Abstract Agonists targeting α2-adrenergic receptors (ARs) are used to treat diverse conditions, including hypertension, attention-deficit/hyperactivity disorder, pain, panic disorders, opioid and alcohol withdrawal symptoms, and cigarette cravings. These receptors transduce signals through heterotrimeric Gi proteins. Here, we elucidated cryo-EM structures that depict α2A-AR in complex with Gi proteins, along with the endogenous agonist epinephrine or the synthetic agonist dexmedetomidine. Molecular dynamics simulations and functional studies reinforce the results of the structural revelations. Our investigation revealed that epinephrine exhibits different conformations when engaging with α-ARs and β-ARs. Furthermore, α2A-AR and β1-AR (primarily coupled to Gs, with secondary associations to Gi) were compared and found to exhibit different interactions with Gi proteins. Notably, the stability of the epinephrine–α2A-AR–Gi complex is greater than that of the dexmedetomidine–α2A-AR–Gi complex. These findings substantiate and improve our knowledge on the intricate signaling mechanisms orchestrated by ARs and concurrently shed light on the regulation of α-ARs and β-ARs by epinephrine.

α1-adrenergic receptors (α1-ARs) play critical roles in the cardiovascular and nervous systems where they regulate blood pressure, cognition, and metabolism. However, the lack of specific agonists for all α1 subtypes has limited our understanding of the physiological roles of different α1-AR subtypes, and led to the stagnancy in agonist-based drug development for these receptors. Here we report cryo-EM structures of α1A-AR in complex with heterotrimeric G-proteins and either the endogenous common agonist epinephrine or the α1A-AR-specific synthetic agonist A61603. These structures provide molecular insights into the mechanisms underlying the discrimination between α1A-AR and α1B-AR by A61603. Guided by the structures and corresponding molecular dynamics simulations, we engineer α1A-AR mutants that are not responsive to A61603, and α1B-AR mutants that can be potently activated by A61603. Together, these findings advance our understanding of the agonist specificity for α1-ARs at the molecular level, opening the possibility of rational design of subtype-specific agonists.

G-protein-coupled receptors (GPCRs) receive signals from ligands with different efficacies, and transduce to heterotrimeric G-proteins to generate different degrees of physiological responses. Previous studies revealed how ligands with different efficacies activate GPCRs. Here, we investigate how a GPCR activates G-proteins upon binding ligands with different efficacies. We report the cryo-EM structures of β1-adrenergic receptor (β1-AR) in complex with Gs (GαsGβ1Gγ2) and a partial agonist or a very weak partial agonist, and compare them to the β1-AR-Gs structure in complex with a full agonist. Analyses reveal similar overall complex architecture, with local conformational differences. Cellular functional studies with mutations of β1-AR residues show effects on the cellular signaling from β1-AR to the cAMP response initiated by the three different ligands, with residue-specific functional differences. Biochemical investigations uncover that the intermediate state complex comprising β1-AR and nucleotide-free Gs is more stable when binding a full agonist than a partial agonist. Molecular dynamics simulations support the local conformational flexibilities and different stabilities among the three complexes. These data provide insights into the ligand efficacy in the activation of GPCRs and G-proteins.

The β 1 -adrenergic receptor (β 1 -AR) can activate two families of G proteins. When coupled to Gs, β 1 -AR increases cardiac output, and coupling to Gi leads to decreased responsiveness in myocardial infarction. By comparative structural analysis of turkey β 1 -AR complexed with either Gi or Gs, we investigate how a single G-protein-coupled receptor simultaneously signals through two G proteins. We find that, although the critical receptor-interacting C-terminal α5-helices on Gα i and Gα s interact similarly with β 1 -AR, the overall interacting modes between β 1 -AR and G proteins vary substantially. Functional studies reveal the importance of the differing interactions and provide evidence that the activation efficacy of G proteins by β 1 -AR is determined by the entire three-dimensional interaction surface, including intracellular loops 2 and 4 (ICL2 and ICL4).

Cardiac disease remains the leading cause of morbidity and mortality worldwide. The β1-adrenergic receptor (β1-AR) is a major regulator of cardiac functions and is downregulated in the majority of heart failure cases. A key physiological process is the activation of heterotrimeric G-protein Gs by β1-ARs, leading to increased heart rate and contractility. Here, we use cryo-electron microscopy and functional studies to investigate the molecular mechanism by which β1-AR activates Gs. We find that the tilting of α5-helix breaks a hydrogen bond between the sidechain of His373 in the C-terminal α5-helix and the backbone carbonyl of Arg38 in the N-terminal αN-helix of Gαs. Together with the disruption of another interacting network involving Gln59 in the α1-helix, Ala352 in the β6-α5 loop, and Thr355 in the α5-helix, these conformational changes might lead to the deformation of the GDP-binding pocket. Our data provide molecular insights into the activation of G-proteins by G-protein-coupled receptors.

Autophagy is a conserved, eukaryotic cell survival pathway, responsible for the degradation and recycling of large and stable macromolecular assemblies, such as protein aggregates, organelles, and intracellular pathogens. BECN1/Beclin 1 is an essential component of the autophagosome nucleation complex that mediates the first committed step in autopahgy. The Golgi‐Associated Plant Pathogenesis Related protein 1 (GAPR‐1) is a mammalian protein that down‐regulates autophagy by binding to BECN11. BECN1 residues 267–284 are required for this interaction1. Further, a cell‐permeable peptide, Tat‐BECN1 (residues 267–284) inhibits the BECN1:GAPR‐1 interaction, thereby inducing autophagy. Autophagy induced by treatment with the Tat‐BECN1 (residues 267–284) peptide reduces certain virus infections and protein aggregation. Therefore, understanding the mechanism of this interaction is important for developing therapies for infectious and neurodegenerative diseases.Our goal was to better understand the mechanism of the BECN1:GAPR‐1 interaction using a combination of structural methods, structure modeling and mutagenesis combined with co‐immunoprecipiitation. GAPR‐1 crystallizes as a dimer, although size‐exclusion chromatography indicates that it is a monomer in solution. To date, we have been unsuccessful in crystallizing the complex of wild‐type (WT) GAPR‐1:Tat‐BECN1 (residues 267–284). Therefore, as an alternative strategy, we modeled the BECN1:GAPR‐1 interaction using the CABS‐dock server that searches for the binding sites by utilizing Replica Exchange Monte Carlo dynamics with 10 replicas spread on the temperature scale and final models are reconstructed from the C‐alpha trace to an all‐atom representation. According to this model, Tat‐BECN1 is bound to a groove located at the dimer interface of the WT GAPR‐1. A model of the BECN1 (248–450):GAPR‐1 dimer complex was constructed by aligning the BECN1 (248–450) structure with the GAPR‐1 dimer:Tat‐BECN1 (248–450) model using PyMol. According to these models, dimeric GAPR‐1 is not likely to bind to BECN1 or even the Tat‐BECN1 (248–450) peptide, since BECN1 binding to a GAPR‐1 dimer would result in extensive steric clashes.Several conserved residues in this groove interact with Tat‐BECN1, so we mutated several combinations of these conserved residues, and tested if mutations abrogate the interaction between GAPR‐1 and BECN1. We find that two sets of GAPR‐1 mutations E86A/G102K/H103A/G138N and H54A/E86A/G102K/H103A/G138N abrogate their interactions with BECN1. Furthermore, we determined the crystal structure of the latter mutant, and compared this structure with WT GAPR‐1. We find that the binding groove of the WT GAPR‐1 is more hydrophobic, while the groove of mutant GAPR‐1 is more positively charged, and therefore may not efficiently bind BECN1 residues 267–284, which include many hydrophobic residues.Support or Funding InformationNIH grants RO3 NS090939 (S.C.S.) and U19AI109725 (S.C.S)NSF grant MCB‐1413525 (S.C.S.);and NSF and North Dakota EPSCoR grant II‐1355466 doctoral dissertation awards for M.S. (PI: S.C.S.)

Mammalian Golgi-associated plant pathogenesis-related protein 1 (GAPR-1) is a negative autophagy regulator that binds Beclin 1, a key component of the autophagosome nucleation complex. Beclin 1 residues 267-284 are required for binding GAPR-1. Here, sequence analyses, structural modeling, mutagenesis combined with pull-down assays, X-ray crystal structure determination and small-angle X-ray scattering were used to investigate the Beclin 1-GAPR-1 interaction. Five conserved residues line an equatorial GAPR-1 surface groove that is large enough to bind a peptide. A model of a peptide comprising Beclin 1 residues 267-284 docked onto GAPR-1, built using the CABS-dock server, indicates that this peptide binds to this GAPR-1 groove. Mutation of the five conserved residues lining this groove, H54A/E86A/G102K/H103A/N138G, abrogates Beclin 1 binding. The 1.27 Å resolution X-ray crystal structure of this pentad mutant GAPR-1 was determined. Comparison with the wild-type (WT) GAPR-1 structure shows that the equatorial groove of the pentad mutant is shallower and more positively charged, and therefore may not efficiently bind Beclin 1 residues 267-284, which include many hydrophobic residues. Both WT and pentad mutant GAPR-1 crystallize as dimers, and in each case the equatorial groove of one subunit is partially occluded by the other subunit, indicating that dimeric GAPR-1 is unlikely to bind Beclin 1. SAXS analysis of WT and pentad mutant GAPR-1 indicates that in solution the WT forms monomers, while the pentad mutant is primarily dimeric. Thus, changes in the structure of the equatorial groove combined with the improved dimerization of pentad mutant GAPR-1 are likely to abrogate binding to Beclin 1.

Autophagy, an essential eukaryotic cellular homeostasis pathway, is responsible for sequestration of unwanted, damaged or harmful cytoplasmic components in vesicles called autophagosomes, followed by their lysosomal degradation, thereby enabling nutrient recycling. Autophagosome nucleation is mediated by Class III phosphatidylinositol 3‐kinase complexes that include two key autophagy proteins, a BECN (also called Beclin, ATG6 or VPS30) homolog and ATG14/BARKOR. BECN1 was amongst the first mammalian autophagy proteins to be identified1. Recently, mammals were shown to encode a second BECN paralog, BECN22. BECN2 shares 57% sequence identity with BECN1 is also involved in autophagy as well as in ligand‐induced endolysosomal degradation of several cellular GPCRs. BECN2 heterozygous knockout mice have defective autophagy, increased levels of brain cannabinoid 1 receptor, elevated food intake, obesity and insulin resistance2. Further, BECN2 regulates endolysosomal degradation and oncogenic signaling of GPCRs encoded by the Kaposi's Sarcoma‐associated herpesvirus3.Here we focus on understanding the structure of the helical, coiled‐coil domain (CCD) of BECN homologs and the similarities and differences in their interaction with ATG14. X‐ray crystal structures of the CCDs of human BECN1 and BECN2 (unpublished data), show that they form similar autophagy‐inactive, anti‐parallel homodimers, which includes six and seven non‐ideal packing interactions respectively, although the BECN2 CCD homodimer is significantly more curved and has a tighter binding affinity than that of BECN1. Conversely, the CCDs of both BECN1 and BECN2 bind the ATG14 CCD to form a parallel, curved heterodimer stabilized by multiple, conserved polar interactions. Isothermal calorimetry experiments demonstrate that compared to BECN1, the BECN2 CCD forms a stronger heterodimer with the ATG14 CCD. Mutation of non‐ideal BECN1/BECN2 interface residues to more ideal pairs improves homodimer self‐association and thermal stability. Many of these BECN1 mutations knock‐out binding to ATG14, but all BECN2 CCD mutants bind ATG14, although more weakly than wild‐type. Thus, polar CCD interface residues of BECN homologs result in a metastable homodimer, facilitating dissociation; but enable better interactions with polar ATG14 residues stabilizing the BECN:ATG14 heterodimer. Further, these structure‐based mechanistic differences in BECN1 and BECN2 homodimerization and heterodimerization likely dictate competitive ATG14 recruitment within the cell.Support or Funding InformationThis work was supported by NIH grants RO3 NS090939 (S.C.S.) and R15 GM113227 (C.L.C.); a National Science Foundation grant MCB‐1413525 (S.C.S.); and NSF and North Dakota EPSCoR grant II‐1355466 doctoral dissertation awards for M.S. (PI: S.C.S.).

ATG14 binding to BECN/Beclin homologs is essential for autophagy, a critical catabolic homeostasis pathway. Here, we show that the α‐helical, coiled‐coil domain (CCD) of BECN2, a recently identified mammalian BECN1 paralog, forms an antiparallel, curved homodimer with seven pairs of nonideal packing interactions, while the BECN2 CCD and ATG14 CCD form a parallel, curved heterodimer stabilized by multiple, conserved polar interactions. Compared to BECN1, the BECN2 CCD forms a weaker homodimer, but binds more tightly to the ATG14 CCD. Mutation of nonideal BECN2 interface residues to more ideal pairs improves homodimer self‐association and thermal stability. Unlike BECN1, all BECN2 CCD mutants bind ATG14, although more weakly than wild type. Thus, polar BECN2 CCD interface residues result in a metastable homodimer, facilitating dissociation, but enable better interactions with polar ATG14 residues stabilizing the BECN2:ATG14 heterodimer. These structure‐based mechanistic differences in BECN1 and BECN2 homodimerization and heterodimerization likely dictate competitive ATG14 recruitment.

BECN1 (Beclin 1), a highly conserved eukaryotic protein, is a key regulator of autophagy, a cellular homeostasis pathway, and also participates in vacuolar protein sorting, endocytic trafficking, and apoptosis. BECN1 is important for embryonic development, the innate immune response, tumor suppression, and protection against neurodegenerative disorders, diabetes, and heart disease. BECN1 mediates autophagy as a core component of the class III phosphatidylinositol 3-kinase complexes. However, the exact mechanism by which it regulates the activity of these complexes, or mediates its other diverse functions is unclear. BECN1 interacts with several diverse protein partners, perhaps serving as a scaffold or interaction hub for autophagy. Based on extensive structural, biophysical and bioinformatics analyses, BECN1 consists of an intrinsically disordered region (IDR), which includes a BH3 homology domain (BH3D); a flexible helical domain (FHD); a coiled-coil domain (CCD); and a β-α-repeated autophagy-specific domain (BARAD). Each of these BECN1 domains mediates multiple diverse interactions that involve concomitant conformational changes. Thus, BECN1 conformational flexibility likely plays a key role in facilitating diverse protein interactions. Further, BECN1 conformation and interactions are also modulated by numerous post-translational modifications. A better structure-based understanding of the interplay between different BECN1 conformational and binding states, and the impact of post-translational modifications will be essential to elucidating the mechanism of its multiple biological roles.

Autophagy, an essential eukaryotic homeostasis pathway, allows the sequestration of unwanted, damaged, or harmful cytoplasmic components in vesicles called autophagosomes, permitting subsequent lysosomal degradation and nutrient recycling. Autophagosome nucleation is mediated by class III phosphatidylinositol-3-kinase complexes that include two key autophagy proteins, BECN1/Beclin 1 and ATG14/BARKOR, which form parallel heterodimers via their coiled-coil domains (CCDs). Here we present the 1.46 Å X-ray crystal structure of the antiparallel, human BECN1 CCD homodimer, which represents BECN1 oligomerization outside the autophagosome nucleation complex. We use circular dichroism and small-angle X-ray scattering (SAXS) to show that the ATG14 CCD is significantly disordered but becomes more helical in the BECN1:ATG14 heterodimer, although it is less well-folded than the BECN1 CCD homodimer. SAXS also indicates that the BECN1:ATG14 heterodimer is more curved than other BECN1-containing CCD dimers, which has important implications for the structure of the autophagosome nucleation complex. A model of the BECN1:ATG14 CCD heterodimer that agrees well with the SAXS data shows that BECN1 residues at the homodimer interface are also responsible for heterodimerization, allowing us to identify ATG14 interface residues. Finally, we verify the role of BECN1 and ATG14 interface residues in binding by assessing the impact of point mutations of these residues on co-immunoprecipitation of the partner and demonstrate that these mutations abrogate starvation-induced upregulation of autophagy but do not impact basal autophagy. Thus, this research provides insights into structures of the BECN1 CCD homodimer and the BECN1:ATG14 CCD heterodimer and identifies interface residues that are important for BECN1:ATG14 heterodimerization and for autophagy.

γ-herpesviruses (γHVs) are common human pathogens that encode homologs of the anti-apoptotic cellular Bcl-2 proteins, which are critical to viral reactivation and oncogenic transformation. Bcl-XL, a cellular Bcl-2 homolog, and the γHV68 Bcl-2 homolog, M11, both bind to a BH3 domain within the key autophagy effector BECN1 with comparable affinities, resulting in the suppression of BECN1-mediated autophagy. Despite this similarity, the different residues lining the binding site of M11 and Bcl-XL dictate varying affinities for BH3 domains from diverse proteins. Here we delineate BECN1 differential specificity determinants for binding to M11 or Bcl-XL by quantifying autophagy levels in cells expressing different BECN1 mutants and either M11 or Bcl-XL, and we show that a G120E+D121A BECN1 mutant selectively prevents suppression of BECN1-mediated autophagy by Bcl-XL, but not by M11. We use isothermal titration calorimetry to identify a BECN1 BH3 domain-derived peptide that selectively binds to M11, but not to Bcl-XL. The X-ray crystal structure of this peptide bound to M11 reveals the mechanism by which the M11 BH3 domain-binding groove accommodates this M11-specific peptide. Lastly, we have used this information to develop a cell-permeable peptide inhibitor that selectively inhibits M11-mediated, but not Bcl-XL-mediated suppression of autophagy. We are now testing the ability of this inhibitor to selectively inhibit M11-mediated suppression of apoptosis. This work was funded by NIH grants P20 RR015566 and P30 GM103332-01, and NSF grant EPS-0814442 to SS and CC; NIH grant R21 AI078108 and NSF grant HRD-0811239 to SS.

Autophagy is a fundamental adaptive response to amino acid starvation orchestrated by conserved gene products, the autophagy (ATG) proteins. However, the cellular cues that activate the function of ATG proteins during amino acid starvation are incompletely understood. Here we show that two related stress-responsive kinases, members of the p38 mitogen-activated protein kinase (MAPK) signaling pathway MAPKAPK2 (MK2) and MAPKAPK3 (MK3), positively regulate starvation-induced autophagy by phosphorylating an essential ATG protein, Beclin 1, at serine 90, and that this phosphorylation site is essential for the tumor suppressor function of Beclin 1. Moreover, MK2/MK3-dependent Beclin 1 phosphorylation (and starvation-induced autophagy) is blocked in vitro and in vivo by BCL2, a negative regulator of Beclin 1. Together, these findings reveal MK2/MK3 as crucial stress-responsive kinases that promote autophagy through Beclin 1 S90 phosphorylation, and identify the blockade of MK2/3-dependent Beclin 1 S90 phosphorylation as a mechanism by which BCL2 inhibits the autophagy function of Beclin 1.

Autophagy, an essential conserved, cell survival pathway in eukaryotes is responsible for degradation and recycling of unwanted, damaged or harmful cytoplasmic components. Our pathway-wide bioinformatics analysis shows that intrinsically disordered regions (IDRs) are the most common structural feature of autophagy proteins and these IDRs may be responsible for many regulatory interactions. BECN1 is a critical component of the autophagy nucleation complex. Aberrant or reduced autophagy, due to BECN1 defects or low levels is implicated in many diseases. Over 20 cellular or viral proteins appear to interact with BECN1 to modulate autophagy. We are using a multi-faceted experimental approach encompassing bioinformatics, computational, structural, biophysical, biochemical, cell biology and molecular biology methods to understand the mechanism by which BECN1 mediates autophagy, and how partners bind to BECN1 to modulate autophagy. Our work reveals a structural and functional dichotomy within the 450-residue human BECN1: residues 42-132 constitute a poorly conserved IDR that includes a BECN1 BH3 domain (BH3D) responsible for binding Bcl-2 proteins, while the highly-conserved C-terminal residues 133-450 comprise three conformationally-flexible domains: the helical domain (HD), the coiled-coil domain and the b-a-repeat in autophagy domain. Our structural and biophysical results show that the BH3D and HD undergo binding-associated disorder-to-structure transitions and have identified residues important for regulating autophagy. Funded by NIH grant R03 NS090939 and NSF grant 1413525.

Background: Cellular and γ-herpesvirus Bcl-2 homologs down-regulate autophagy. Results: A peptide designed to bind to the γ-herpesvirus68 Bcl-2, M11, but not cellular Bcl-2 homologs, abrogates M11-mediated down-regulation of autophagy. Conclusion: This peptide is a selective M11 inhibitor. Significance: Such selective inhibitors are important for understanding the role of γ-herpesvirus Bcl-2 homologs in viral reactivation and oncogenic transformation of host cells. γ-herpesviruses (γHVs) are common human pathogens that encode homologs of the anti-apoptotic cellular Bcl-2 proteins, which are critical to viral reactivation and oncogenic transformation. The murine γHV68 provides a tractable in vivo model for understanding general features of these important human pathogens. Bcl-XL, a cellular Bcl-2 homolog, and the murine γHV68 Bcl-2 homolog, M11, both bind to a BH3 domain within the key autophagy effector Beclin 1 with comparable affinities, resulting in the down-regulation of Beclin 1-mediated autophagy. Despite this similarity, differences in residues lining the binding site of M11 and Bcl-XL dictate varying affinities for the different BH3 domain-containing proteins. Here we delineate Beclin 1 differential specificity determinants for binding to M11 or Bcl-XL by quantifying autophagy levels in cells expressing different Beclin 1 mutants and either M11 or Bcl-XL, and we show that a G120E/D121A Beclin 1 mutant selectively prevents down-regulation of Beclin 1-mediated autophagy by Bcl-XL, but not by M11. We use isothermal titration calorimetry to identify a Beclin 1 BH3 domain-derived peptide that selectively binds to M11, but not to Bcl-XL. The x-ray crystal structure of this peptide bound to M11 reveals the mechanism by which the M11 BH3 domain-binding groove accommodates this M11-specific peptide. This information was used to develop a cell-permeable peptide inhibitor that selectively inhibits M11-mediated, but not Bcl-XL-mediated, down-regulation of autophagy.

ABSTRACT Autophagy is an essential eukaryotic pathway required for cellular homeostasis. Numerous key autophagy effectors and regulators have been identified, but the mechanism by which they carry out their function in autophagy is not fully understood. Our rigorous bioinformatic analysis shows that the majority of key human autophagy proteins include i ntrinsically d isordered r egions (IDRs), which are sequences lacking stable secondary and tertiary structure; suggesting that IDRs play an important, yet hitherto uninvestigated, role in autophagy. Available crystal structures corroborate the absence of structure in some of these predicted IDRs. Regions of orthologs equivalent to the IDRs predicted in the human autophagy proteins are poorly conserved, indicating that these regions may have diverse functions in different homologs. We also show that IDRs predicted in human proteins contain several regions predicted to facilitate protein–protein interactions, and delineate the network of proteins that interact with each predicted IDR‐containing autophagy protein, suggesting that many of these interactions may involve IDRs. Lastly, we experimentally show that a BCL2 homology 3 domain (BH3D), within the key autophagy effector BECN1 is an IDR. This BH3D undergoes a dramatic conformational change from coil to α‐helix upon binding to BCL2s, with the C‐terminal half of this BH3D constituting a binding motif, which serves to anchor the interaction of the BH3D to BCL2s. The information presented here will help inform future in‐depth investigations of the biological role and mechanism of IDRs in autophagy proteins. Proteins 2014; 82:565–578. © 2013 Wiley Periodicals, Inc.

Autophagy and apoptosis are catabolic pathways essential for organismal homeostasis. Autophagy is normally a cell-survival pathway involving the degradation and recycling of obsolete, damaged, or harmful macromolecular assemblies; however, excess autophagy has been implicated in type II cell death. Apoptosis is the canonical programmed cell death pathway. Autophagy and apoptosis have now been shown to be interconnected by several molecular nodes of crosstalk, enabling the coordinate regulation of degradation by these pathways. Normally, autophagy and apoptosis are both tumor suppressor pathways. Autophagy fulfils this role as it facilitates the degradation of oncogenic molecules, preventing development of cancers, while apoptosis prevents the survival of cancer cells. Consequently, defective or inadequate levels of either autophagy or apoptosis can lead to cancer. However, autophagy appears to have a dual role in cancer, as it has now been shown that autophagy also facilitates the survival of tumor cells in stress conditions such as hypoxic or low-nutrition environments. Here we review the multiple molecular mechanisms of coordination of autophagy and apoptosis and the role of the proteins involved in this crosstalk in cancer. A comprehensive understanding of the interconnectivity of autophagy and apoptosis is essential for the development of effective cancer therapeutics.