modulators offer new avenues for the regulation of GPCR function with potential therapeutic benefits. Recent advances in the structure determination of GPCRs bound to different types of allosteric modulators have led to the identification of multiple allosteric sites and significantly enhanced our understanding of how allosteric ligands interact with receptors. These structural insights, together with the plethora of GPCR structures available today, will facilitate structure-based discovery and development of allosteric modulators as novel therapeutic candidates. In this review, we provide a systematic analysis of the currently available GPCR structures in complex with small-molecule allosteric ligands in terms of the location of allosteric pockets, receptor-ligand interactions, and the chemical features of the allosteric modulators. In addition, we summarize current strategies for the identification of allosteric sites as well as ligand-based and structure-based drug discovery and design.

The β2 adrenergic receptor (β2AR) signals through both Gs and Gi in cardiac myocytes, and the Gi pathway counteracts the Gs pathway. However, Gi coupling is much less efficient than Gs coupling in most cell-based and biochemical assays, making it difficult to study β2AR−Gi interactions. Here we investigate the role of phospholipid composition on Gs and Gi coupling. While negatively charged phospholipids are known to enhance agonist affinity and stabilize an active state of the β2AR, we find that they impair coupling to Gi3 and facilitate coupling to Gs. Positively charged Ca2+ and Mg2+, known to interact with the negative charge on phospholipids, facilitates Gi3 coupling. Mutational analysis suggests that Ca2+ coordinates an interaction between phospholipid and the negatively charged EDGE motif on the amino terminal helix of Gi3. Taken together, our observations suggest that local membrane charge modulates the interaction between β2AR and competing G protein subtypes.

SUMMARYThe melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a G-protein coupled receptor and a prime target for the treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R− Gs-protein complexes with two recently FDA-approved drugs, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6 regulated receptor activation. In both structures, different ligand binding modes of NDP-α-MSH, a high-affinity variant of the endogenous agonist, and setmelanotide, an anti-obesity drug with biased signaling, underline the key role of TM3 for ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-TM3 interplay subsequently impacts the receptor− Gs-protein interfaces, mainly at intracellular loop 2. These structures reveal mechanistic details of MC4R activation or inhibition and provide important insights into receptor selectivity that will facilitate the development of tailored anti-obesity drugs.

The β(2) adrenergic receptor (β(2)AR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 Å) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The β(2)AR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free β(2)AR. Here we employ spin-labeling and pressure-resolved double electron–electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in β(2)AR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.

In this work, we present a novel method for the mesh update in flow problems with moving boundaries, the phantom domain deformation mesh update method (PD-DMUM). The PD-DMUM is designed to avoid remeshing; even in the event of large, unidirectional displacements of boundaries. The method combines the concept of two mesh adaptation approaches: (1) The virtual ring shear-slip mesh update method (VR-SSMUM); and (2) the elastic mesh update method (EMUM). As in the VR-SSMUM, the PD-DMUM extends the fluid domain by a phantom domain; the PD-DMUM can thus locally adapt the element density. Combined with the EMUM, the PD-DMUM allows the consideration of arbitrary boundary movements. In this work, we apply the PD-DMUM in two test cases. Within the first test case, we validate the PD-DMUM in a 2D Poiseuille flow on a moving background mesh. Subsequently the fluid-structure interaction (FSI) problem serves as a proof of concept. Within the FSI problem, isogeometric analysis and NURBS-enhanced finite elements are employed to ensure an accurate description of the moving boundaries and a consistent coupling along the FSI boundary. Moreover, we stress the advantages of the novel method as compared to conventional mesh update approaches.

INTRODUCTIONTransmembrane signaling of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) is mediated by ligand-dependent conformational changes that are transmitted from the extracellular ligand binding site to the intracellular side of the receptor to allow coupling with transducers. One hallmark of GPCR activation is the outward movement of the cytoplasmic end of transmembrane domain 6 (TM6) that opens up an intracellular cavity to accommodate the Gα subunit, leading to nucleotide exchange and activation of the G protein. Comparison of family A and family B receptor-Gs protein complex structures has revealed substantial differences in the conformational changes of TM6 upon activation. In family B GPCRs, TM6 shows a disruption of the helical fold and the formation of a sharp kink. This differs from the gradual bending in TM6 observed in family A GPCRs.RATIONALEDespite the recent surge of determined GPCR–G protein complex structures, the activation mechanism of family B receptors remains poorly understood. The missing conserved structural motifs found in family A GPCRs together with the marked differences in the conformation of TM6 in the active state suggest distinct activation mechanisms between family B and family A GPCRs. In particular, the disruption of the helical fold and the unraveling of the extracellular end of TM6 suggest that the energy required to produce the fully active-state of family B GPCRs is higher than for family A GPCRs. We investigated the functional impact of these structural differences by comparing the structure and function of a prototypical family B receptor, the glucagon receptor (GCGR), with the β2 adrenergic receptor (β2AR), a family A GPCR.RESULTSWe present the cryo–electron microscopy structure of the GCGR-Gs complex bound to an engineered soluble glucagon derivative. The structure shows that full activation of GCGR leads to a disruption in the α-helix of TM6 typical for family B GPCRs. Analysis of the functional consequence of this helix break on receptor-mediated G protein dissociation and guanosine triphosphate (GTP) turnover reveals that GCGR exhibits a substantially lower guanine nucleotide exchange factor (GEF) activity in comparison with the family A receptor β2AR. Characterization of G protein association, guanosine diphosphate (GDP) release, and GTP binding kinetics shows that the receptor-mediated GDP dissociation and GTP binding of Gs are slower for GCGR than for β2AR. Measurements of ligand-dependent conformational alterations of GCGR by means of fluorescence and double electron-electron resonance spectroscopy show that agonist binding alone is insufficient to promote TM6 opening, in contrast to previously studied family A GPCRs, including β2AR. The outward movement of TM6 of GCGR is only observed upon interaction with Gs, suggesting that TM6 activation is only triggered by the engagement of the α5 helix of Gαs. Furthermore, TM6 of GCGR remains in the active state for a prolonged time after disengagement of Gs, which might contribute to the persistent and sustained cyclic adenosine monophosphate (cAMP) signaling previously observed for this receptor. A comprehensive comparison of the G protein activation kinetics for a number of other family A and family B GPCRs shows that family B receptors are in general less efficient than family A GPCRs in triggering G protein signaling.CONCLUSIONOur findings provide evidence for distinct activation mechanisms between family A and family B GPCRs. We propose that formation of the helix break and the sharp kink in TM6 of GCGR requires overcoming a higher energy barrier than the bending and outward movement of TM6 in family A receptors. Because of this kinetic barrier, ligand binding alone is not sufficient to stabilize the outward movement of TM6 but promotes the initial formation of the receptor–G protein complex and subsequent full engagement of the G protein at a slower time scale. Once activated by the insertion of the α5 helix of Gαs into the receptor core, as seen in the nucleotide-free complex structure, TM6 stays in the active conformation long after full disengagement of the G protein. This may be responsible for the previously described sustained and prolonged signaling of GCGR. Our comprehensive comparison of the G protein activation kinetics of family A and family B receptors suggests that the activation mechanism described for GCGR is generalizable to other family B GPCRs.

Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behaviour studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anion channels, but also to create next-generation optogenetic tools. Here we report crystal structures of the dACR iC++, along with spectroscopic, electrophysiological and computational analyses that provide unexpected insights into pH dependence, substrate recognition, channel gating and ion selectivity of both dACRs and nACRs. These results enabled us to create an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.

G-protein-coupled receptors (GPCRs) relay numerous extracellular signals by triggering intracellular signaling through coupling with G proteins and arrestins. Recent breakthroughs in the structural determination of GPCRs and GPCR-transducer complexes represent important steps toward deciphering GPCR signal transduction at a molecular level. A full understanding of the molecular basis of GPCR-mediated signaling requires elucidation of the dynamics of receptors and their transducer complexes as well as their energy landscapes and conformational transition rates. Here, we summarize current insights into the structural plasticity of GPCR-G-protein and GPCR-arrestin complexes that underlies the regulation of the receptor's intracellular signaling profile.

SUMMARY G protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signaling responses remain poorly understood. In Class A GPCRs, receptor activation and G protein coupling entail outward movements of transmembrane segment 6 (TM6). Using single-molecule Fluorescence Resonance Energy Transfer (smFRET) imaging, we examine TM6 motions in the β2 adrenergic receptor (β2AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the Gs heterotrimer. We show that partial and full agonists affect TM6 motions in a manner that differentially regulates the rate at which GDP-bound β2AR-Gs complexes are formed and the efficiency of nucleotide exchange leading to Gs activation. These data also reveal transient nucleotide-bound β2AR-Gs species distinct from known structures and single-molecule perspectives on the allosteric link between ligand and nucleotide binding pockets that shed new light on the G protein activation mechanism.

The number of structures and molecular dynamics simulations of proteins is exploding owing to dramatic advances in cryo-electron microscopy, crystallography, and computing. One of the most powerful ways to analyze structural information involves comparisons of interatomic interactions across different structures or simulations of the same protein or related proteins from the same family (e.g.different GPCRs). Such comparative analyses are of interest to a wide range of researchers but currently prove challenging for all but a few. To facilitate comparative structural analyses, we have developed tools for (i) rapidly computing and comparing interatomic interactions and (ii) interactively visualizing interactions to enable structure-based interpretations. Using these tools, we have developed the Contact Comparison Atlas, a web-based resource for the comparative analysis of interactions in structures and simulations of proteins. Using the Contact Comparison Atlas and our tools, we have identified patterns of interactions with functional implications in structures of G-protein-coupled receptors, G proteins and kinases and in the dynamics of muscarinic receptors. The Contact Comparison Atlas can be used to enable structure modeling, drug discovery, protein engineering, and the prediction of disease-associated mutations.Contact Comparison Atlas website:https://getcontacts.github.io/atlas/

The glucagon receptor family comprises Class B G protein-coupled receptors (GPCRs) that play a crucial role in regulating blood sugar levels. Receptors of this family represent important therapeutic targets for the treatment of diabetes and obesity. Despite intensive structural studies, we only have a poor understanding of the mechanism of peptide hormone-induced Class B receptor activation. This process involves the formation of a sharp kink in transmembrane helix 6 that moves out to allow formation of the nucleotide-free G protein complex. Here, we present the cryo-EM structure of the glucagon receptor (GCGR), a prototypical Class B GPCR, in complex with an engineered soluble glucagon derivative and the heterotrimeric G-protein, Gs. Comparison with the previously determined crystal structures of GCGR bound to a partial agonist reveals a structural framework to explain the molecular basis of ligand efficacy that is further supported by mutagenesis data.

Secondary transporters exist as monomers, dimers or higher state oligomers. The significance of the oligomeric state is only partially understood. Here, the significance of the trimeric state of the L-carnitine/γ-butyrobetaine antiporter CaiT of Escherichia coli was investigated. Amino acids important for trimer stability were identified and experimentally verified. Among others, CaiT-D288A and -D288R proved to be mostly monomeric in detergent solution and after reconstitution into proteoliposomes, as shown by blue native gel electrophoresis, gel filtration, and determination of intermolecular distances. CaiT-D288A was fully functional with kinetic parameters similar to the trimeric wild-type. Significant differences in amount and stability in the cell membrane between monomeric and trimeric CaiT were not observed. Contrary to trimeric CaiT, addition of substrate had no or only a minor effect on the tryptophan fluorescence of monomeric CaiT. The results suggest that physical contacts between protomers are important for the substrate-induced changes in protein fluorescence and the underlying conformational alterations., Scientific Reports, 9 (1), ISSN:2045-2322

The activation of G proteins by G protein-coupled receptors (GPCRs) underlies the majority of transmembrane signaling by hormones and neurotransmitters. Recent structures of GPCR-G protein complexes obtained by crystallography and cryo-electron microscopy (cryo-EM) reveal similar interactions between GPCRs and the alpha subunit of different G protein isoforms. While some G protein subtype-specific differences are observed, there is no clear structural explanation for G protein subtype-selectivity. All of these complexes are stabilized in the nucleotide-free state, a condition that does not exist in living cells. In an effort to better understand the structural basis of coupling specificity, we used time-resolved structural mass spectrometry techniques to investigate GPCR-G protein complex formation and G-protein activation. Our results suggest that coupling specificity is determined by one or more transient intermediate states that serve as selectivity filters and precede the formation of the stable nucleotide-free GPCR-G protein complexes observed in crystal and cryo-EM structures.

Single-particle cryo-electron microscopy (cryo-EM) has recently enabled high-resolution structure determination of numerous biological macromolecular complexes. Despite this progress, the application of high-resolution cryo-EM to G protein coupled receptors (GPCRs) in complex with heterotrimeric G proteins remains challenging, owning to both the relative small size and the limited stability of these assemblies. Here we describe the development of antibody fragments that bind and stabilize GPCR-G protein complexes for the application of high-resolution cryo-EM. One antibody in particular, mAb16, stabilizes GPCR/G-protein complexes by recognizing an interface between Gα and Gβγ subunits in the heterotrimer, and confers resistance to GTPγS-triggered dissociation. The unique recognition mode of this antibody makes it possible to transfer its binding and stabilizing effect to other G-protein subtypes through minimal protein engineering. This antibody fragment is thus a broadly applicable tool for structural studies of GPCR/G-protein complexes.

G protein coupled receptors (GPCRs) are transmembrane receptors that signal through heterotrimeric G proteins. Lipid modifications anchor G proteins to the plasma membrane; however, little is known about the effect of phospholipid composition on GPCR-G protein coupling. The β2adrenergic receptor (β2AR) signals through both Gsand Giin cardiac myocytes where studies suggest that Gisignaling may be cardioprotective. However, Gicoupling is much less efficient than Gscoupling in most cell-based and biochemical assays, making it difficult to study β2AR-Giinteractions. To investigate the role of phospholipid composition on Gsand Gicoupling, we reconstituted β2AR in detergent/lipid mixed micelles and found that negatively charged phospholipids (PS and PG) inhibit β2AR-Gi3coupling. Replacing negatively charged lipids with neutral lipids (PC or PE) facilitated the formation of a functional β2AR-Gi3interaction that activated Gi3. Ca2+, known to interact with negatively charged PS, facilitated β2AR-Gi3interaction in PS. Mutational analysis suggested that Ca2+interacts with the negatively charged EDGE motif on the carboxyl-terminal end of the αN helix of Gi3and coordinates an EDGE-PS interaction. These results were confirmed in β2AR reconstituted into nanodisc phospholipid bilayers. β2AR-Gi3interaction was favored in neutral lipids (PE and PC) over negatively charged lipids (PG and PS). In contrast, basal β2AR-Gsinteraction was favored in negatively charged lipids over neutral lipids. In negatively-charged lipids, Ca2+and Mg2+facilitated β2AR-Gi3interaction. Taken together, our observations suggest that local membrane charge modulates the interaction between β2AR and competing G protein subtypes.

How a receptor transmits a signal G protein–coupled receptors (GPCRs) transmit diverse external signals into the cell. When activated by an outside stimulus, they bind to a G protein inside the cell and accelerate exchange of a bound guanosine diphosphate (GDP) nucleotide for guanosine triphosphate, which initiates intercellular signaling. Dror et al. used atomic-level molecular dynamics simulations to show how GPCRs enhance GDP release. The G protein is dynamic and frequently adopts a conformation that exposes GDP even without the receptor bound. GPCR binding to this conformation favors an additional structural rearrangement that favors GDP release. The authors confirmed these predictions experimentally using double electron-electron resonance spectroscopy. Science , this issue p. 1361

Camelid single-domain antibody fragments (‘nanobodies’) provide the remarkable specificity of antibodies within a single 15-kDa immunoglobulin VHH domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G-protein-coupled receptors (GPCRs). Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover conformationally selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for nonprofit research.

Camelid single-domain antibody fragments (“nanobodies”) provide the remarkable specificity of antibodies within a single immunoglobulin VHH domain. This unique feature enables applications ranging from their use as biochemical tools to therapeutic agents. Virtually all nanobodies reported to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we developed a fully in vitro platform for nanobody discovery based on yeast surface display of a synthetic nanobody scaffold. This platform provides a facile and cost-effective method for rapidly isolating nanobodies targeting a diverse range of antigens. We provide a blueprint for identifying nanobodies starting from both purified and non-purified antigens, and in addition, we demonstrate application of the platform to discover rare conformationally-selective nanobodies to a lipid flippase and a G protein-coupled receptor. To facilitate broad deployment of this platform, we have made the library and all associated protocols publicly available.

The crystal structure of the β2-adrenergic receptor (β2AR) bound to the G protein adenylyl cyclase stimulatory G protein (Gs) captured the complex in a nucleotide-free state (β2AR-Gsempty). Unfortunately, the β2AR-Gsempty complex does not provide a clear explanation for G protein coupling specificity. Evidence from several sources suggests the existence of a transient complex between the β2AR and GDP-bound Gs protein (β2AR-GsGDP) that may represent an intermediate on the way to the formation of β2AR-Gsempty and may contribute to coupling specificity. Here we present a structure of the β2AR in complex with the carboxyl terminal 14 amino acids from Gαs along with the structure of the GDP-bound Gs heterotrimer. These structures provide evidence for an alternate interaction between the β2AR and Gs that may represent an intermediate that contributes to Gs coupling specificity.

Cannabis elicits its mood-enhancing and analgesic effects through the cannabinoid receptor 1 (CB1), a G protein coupled receptor (GPCR) that signals primarily through the adenylyl cyclase-inhibiting heterotrimeric G protein G(i). Activation of CB1-G(i) signaling pathways holds potential for treating a number of neurological disorders, and is thus crucial to understand the mechanism of G(i) activation by CB1. Here we present the structure of the CB1-G(i) signaling complex bound to the highly potent agonist MDMB-Fubinaca (FUB), a recently emerged illicit synthetic cannabinoid infused in street drugs that have been associated with numerous overdoses and fatalities. The structure illustrates how FUB stabilizes the receptor in an active state to facilitate nucleotide exchange in G(i). The results compose the structural framework to explain CB1 activation by different classes of ligands and provide insights into the G protein coupling and selectivity mechanisms adopted by the receptor.

“Biased” G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or β-arrestins. Here we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between ten pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an “open” conformation with an accessible transducer-binding site. β-Arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.

Na(+)/solute symporters are essential membrane integrated proteins that couple the flow of Na(+) ions driven by electrochemical Na(+) gradients to the transport of solutes across biological membranes. Here, we used a combination of molecular modeling techniques and evolutionary conservation analysis to construct and validate a first model of the Na(+)/proline symporter PutP of Escherichia coli based on the crystal structure of the bacterial Na(+)/galactose symporter vSGLT. Ligand docking experiments were employed to gain information about residues involved in proline binding. The proposed model is consistent with the available experimental data and was further validated by amino acid substitutions and kinetic and protein chemical analyses. Combination of the results of molecular modeling and functional studies predicts the location and organization of the Na(+) and proline binding sites. Remarkably, as proposed computationally and discovered here experimentally, residues Y140, W244, and Y248 of transmembrane segments 4 and 7 are found to be particularly important for PutP function and suggested to participate in proline binding and/or gating.

The backbone structure is determined by site-directed spin labeling, double electron electron resonance measurements of distances, and modeling in terms of a helix-loop-helix construct for a transmembrane domain that is supposed to line the translocation pathway in the 54.3 kDa Na+/proline symporter PutP of Escherichia coli. The conformational distribution of the spin labels is accounted for by a rotamer library. An ensemble of backbone models with a root mean-square deviation of less than 2 Å is obtained. These models exhibit a pronounced kink near residue T341, which is involved in substrate binding. The kink may be associated with a hinge that allows the protein to open and close an inwardly oriented cavity.

Selected residues of transmembrane domain (TM) IX were previously shown to play key roles in ligand binding and transport in members of the Na(+)/solute symporter family. Using the Na(+)/proline transporter PutP as a model, a complete Cys scanning mutagenesis of TM IX (positions 324 to 351) was performed here to further investigate the functional significance of the domain. G328, S332, Q345, and L346 were newly identified as important for Na(+)-coupled proline uptake. Placement of Cys at one of these positions altered K(m(pro)) (S332C and L346C, 3- and 21-fold decreased, respectively; Q345C, 38-fold increased), K(0.5(Na+)) (S332C, 13-fold decreased; Q345C, 19-fold increased), and/or V(max) [G328C, S332C, Q345C, and L346C, 3-, 22-, 2-, and 8-fold decreased compared to PutP(wild type), respectively]. Membrane-permeant N-ethylmaleimide inhibited proline uptake into cells containing PutP with Cys at distinct positions in the middle (T341C) and cytoplasmic half of TM IX (C344, L347C, V348C, and S351C) and had little or no effect on all other single Cys PutP variants. The inhibition pattern was in agreement with the pattern of labeling with fluorescein-5-maleimide. In addition, Cys placed into the cytoplasmic half of TM IX (C344, L347C, V348C, and S351C) was protected from fluorescein-5-maleimide labeling by proline while Na(+) alone had no effect. Membrane-impermeant methanethiosulfonate ethyltrimethylammonium modified Cys in the middle (A337C and T341C) and periplasmic half (L331C) but not in the cytoplasmic half of TM IX in intact cells. Furthermore, Cys at the latter positions was partially protected by Na(+) but not by proline. Based on these results, a model is discussed according to which residues of TM IX participate in the formation of ligand-sensitive, hydrophilic cavities in the protein that may reconstitute part of the Na(+) and/or proline translocation pathway of PutP.

The Na+/solute symporter family comprises more than 400 members of pro- and eukaryotic origin. Using the Na+/proline transporter PutP of Escherichia coli as a model, the role of two conserved residues, Ser-340 and Thr-341, is investigated to obtain insights into the mechanism of transport catalyzed by members of this family. Substitution of these amino acids alters the transport kinetics of cells and proteoliposomes containing the PutP variants significantly. In particular, the apparent affinities for Na+ and Li+ are reduced by 2 orders of magnitude or more. Also proline binding is affected, albeit to a lesser extent than ion binding. Thereby, the presence of a hydroxyl group at position 341 is essential for high affinity ligand binding. Furthermore, Cys placed at position 340 or 341 reacts with sulfhydryl reagents of different polarity, indicating accessibility from the water phase. In addition, Cys cross-linking suggests proximity of the residues to other amino acids previously shown to be crucial for ligand binding. For these reasons it is suggested that Ser-340 and Thr-341 are located in a ligand translocation pathway. Furthermore, it is proposed that the side chain of Thr-341 directly participates in Na+ binding.

Transient or partial formation of complexes between biomacromolecules is a general mechanism used to control cellular functions. Several of these complexes escape structure determination by crystallographic means. We developed a new approach for determining the structure of protein dimers in the native environment (e.g., in the membrane) with high resolution in cases where the structure of the two monomers is known. The approach is based on measurements of distance distributions between spin labels in the range between 2 and 6 nanometers by a pulsed electron paramagnetic resonance technique and explicit modeling of spin label conformations. By applying this method to the membrane protein homodimer of the Na+/H+ antiporter NhaA of Escherichia coli, the structure of the presumably physiological dimer was determined. It reveals two points of contact between the two monomers, with one of them confirming results of earlier cross-linking experiments.

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.

Amino acid transport is a ubiquitous phenomenon and serves a variety of functions in prokaryotes, including supply of carbon and nitrogen for catabolic and anabolic processes, pH homeostasis, osmoprotection, virulence, detoxification, signal transduction and generation of electrochemical ion gradients. Many of the participating proteins have eukaryotic relatives and are successfully used as model systems for exploration of transporter structure and function. Distribution, physiological roles, functional properties, and structure-function relationships of prokaryotic alpha-amino acid transporters are discussed.

Summary The Na + /proline symporter (PutP), like several other Na + -coupled symporters, belongs to the so-called LeuT-fold structural family, which features ten core transmembrane domains (cTMs) connected by extra- and intracellular loops. The role of these loops has been discussed in context with the gating function in the alternating access model of secondary active transport processes. Here we report the complete spin-labeling site scan of extracellular loop 4 (eL4) in PutP that reveals the presence of two α-helical segments, eL4a and eL4b. Among the eL4 residues that are directly implicated in the functional dynamics of the transporter, Phe314 in eL4b anchors the loop by means of hydrophobic contacts to cTM1 close to the ligand binding sites. We propose that ligand-induced conformational changes at the binding sites are transmitted via the anchoring residue to eL4 and through eL4 further to adjacent cTMs, leading to closure of the extracellular gate.

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of β-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin-1. The structure of the β-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in β-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of β-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on β-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

The functions of the ubiquitous family of G-protein coupled receptors (GPCRs), are mediated and critically modulated by beta arrestins. Beta arrestins bind to phosphorylated receptors and lead to r...

Helicobacter pylori is cause of chronic gastritis, duodenal ulcer and gastric carcinoma in humans. L-proline is a preferred energy source of the microaerophilic bacterium. Previous analyses revealed that HpputP and HpputA, the genes that are predicted to play a central role in proline metabolism as they encode for the proline transporter and proline dehydrogenase, respectively, are essential for stomach colonization. Here, the molecular basis of proline transport in H. pylori by HpPutP was investigated experimentally for the first time. Measuring radiolabeled substrate transport in H. pylori and E. coli heterologously expressing HpputP as well as in proteoliposomes reconstituted with HpPutP, we demonstrate that the observed proline transport in H. pylori is mediated by HpPutP. HpPutP is specific and exhibits a high affinity for L-proline. Notably, L-proline transport is exclusively dependent on Na(+) as coupling ion, i.e., Na(+)/L-proline symport, reminiscent to the properties of PutP of E. coli even though H. pylori lives in a more acidic environment. Homology model-based structural comparisons and substitution analyses identified amino acids crucial for function. HpPutP-catalyzed proline uptake was efficiently inhibited by the known proline analogs 3,4-dehydro-D,L-proline and L-azetidine-2-carboxylic acid.

In the cyanobacterium Synechocystis sp PCC 6803, early steps in thylakoid membrane (TM) biogenesis are considered to take place in specialized membrane fractions resembling an interface between the plasma membrane (PM) and TM. This region (the PratA-defined membrane) is defined by the presence of the photosystem II (PSII) assembly factor PratA (for processing-associated TPR protein) and the precursor of the D1 protein (pD1). Here, we show that PratA is a Mn(2+) binding protein that contains a high affinity Mn(2+) binding site (K(d) = 73 μM) and that PratA is required for efficient delivery of Mn(2+) to PSII in vivo, as Mn(2+) transport is retarded in pratA(-). Furthermore, ultrastructural analyses of pratA(-) depict changes in membrane organization in comparison to the wild type, especially a semicircle-shaped structure, which appears to connect PM and TM, is lacking in pratA(-). Immunogold labeling located PratA and pD1 to these distinct regions at the cell periphery. Thus, PratA is necessary for efficient delivery of Mn(2+) to PSII, leading to Mn(2+) preloading of PSII in the periplasm. We propose an extended model for the spatial organization of Mn(2+) transport to PSII, which is suggested to take place concomitantly with early steps of PSII assembly in biogenesis centers at the cell periphery.

The Na⁺/L-proline transporter PutP is a member of the Na⁺/solute symporter family (TC 2A.21, SLC5), which contains several hundred proteins of pro- and eukaryotic origin. Within the family, the capability of L-proline uptake is restricted to proteins of prokaryotes. PutP contributes to the use of L-proline as a nutrient. In addition, the transporter may supply cells with compatible solute during adaptation to osmotic stress. Based on these and other functions, PutP is of significance for various bacteria-host interactions including the virulence of human pathogens. A homology model of Escherichia coli PutP was generated based on the crystal structure of the Vibrio parahaemolyticus Na+/galactose symporter. According to the model, PutP has a core structure of five plus five transmembrane domains forming an inverted repeat similar as originally revealed by the crystal structure of the Na+/leucine transporter LeuT. The homology model is experimentally verified by Cys cross-linking and site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy. The putative sites of Na⁺ and L-proline binding are described, and a putative transport mechanism is discussed.