Multiple Active Conformations Research Articles

Composite hydrogel derived iron/nitrogen co-doped carbon for bisphenol A removal

Conventional carbon-based adsorbents encounter insufficient active sites, low adsorption efficiency and poor anti-interference ability for bisphenol A (BPA) removal. Herein, we developed a cellulose nanocrystal/polyacrylic acid hydrogel derived strategy for the preparation of a novel iron/nitrogen co-doped carbon (FeN@CP800) via simple immersion of iron (III) acetylacetonate and melamine in hydrogel precursors and further carbonization at 800 °C. The precursor scaffold interacted with active species source by swelling behavior to form hydrogen bonds and Fe coordination, thereby facilitating subsequent growth of multiple active conformations without aggregation. The hierarchical porous surface morphology, abundant N doping, multi-active site structure and large specific surface area significantly improved the accessible BPA adsorption of FeN@CP800. The maximum adsorption capacity calculated by Langmuir model was 309.17 mg·g−1, 4.5 times higher than that of bare CP. Benefited from pore filling, hydrogen bonds, π-π stacking and hydrophobic interactions, the excellent adsorption performance of FeN@CP800 was retained under varied coexisting ions species, ionic strength and solution pH with 84 % capacity retention after 5 cycles. DFT calculations disclosed the Fe4N component was the predominant active sites to improve the adsorption and interfacial transfer process. Thus, these findings provide a new strategy to design high-performance adsorbent for phenolic pollutants removal in water.

Revisiting degron motifs in human AURKA required for its targeting by APC/CFZR1.

Mitotic kinase Aurora A (AURKA) diverges from other kinases in its multiple active conformations that may explain its interphase roles and the limited efficacy of drugs targeting the kinase pocket. Regulation of AURKA activity by the cell is critically dependent on destruction mediated by the anaphase-promoting complex (APC/CFZR1) during mitotic exit and G1 phase and requires an atypical N-terminal degron in AURKA called the "A-box" in addition to a reported canonical D-box degron in the C-terminus. Here, we find that the reported C-terminal D-box of AURKA does not act as a degron and instead mediates essential structural features of the protein. In living cells, the N-terminal intrinsically disordered region of AURKA containing the A-box is sufficient to confer FZR1-dependent mitotic degradation. Both in silico and in cellulo assays predict the QRVL short linear interacting motif of the A-box to be a phospho-regulated D-box. We propose that degradation of full-length AURKA also depends on an intact C-terminal domain because of critical conformational parameters permissive for both activity and mitotic degradation of AURKA.

Determination of the molecular reach of the protein tyrosine phosphatase SHP-1

Immune receptors signal by recruiting (or tethering) enzymes to their cytoplasmic tails to catalyze reactions on substrates within reach. This is the case for the phosphatase SHP-1, which, upon tethering to inhibitory receptors, dephosphorylates diverse substrates to control T cell activation. Precisely how tethering regulates SHP-1 activity is incompletely understood. Here, we measure binding, catalysis, and molecular reach for tethered SHP-1 reactions. We determine the molecular reach of SHP-1 to be 13.0 nm, which is longer than the estimate from the allosterically active structure (5.3 nm), suggesting that SHP-1 can achieve a longer reach by exploring multiple active conformations. Using modeling, we show that when uniformly distributed, receptor-SHP-1 complexes can only reach 15% of substrates, but this increases to 90% when they are coclustered. When within reach, we show that membrane recruitment increases the activity of SHP-1 by a 1000-fold increase in local concentration. The work highlights how molecular reach regulates the activity of membrane-recruited SHP-1 with insights applicable to other membrane-tethered reactions.

Position Review: Functional Selectivity in Mammalian Olfactory Receptors.

There is increasing appreciation that G-protein-coupled receptors (GPCRs) can initiate diverse cellular responses by activating multiple G proteins, arrestins, and other biochemical effectors. Structurally different ligands targeting the same receptor are thought to stabilize the receptor in multiple distinct active conformations such that specific subsets of signaling effectors are engaged at the exclusion of others, creating a bias toward a particular outcome, which has been referred to as ligand-induced selective signaling, biased agonism, ligand-directed signaling, and functional selectivity, among others. The potential involvement of functional selectivity in mammalian olfactory signal transduction has received little attention, notwithstanding the fact that mammalian olfactory receptors comprise the largest family of mammalian GPCRs. This position review considers the possibility that, although such complexity in G-protein function may have been lost in the specialization of olfactory receptors to serve as sensory receptors, the ability of olfactory receptor neurons (ORNs) to function as signal integrators and growing appreciation that this functionality is widespread in the receptor population suggest otherwise. We pose that functional selectivity driving 2 opponent inputs have the potential to generate an output that reflects the balance of ligand-dependent signaling, the direction of which could be either suppressive or synergistic and, as such, needs to be considered as a mechanistic basis for signal integration in mammalian ORNs.

Csk-homologous kinase (Chk) is an efficient inhibitor of Src-family kinases but a poor catalyst of phosphorylation of their C-terminal regulatory tyrosine

BackgroundC-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are the major endogenous inhibitors of Src-family kinases (SFKs). They employ two mechanisms to inhibit SFKs. First, they phosphorylate the C-terminal tail tyrosine which stabilizes SFKs in a closed inactive conformation by engaging the SH2 domain in cis. Second, they employ a non-catalytic inhibitory mechanism involving direct binding of Csk and Chk to the active forms of SFKs that is independent of phosphorylation of their C-terminal tail. Csk and Chk are co-expressed in many cell types. Contributions of the two mechanisms towards the inhibitory activity of Csk and Chk are not fully clear. Furthermore, the determinants in Csk and Chk governing their inhibition of SFKs by the non-catalytic inhibitory mechanism are yet to be defined.MethodsWe determined the contributions of the two mechanisms towards the inhibitory activity of Csk and Chk both in vitro and in transduced colorectal cancer cells. Specifically, we assayed the catalytic activities of Csk and Chk in phosphorylating a specific peptide substrate and a recombinant SFK member Src. We employed surface plasmon resonance spectroscopy to measure the kinetic parameters of binding of Csk, Chk and their mutants to a constitutively active mutant of the SFK member Hck. Finally, we determined the effects of expression of recombinant Chk on anchorage-independent growth and SFK catalytic activity in Chk-deficient colorectal cancer cells.ResultsOur results revealed Csk as a robust enzyme catalysing phosphorylation of the C-terminal tail tyrosine of SFKs but a weak non-catalytic inhibitor of SFKs. In contrast, Chk is a poor catalyst of SFK tail phosphorylation but binds SFKs with high affinity, enabling it to efficiently inhibit SFKs with the non-catalytic inhibitory mechanism both in vitro and in transduced colorectal cancer cells. Further analyses mapped some of the determinants governing this non-catalytic inhibitory mechanism of Chk to its kinase domain.ConclusionsSFKs are activated by different upstream signals to adopt multiple active conformations in cells. SFKs adopting these conformations can effectively be constrained by the two complementary inhibitory mechanisms of Csk and Chk. Furthermore, the lack of this non-catalytic inhibitory mechanism accounts for SFK overactivation in the Chk-deficient colorectal cancer cells.

Mechanism of Allosteric Communication in GPCR Activation from Microsecond Scale Molecular Dynamics Simulations

G protein coupled receptors (GPCRs) are membrane proteins that signal through effector proteins bound at the intracellular (IC) interface. Under physiological conditions, GPCRs adopt multiple inactive and active conformations, thereby signaling through diverse signaling pathways. Binding of agonists at the extracellular (EC) domain initiates a conformational change in the receptor leading to activation. Antagonists and inverse agonists suppress activation by stabilizing one of the inactive states. The communication between the agonist/antagonist binding site and the IC interface takes place through allosteric coupling of distant receptor domains. However, the detailed mechanism of allosteric communication in GPCR signaling is not well understood. Using ensembles of molecular dynamics (MD) simulations on eight class A GPCRs, we showed that allosteric communication between the antagonist binding site and the IC interface stabilizes the inactive state, and disruption of this allosteric communication due to agonist binding leads to activation. Furthermore, the fundamental framework and mechanism of allosteric communication are conserved among multiple GPCRs, as is evident from the conserved allosteric hubs (residues that mediate multiple allosteric pathways) in these receptors. Mutating the allosteric hubs either suppress activation or impart constitutive activity, suggesting the key role of these residues in GPCR function. We have shown that, besides stabilizing GPCR functional states, allosteric communication is also responsible for transitions among functional states. Analysis of the deactivation dynamics of two class A GPCRs (β2 adrenergic receptor and neurotensin receptor 1) shows that several allosteric hubs in the middle of the transmembrane domain undergo concerted dihedral changes during the transition from the active to the inactive state. These observations provide valuable insights into the complex mechanism of GPCR activation and membrane protein function in general.

Identifying multiple active conformations in the G protein-coupled receptor activation landscape using computational methods.

G protein-coupled receptors (GPCRs) are membrane proteins critical in cellular signaling, making them important targets for therapeutics. The activation of GPCRs is central to their function, requiring multiple conformations of the GPCRs in their activation landscape. To enable rational design of GPCR-targeting drugs, it is essential to obtain the ensemble of atomistic structures of GPCRs along their activation pathways. This is most challenging for structure determination experiments, making it valuable to develop reliable computational structure prediction methods. In particular, since the active-state conformations are higher in energy (less stable) than inactive-state conformations, they are difficult to stabilize. In addition, the computational methods are generally biased toward lowest energy structures by design and miss these high energy but functionally important conformations. To address this problem, we have developed a computationally efficient ActiveGEnSeMBLE method that systematically predicts multiple conformations that are likely in the GPCR activation landscape, including multiple active- and inactive-state conformations. ActiveGEnSeMBLE starts with a systematic coarse grid sampling of helix tilts/rotations (~13 trillion transmembrane domain conformations) and identifies multiple potential active-state energy wells, using the TM3-TM6 intracellular distance as a surrogate activation coordinate. These energy wells are then sampled locally using a finer grid in conformational space to find a locally minimized conformation in each energy well, which can be further relaxed using molecular dynamics (MD) simulations. This method, combining homology modeling, hierarchical complete conformational sampling, and nanosecond scale MD, provides one of the very few computational methods that predict multiple candidates for active-state conformations and is one of the most computationally affordable.

Molecular Pharmacology of δ-Opioid Receptors.

Opioids are among the most effective analgesics available and are the first choice in the treatment of acute severe pain. However, partial efficacy, a tendency to produce tolerance, and a host of ill-tolerated side effects make clinically available opioids less effective in the management of chronic pain syndromes. Given that most therapeutic opioids produce their actions via µ-opioid receptors (MOPrs), other targets are constantly being explored, among which δ-opioid receptors (DOPrs) are being increasingly considered as promising alternatives. This review addresses DOPrs from the perspective of cellular and molecular determinants of their pharmacological diversity. Thus, DOPr ligands are examined in terms of structural and functional variety, DOPrs' capacity to engage a multiplicity of canonical and noncanonical G protein-dependent responses is surveyed, and evidence supporting ligand-specific signaling and regulation is analyzed. Pharmacological DOPr subtypes are examined in light of the ability of DOPr to organize into multimeric arrays and to adopt multiple active conformations as well as differences in ligand kinetics. Current knowledge on DOPr targeting to the membrane is examined as a means of understanding how these receptors are especially active in chronic pain management. Insight into cellular and molecular mechanisms of pharmacological diversity should guide the rational design of more effective, longer-lasting, and better-tolerated opioid analgesics for chronic pain management.

Funktionelle Selektivität am Beispiel des β2-Adrenozeptors

Originally, G protein-coupled receptors were assumed to act as on-off switches, adopting either an active (R*) or inactive (R) conformation. However, pharmacological and biophysical studies over the past 15–20 years have now unequivocally shown that receptors can exist in multiple active conformations with distinct capabilities to activate effectors. This concept is referred to as functional selectivity and has important implications for future drug design. Here, we discuss this fundamental pharmacological concept using the β2-adrenoceptor as paradigm.

Structural Studies of Yeast Δ1-Pyrroline-5-carboxylate Dehydrogenase (ALDH4A1): Active Site Flexibility and Oligomeric State

The proline catabolic enzyme Δ1-pyrroline-5-carboxylate dehydrogenase (ALDH4A1) catalyzes the NAD+-dependent oxidation of γ-glutamate semialdehyde to l-glutamate. In Saccharomyces cerevisiae, ALDH4A1 is encoded by the PUT2 gene and known as Put2p. Here we report the steady-state kinetic parameters of the purified recombinant enzyme, two crystal structures of Put2p, and the determination of the oligomeric state and quaternary structure from small-angle X-ray scattering and sedimentation velocity. Using Δ1-pyrroline-5-carboxylate as the substrate, catalytic parameters kcat and Km were determined to be 1.5 s–1 and 104 μM, respectively, with a catalytic efficiency of 14000 M–1 s–1. Although Put2p exhibits the expected aldehyde dehydrogenase superfamily fold, a large portion of the active site is disordered in the crystal structure. Electron density for the 23-residue aldehyde substrate-binding loop is absent, implying substantial conformational flexibility in solution. We furthermore report a new crystal form of human ALDH4A1 (42% identical to Put2p) that also shows disorder in this loop. The crystal structures provide evidence of multiple active site conformations in the substrate-free form of the enzyme, which is consistent with a conformational selection mechanism of substrate binding. We also show that Put2p forms a trimer-of-dimers hexamer in solution. This result is unexpected because human ALDH4A1 is dimeric, whereas some bacterial ALDH4A1s are hexameric. Thus, global sequence identity and domain of life are poor predictors of the oligomeric states of ALDH4A1. Mutation of a single Trp residue that forms knob-in-hole interactions across the dimer–dimer interface abrogates hexamer formation, suggesting that this residue is the center of a protein–protein association hot spot.

Gonadotropin-Releasing Hormone Receptor Signaling: Biased and Unbiased

Gonadotropin-releasing hormone is a neuropeptide that acts via Gq coupled G-protein coupled receptors in the pituitary that mediate central control of reproduction. GnRH receptors (GnRHR) and GnRH ligands are also found in extra-pituitary sites including the CNS as well as reproductive tissues and cancer cells derived from such tissues. Much of the interest in the extra-pituitary receptors stems from the fact that they mediate anti-proliferative and/or pro-apoptotic effects and may therefore be directly targeted for cancer therapy. Type I mammalian GnRHR are atypical in that they do not bind to (or signal via) arrestins. In spite of this restriction on their signaling repertoire, there is good evidence for existence of multiple active GnRHR conformations and for activation of multiple upstream effectors (heterotrimeric and monomeric G-proteins). In this review GnRHR signaling is described, with emphasis on the relevance of functional selectivity for pharmacological characterization of GnRHR ligands, as well as its possible contribution to contextdependent GnRHR signaling and relevance for GnRHR-mediated effects on cell fate as well as GnRHR trafficking.

Conformational Selection and Allosteric Regulation upon Ligand Binding in G-Protein Coupled Receptors

G-protein coupled receptors (GPCRs) are allosteric membrane proteins mediating cellular signaling. GPCRs exhibit multiple inactive and active conformations, and the population balance between these conformations is altered upon binding of signaling molecules (or ligands). However, the nature of the conformational ensemble or the mechanism of the conformational transitions is not well understood. We have applied a multiscale computational approach combining a coarse-grained discrete conformational sampling method with fine-grained molecular dynamics to investigate the effect of various ligands binding on the ensemble of conformations sampled by human β2-adrenergic receptor (β2AR). We show that the receptor, in the absence of any ligand, samples an extensive conformational space that includes breathing of the orthosteric ligand binding site and shear motion of the transmembrane helices 5 and 6 against the other helices. The shear motion is similar to the reorganization of the intracellular regions of TM3, TM5, and TM6 observed in the crystal structure of the active state of GPCRs. Upon ligand binding a shift in population density, as well as a reduction in the number of conformations sampled by the receptor is observed. Binding of agonist norepinephrine or partial agonist salbutamol leads to the selection of a subset of conformations that include both active and inactive state conformations, while inverse agonist carazolol selects only the inactive state conformation. Possible mechanisms for the allosteric regulation of GPCR activity by ligand binding were identified by correlating receptor-ligand interactions with receptor sidechain orientation near the G-Protein coupling region.

Biased agonism of the calcium-sensing receptor

After the discovery of molecules modulating G protein-coupled receptors (GPCRs) that are able to selectively affect one signaling pathway over others for a specific GPCR, thereby “biasing” the signaling, it has become obvious that the original model of GPCRs existing in either an “on” or “off” conformation is too simple. The current explanation for this biased agonism is that GPCRs can adopt multiple active conformations stabilized by different molecules, and that each conformation affects intracellular signaling in a different way. In the present study we sought to investigate biased agonism of the calcium-sensing receptor (CaSR), by looking at 12 well-known orthosteric CaSR agonists in 3 different CaSR signaling pathways: Gq/11 protein, Gi/o protein, and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Here we show that apart from Gq/11 and Gi/o signaling, ERK1/2 is activated through recruitment of β-arrestins. Next, by measuring activity of all three signaling pathways we found that barium, spermine, neomycin, and tobramycin act as biased agonist in terms of efficacy and/or potency. Finally, polyamines and aminoglycosides in general were biased in their potencies toward ERK1/2 signaling. In conclusion, the results of this study indicate that several active conformations of CaSR, stabilized by different molecules, exist, which affect intracellular signaling distinctly.

Non-Canonical Cleavage of Protease Activated Receptor 1 (PAR1) by Activated Protein C Provides Novel Insights Into the Repertoire of Cytoprotective and Proinflammatory PAR1 Signaling

Abstract 534 Activated protein C (APC) exerts direct cytoprotective activities on cells that are required for APC's ability to reduce mortality in murine sepsis models and that likely have potential contributions to mortality reduction in patients with severe sepsis. Although multiple receptors have been implicated to promote APC direct cytoprotective activities on various cell types, the endothelial protein C receptor (EPCR) and PAR1 play pivotal roles in our current understanding of the molecular mechanisms for APC's direct effects on cells. According to the current generally accepted model, binding of APC to EPCR on the endothelial cell membrane facilitates activation of PAR1 and induction of signaling that ultimately results in cytoprotective effects. However, this model raises the dilemma of how thrombin and APC can often have seemingly opposing effects when activating PAR1. To gain insights into mechanisms for the contrasting effects of APC vs. thrombin signaling, PAR1 activation by APC was studied in greater detail. We hypothesized that APC might cleave PAR1 not only at Arg41 but also at Arg46 with distinctly different consequences than caused by Arg41 cleavage and that this alternative cleavage could distinguish APC's from thrombin's effects. Previously we found that, in the presence of EDTA, APC cleaved a synthetic PAR1 N-terminal peptide (TR33-66) at Arg41. Now we have found that, in buffers containing CaCl2, APC cleaved the PAR1 TR33-66 peptide at Arg41 and also at an additional site distal from Arg41. Proteolysis of the TR33-66 peptide with APC resulted in fragments corresponding to TR33-41 and TR42-66 similar to thrombin. But in contrast to thrombin, a third fragment was generated by APC and the TR42-66 fragment disappeared over time with the concomitant accumulation of a novel peptide. Furthermore, incubation of thrombin-cleaved TR33-66 with APC resulted in additional proteolysis of TR42-66 and generation of the same novel fragment, indicating the possibility of a second APC cleavage site in PAR1 that was distinct and distal from Arg41. Isolation of the novel proteolytic fragments and their MALDI-TOF analysis identified Arg46 as the second APC cleavage site in the TR33-66 peptide. When cells containing wild-type EPCR were transfected with SEAP-PAR1 wild type and mutant constructs, both thrombin and APC cleaved wt-PAR1. As anticipated, efficient cleavage by thrombin was observed for R46Q-PAR1 but not R41Q-PAR1 or R41Q/R46Q-PAR1. In contrast, APC readily cleaved both R41Q-PAR1 and R46Q-PAR1 whereas cleavage of R41Q/R46Q-PAR1 by APC was negligible. APC mediated cleavage of R41Q-PAR1 and R46Q-PAR1 required the presence of functional EPCR and was not supported by the APC-binding-defective E86A-EPCR mutant. These results indicate that on cells Arg46 in PAR1 can serve as a second cleavage site for APC. Since the new PAR1 N-terminus after proteolysis acts as a tethered ligand for receptor activation, cleavage at Arg41 vs. Arg46 could potentially create structurally distinct agonists, which might help explain the divergent patterns for PAR1-mediated cytoprotective APC signaling vs. proinflammatory IIa signaling. In studies to see if the APC-induced new PAR1 N-terminus starting at Asn47 could promote signaling, we found that a synthetic peptide with the PAR1 47–66 N-terminal sequence (NPND-peptide) increased Akt phosphorylation at Ser473 in endothelial cells over 30 min whereas neither a control scrambled sequence (47-66)-peptide nor a TRAP peptide had a similar remarkable effect on Akt phosphorylation. Moreover, the NPND-peptide, but neither the scrambled sequence-related peptide nor a TRAP peptide, inhibited staurosporine-induced endothelial cell apoptosis. Thus, it appears that the new N-terminus generated by APC's cleavage at Arg46 in PAR1 generates a novel tethered ligand that can induce cytoprotective APC-like but not thrombin-like signaling characteristics. In summary, APC is capable of a unique, functionally significant cleavage of PAR1 at Arg46 that can initiate distinctive signaling compared to canonical cleavage of PAR1 at Arg41. This implies activated PAR1 is a GPCR with multiple active conformations capable of multiple, agonist selective activity profiles which may help explain the divergent patterns for cytoprotective APC signaling vs. proinflammatory thrombin signaling. Disclosures: Mosnier: Scripps Research institute: Employment, Patents & Royalties. Griffin:ZZBiotech LLC: Consultancy, Membership on an entity's Board of Directors or advisory committees; Scripps Research Institute: Employment, Patents & Royalties.

Molecular Mechanism of β-Arrestin-Biased Agonism at Seven-Transmembrane Receptors

The concept of biased agonism has recently come to the fore with the realization that seven-transmembrane receptors (7TMRs, also known as G protein-coupled receptors, or GPCRs) activate complex signaling networks and can adopt multiple active conformations upon agonist binding. As a consequence, the "efficacy" of receptors, which was classically considered linear, is now recognized as pluridimensional. Biased agonists selectively stabilize only a subset of receptor conformations induced by the natural "unbiased" ligand, thus preferentially activating certain signaling mechanisms. Such agonists thus reveal the intriguing possibility that one can direct cellular signaling with unprecedented precision and specificity and support the notion that biased agonists may identify new classes of therapeutic agents that have fewer side effects. This review focuses on one particular class of biased ligands that has the ability to alter the balance between G protein-dependent and β-arrestin-dependent signal transduction.

The Role of Conformational Ensembles in Ligand Recognition in G-Protein Coupled Receptors

G-protein coupled receptors (GPCRs) are allosteric membrane proteins mediating cellular signaling. GPCRs exhibit multiple inactive and active conformations, and the population balance between these conformations is altered upon binding of signaling molecules (or ligands). However, the nature of the conformational ensemble or the mechanism of the conformational transitions is not well understood. We present a multiscale computational approach combining a coarse-grained discrete conformational sampling method with fine-grained molecular dynamics investigating the effect of various ligands binding on the ensemble of conformations sampled by human β2-adrenergic receptor (β2AR). We show that the receptor, in the absence of any ligand, samples an extensive conformational space that includes breathing of the orthosteric ligand binding site and shear motion of the transmembrane helices 5 and 6 against the other helices. The shear motion is similar to the reorganization of the intracellular regions of TM3, TM5, and TM6 observed in the crystal structure of the active state of GPCRs. The binding of agonist norepinephrine or partial agonist salbutamol leads to the selection of a subset of conformations including active and inactive state conformations, while inverse agonist carazolol selects only inactive state conformations. The dynamics of water observed during the simulations provides an explanation for the conformational changes observed in the solution-based fluorescence spectroscopic measurements on agonist activated β2AR, which could not be explained by the agonist bound β2AR crystal structure. This study shows that the receptor activation depends on both the low energy states and the range of the conformations sampled by the receptor.

Evidence for multiple active forms of the DNA damage response protein UmuD

All organisms experience DNA damage and have evolved multiple pathways to maintain genomic stability. In Escherichia coli, the SOS response to DNA damage involves the induction of 57 genes including the umuD gene products. The UmuD homodimer of 139‐amino acid subunits binds a RecA/ssDNA nucleoprotein filament which stimulates the latent ability of UmuD to cleave its N‐terminal 24‐amino acids, producing UmuD′2. UmuD2 and UmuD′2 play differential roles in the DNA damage response. Several models have been proposed for the conformations of UmuD2 including two versions with the N‐terminal arms in trans and two versions with the arms in cis (Beuning, et al. 2006 J. Biol. Chem 281:9633). These conformations expose different binding surfaces that may interact with other proteins. The goal of our research is to determine the conformation and dynamics of the umuD gene products in order to understand how their differential protein‐protein interactions regulate cellular responses to DNA damage. Our preliminary results suggest that a single amino acid substitution, N41G, weakens the KD for dimerization by over five orders of magnitude. This N41G mutation yields a UmuD monomer that is cleavable in the presence and absence of RecA/ssDNA. These results suggest that UmuD2 exists in multiple active conformations and that the cis conformation may be physiologically relevant. Supported by NSF Career Award MCB‐0845033.

Multiple native states reveal persistent ruggedness of an RNA folding landscape

According to the “thermodynamic hypothesis”, the sequence of a biological macromolecule defines its folded, active structure as a global energy minimum on the folding landscape.1,2 But the enormous complexity of folding landscapes of large macromolecules raises a question: Is there indeed a unique global energy minimum corresponding to a unique native conformation, or are there deep local minima corresponding to alternative active conformations?3 Folding of many proteins is well described by two-state models, leading to highly simplified representations of protein folding landscapes with a single native conformation.4,5 Nevertheless, accumulating experimental evidence suggests a more complex topology of folding landscapes with multiple active conformations that can take seconds or longer to interconvert.6,7,8 Here we employ single molecule experiments to demonstrate that an RNA enzyme folds into multiple distinct native states that interconvert much slower than the time scale of catalysis. These data demonstrate that the severe ruggedness of RNA folding landscapes extends into conformational space occupied by native conformations.

Smoothened Adopts Multiple Active and Inactive Conformations Capable of Trafficking to the Primary Cilium

Activation of Hedgehog (Hh) signaling requires the transmembrane protein Smoothened (Smo), a member of the G-protein coupled receptor superfamily. In mammals, Smo translocates to the primary cilium upon binding of Hh ligands to their receptor, Patched (Ptch1), but it is unclear if ciliary trafficking of Smo is sufficient for pathway activation. Here, we demonstrate that cyclopamine and jervine, two structurally related inhibitors of Smo, force ciliary translocation of Smo. Treatment with SANT-1, an unrelated Smo antagonist, abrogates cyclopamine- and jervine-mediated Smo translocation. Further, activation of protein kinase A, either directly or through activation of Gαs, causes Smo to translocate to a proximal region of the primary cilium. We propose that Smo adopts multiple inactive and active conformations, which influence its localization and trafficking on the primary cilium.

Evidence for Distinct Antagonist-Revealed Functional States of 5-Hydroxytryptamine2AReceptor Homodimers

The serotonin (5-hydroxytryptamine; 5-HT) 2A receptor is a cell surface class A G protein-coupled receptor that regulates a multitude of physiological functions of the body and is a target for antipsychotic drugs. Here we found by means of fluorescence resonance energy transfer and immunoprecipitation studies that the 5-HT(2A)-receptor homodimerized in live cells, which we linked with its antagonist-dependent fingerprint in both binding and receptor signaling. Some antagonists, like the atypical antipsychotics clozapine and risperidone, differentiate themselves from others, like the typical antipsychotic haloperidol, antagonizing these 5-HT(2A) receptor-mediated functions in a pathway-specific manner, explained here by a new model of multiple active interconvertible conformations at dimeric receptors.