Ternary Protein Complex Research Articles

Current Status of A 1 Adenosine Receptor Allosteric Enhancers

Adenosine is an ubiquitous nucleoside involved in various physiological and pathological functions by stimulating A1, A2A, A2B and A3 adenosine receptors (ARs). Allosteric enhancers to A1ARs may represent novel therapeutic agents because they increase the activity of these receptors by mediating a shift to their active form in the A1AR-G protein ternary complex. In this manner, they are able to amplify the action of endogenous adenosine, which is produced in high concentrations under conditions of metabolic stress. A1AR allosteric enhancers could be used as a justifiable alternative to the exogenous agonists that are characterized by receptor desensitization and downregulation. In this review, an analysis of some of the most interesting allosteric modulators of A1ARs has been reported.

Visualization and translocation of ternary Calcineurin‐A/Calcineurin‐B/Calmodulin‐2 protein complexes by dual‐color trimolecular fluorescence complementation

Fluorescence complementation (FC) techniques are expedient for analyzing bimolecular protein-protein interactions. Here we aimed to develop a method for visualization of ternary protein complexes using dual-color trimolecular fluorescence complementation (TriFC). Dual-color TriFC combines protein fragments of mCherry and mVenus, in which a scaffold protein is bilaterally fused to C-terminal fragments of both fluorescent proteins and combined with potential interacting proteins fused to an N-terminal fluorescent protein fragment. For efficient visual verification of ternary complex formation, TriFC was combined with a cytoplasm to plasma membrane translocation assay. Modular vector sets were designed which are fully compatible with previously reported bimolecular fluorescence complementation (BiFC) vectors. As a proof-of-principle, the ternary complex formation of the PP2B protein phosphatase Calcineurin-A/Calcineurin-B with Calmodulin-2 was investigated in transiently transformed Nicotiana benthamiana leaf epidermal cells. The results indicate a Calcineurin-B-induced interaction of Calmodulin-2 with Calcineurin-A. TriFC and the translocation of TriFC complexes provide a novel tool to investigate ternary complex formations with the simplicity of a BiFC approach. The robustness of FC applications and the opportunity to quantify fluorescence complementation render this assay suitable for a broad range of interaction analyses.

An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism.

The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary “hitchhiker” mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.

Structural and biophysical methods to analyze clock function and mechanism.

Structural approaches have provided insight into the mechanisms of circadian clock oscillators. This review focuses upon the myriad structural methods that have been applied to the molecular architecture of cyanobacterial circadian proteins, their interactions with each other, and the mechanism of the KaiABC posttranslational oscillator. X-ray crystallography and solution NMR were deployed to gain an understanding of the three-dimensional structures of the three proteins KaiA, KaiB, and KaiC that make up the inner timer in cyanobacteria. A hybrid structural biology approach including crystallography, electron microscopy, and solution scattering has shed light on the shapes of binary and ternary Kai protein complexes. Structural studies of the cyanobacterial oscillator demonstrate both the strengths and the limitations of the divide-and-conquer strategy. Thus, investigations of complexes involving domains and/or peptides have afforded valuable information into Kai protein interactions. However, high-resolution structural data are still needed at the level of complexes between the 360-kDa KaiC hexamer that forms the heart of the clock and its KaiA and KaiB partners.

Choline dehydrogenase interacts with SQSTM1/p62 to recruit LC3 and stimulate mitophagy

CHDH (choline dehydrogenase) is an enzyme catalyzing the dehydrogenation of choline to betaine aldehyde in mitochondria. Apart from this well-known activity, we report here a pivotal role of CHDH in mitophagy. Knockdown of CHDH expression impairs CCCP-induced mitophagy and PARK2/parkin-mediated clearance of mitochondria in mammalian cells, including HeLa cells and SN4741 dopaminergic neuronal cells. Conversely, overexpression of CHDH accelerates PARK2-mediated mitophagy. CHDH is found on both the outer and inner membranes of mitochondria in resting cells. Interestingly, upon induction of mitophagy, CHDH accumulates on the outer membrane in a mitochondrial potential-dependent manner. We found that CHDH is not a substrate of PARK2 but interacts with SQSTM1 independently of PARK2 to recruit SQSTM1 into depolarized mitochondria. The FB1 domain of CHDH is exposed to the cytosol and is required for the interaction with SQSTM1, and overexpression of the FB1 domain only in cytosol reduces CCCP-induced mitochondrial degradation via competitive interaction with SQSTM1. In addition, CHDH, but not the CHDH FB1 deletion mutant, forms a ternary protein complex with SQSTM1 and MAP1LC3 (LC3), leading to loading of LC3 onto the damaged mitochondria via SQSTM1. Further, CHDH is crucial to the mitophagy induced by MPP+ in SN4741 cells. Overall, our results suggest that CHDH is required for PARK2-mediated mitophagy for the recruitment of SQSTM1 and LC3 onto the mitochondria for cargo recognition.

Interaction between Na-K-ATPase and Bcl-2 proteins BclXL and Bak.

In silico analysis predicts interaction between Na-K-ATPase (NKA) and Bcl-2 protein canonical BH3- and BH1-like motifs, consistent with NKA inhibition by the benzo-phenanthridine alkaloid chelerythrine, a BH3 mimetic, in fetal human lens epithelial cells (FHLCs) (Lauf PK, Heiny J, Meller J, Lepera MA, Koikov L, Alter GM, Brown TL, Adragna NC. Cell Physiol Biochem 31: 257-276, 2013). This report establishes proof of concept: coimmunoprecipitation and immunocolocalization showed unequivocal and direct physical interaction between NKA and Bcl-2 proteins. Specifically, NKA antibodies (ABs) coimmunoprecipitated BclXL (B-cell lymphoma extra large) and BAK (Bcl-2 antagonist killer) proteins in FHLCs and A549 lung cancer cells. In contrast, both anti-Bcl-2 ABs failed to pull down NKA. Notably, the molecular mass of BAK1 proteins pulled down by NKA and BclXL ABs appeared to be some 4-kDa larger than found in input monomers. In silico analysis predicts these higher molecular mass BAK1 proteins as alternative splicing variants, encoding 42 amino acid (aa) larger proteins than the known 211-aa long canonical BAK1 protein. These BAK1 variants may constitute a pool separate from that forming mitochondrial pores by specifically interacting with NKA and BclXL proteins. We propose a NKA-Bcl-2 protein ternary complex supporting our hypothesis for a special sensor role of NKA in Bcl-2 protein control of cell survival and apoptosis.

The powdery mildew resistance protein RPW8.2 is carried on VAMP721/722 vesicles to the extrahaustorial membrane of haustorial complexes.

Plants employ multiple cell-autonomous defense mechanisms to impede pathogenesis of microbial intruders. Previously we identified an exocytosis defense mechanism in Arabidopsis against pathogenic powdery mildew fungi. This pre-invasive defense mechanism depends on the formation of ternary protein complexes consisting of the plasma membrane-localized PEN1 syntaxin, the adaptor protein SNAP33 and closely sequence-related vesicle-resident VAMP721 or VAMP722 proteins. The Arabidopsis thaliana resistance to powdery mildew 8.2 protein (RPW8.2) confers disease resistance against powdery mildews upon fungal entry into host cells and is specifically targeted to the extrahaustorial membrane (EHM), which envelops the haustorial complex of the fungus. However, the secretory machinery involved in trafficking RPW8.2 to the EHM is unknown. Here we report that RPW8.2 is transiently located on VAMP721/722 vesicles, and later incorporated into the EHM of mature haustoria. Resistance activity of RPW8.2 against the powdery mildew Golovinomyces orontii is greatly diminished in the absence of VAMP721 but only slightly so in the absence of VAMP722. Consistent with this result, trafficking of RPW8.2 to the EHM is delayed in the absence of VAMP721. These findings implicate VAMP721/722 vesicles as key components of the secretory machinery for carrying RPW8.2 to the plant-fungal interface. Quantitative fluorescence recovery after photobleaching suggests that vesicle-mediated trafficking of RPW8.2-yellow fluorescent protein (YFP) to the EHM occurs transiently during early haustorial development and that lateral diffusion of RPW8.2-YFP within the EHM exceeds vesicle-mediated replenishment of RPW8.2-YFP in mature haustoria. Our findings imply the engagement of VAMP721/722 in a bifurcated trafficking pathway for pre-invasive defense at the cell periphery and post-invasive defense at the EHM.

Motor Impairments, Striatal Degeneration, and Altered Dopamine-Glutamate Interplay in Mice Lacking PSD-95

Excessive activation of the N-methyl-d-aspartate (NMDA) receptor and the neurotransmitter dopamine (DA) mediate neurotoxicity and neurodegeneration under many neurological conditions, including Huntington's disease (HD), an autosomal dominant neurodegenerative disease characterized by the preferential loss of medium spiny projection neurons (MSNs) in the striatum. PSD-95 is a major scaffolding protein in the postsynaptic density (PSD) of dendritic spines, where a classical role for PSD-95 is to stabilize glutamate receptors at sites of synaptic transmission. Our recent studies indicate that PSD-95 also interacts with the D1 DA receptor localized in spines and negatively regulates spine D1 signaling. Moreover, PSD-95 forms ternary protein complexes with D1 and NMDA receptors, and plays a role in limiting the reciprocal potentiation between both receptors from being escalated. These studies suggest a neuroprotective role for PSD-95. Here we show that mice lacking PSD-95, resulting from genetic deletion of the GK domain of PSD-95 (PSD-95-ΔGK mice), sporadically develop progressive neurological impairments characterized by hypolocomotion, limb clasping, and loss of DARPP-32–positive MSNs. Electrophysiological experiments indicated that NMDA receptors in mutant MSNs were overactive, suggested by larger, NMDA receptor–mediated miniature excitatory postsynaptic currents (EPSCs) and higher ratios of NMDA- to AMPA-mediated corticostriatal synaptic transmission. In addition, NMDA receptor currents in mutant cortical neurons were more sensitive to potentiation by the D1 receptor agonist SKF81297. Finally, repeated administration of the psychostimulant cocaine at a dose regimen not producing overt toxicity-related phenotypes in normal mice reliably converted asymptomatic mutant mice to clasping symptomatic mice. These results support the hypothesis that deletion of PSD-95 in mutant mice produces concomitant overactivation of both D1 and NMDA receptors that makes neurons more susceptible to NMDA excitotoxicity, causing neuronal damage and neurological impairments. Understanding PSD-95–dependent neuroprotective mechanisms may help elucidate processes underlying neurodegeneration in HD and other neurological disorders.

Fluorescence Fluctuation Spectroscopy of Ternary Protein Interactions in Living Cells

Because many proteins contain multiple interaction sites, the formation of ternary or higher-order protein complexes are frequently encountered and play a critical role in many cellular processes. Fluorescence fluctuation spectroscopy (FFS) characterizes the brightness of fluorescently-labeled proteins, which provides a quantitative and noninvasive method to characterize protein interactions in living cells. Here we focus on extending two-photon brightness microscopy to the characterization of three interacting protein species, with each species carrying a differently colored tag. The fluorescence signal is split into three detection channels by optical filters. We discuss the choice of tags and filters that optimize the signal-to-noise ratio of resolving species, and the influence of spectral cross-talk on data analysis by time-integrated cumulants of the photon counts. While proof of principal experiments with dyes are useful, our main challenge remains the limited choice of fluorescence proteins that have simple photophysics and good color separation for cellular applications. We characterize different combinations of fluorescent proteins and evaluate their potential for three-color FFS studies of ternary protein interactions. This work has been supported by grants from the National Institutes of Health (GM64589) and the National Science Foundation (PHY 0346782).

ADAM12 redistributes and activates MMP-14, resulting in gelatin degradation, reduced apoptosis, and increased tumor growth

Matrix metalloproteinases (MMPs), in particular MMP-2, MMP-9 and MMP-14, play a key role in various aspects of cancer pathology. Likewise, ADAMs (a disintegrin and metalloproteinases), including ADAM12, are upregulated in malignant tumors and contribute to the pathology of cancers. Here, we show that there is a positive correlation between MMP-14 and ADAM12 expression in human breast cancer. We demonstrated that in 293-VnR and human breast cancer cells expressing ADAM12 at the cell surface, endogenous MMP-14 was recruited to the cell surface, resulting in its activation. Subsequent to this activation, gelatin degradation was stimulated and tumor cell apoptosis was decreased, with reduced expression of the pro-apoptotic proteins BCL2L11 and BIK. The effect on gelatin degradation was abrogated by inhibition of the MMP-14 activity and appeared to be dependent on cell surface αVβ3 integrin localization, but neither the catalytic activity of ADAM12 nor the cytoplasmic tail of ADAM12 were required. The significance of ADAM12-induced activation of MMP-14 was underscored by a reduction in MMP-14-mediated gelatin degradation and abolition of apoptosis-protective effects by specific monoclonal antibodies against ADAM12. Furthermore, orthotopic implantation of ADAM12-expressing MCF7 cells in nude mice produced tumors with increased levels of activated MMP-14 and confirmed that ADAM12 protects tumor cells against apoptosis, leading to increased tumor progression. In conclusion, our data suggest that a ternary protein complex composed of ADAM12, αVβ3 integrin and MMP-14 at the tumor cell surface regulates the function of MMP-14. This interaction might point to a novel concept for the development of MMP-14-targeting drugs in treating cancer.

Identification and characterization of MYB‐bHLH‐WD40 regulatory complexes controlling proanthocyanidin biosynthesis in strawberry (Fragaria × ananassa) fruits

Strawberry (Fragaria×ananassa) fruits contain high concentrations of flavonoids. In unripe strawberries, the flavonoids are mainly represented by proanthocyanidins (PAs), while in ripe fruits the red-coloured anthocyanins also accumulate. Most of the structural genes leading to PA biosynthesis in strawberry have been characterized, but no information is available on their transcriptional regulation. In Arabidopsis thaliana the expression of the PA biosynthetic genes is specifically induced by a ternary protein complex, composed of AtTT2 (AtMYB123), AtTT8 (AtbHLH042) and AtTTG1 (WD40-repeat protein). A strategy combining yeast-two-hybrid screening and agglomerative hierarchical clustering of transcriptomic and metabolomic data was undertaken to identify strawberry PA regulators. Among the candidate genes isolated, four were similar to AtTT2, AtTT8 and AtTTG1 (FaMYB9/FaMYB11, FabHLH3 and FaTTG1, respectively) and two encode putative negative regulators (FaMYB5 and FabHLH3∆). Interestingly, FaMYB9/FaMYB11, FabHLH3 and FaTTG1 were found to complement the tt2-1, tt8-3 and ttg1-1 transparent testa mutants, respectively. In addition, they interacted in yeast and activated the Arabidopsis BANYULS (anthocyanidin reductase) gene promoter when coexpressed in Physcomitrella patens protoplasts. Taken together, these results demonstrated that FaMYB9/FaMYB11, FabHLH3 and FaTTG1 are the respective functional homologues of AtTT2, AtTT8 and AtTTG1, providing new tools for modifying PA content and strawberry fruit quality.

Supramolecularly Oriented Immobilization of Proteins Using Cucurbit[8]uril

A supramolecular strategy is used for oriented positioning of proteins on surfaces. A viologen-based guest molecule is attached to the surface, while a naphthol guest moiety is chemoselectively ligated to a yellow fluorescent protein. Cucurbit[8]uril (CB[8]) is used to link the proteins onto surfaces through specific charge-transfer interactions between naphthol and viologen inside the CB cavity. The assembly process is characterized using fluorescence and atomic force microscopy, surface plasmon resonance, IR-reflective absorption, and X-ray photoelectron spectroscopy measurements. Two different immobilization routes are followed to form patterns of the protein ternary complexes on the surfaces. Each immobilization route consists of three steps: (i) attaching the viologen to the glass using microcontact chemistry, (ii) blocking, and (iii) either incubation or microcontact printing of CB[8] and naphthol guests. In both cases uniform and stable fluorescent patterns are fabricated with a high signal-to-noise ratio. Control experiments confirm that CB[8] serves as a selective linking unit to form stable and homogeneous ternary surface-bound complexes as envisioned. The attachment of the yellow fluorescent protein complexes is shown to be reversible and reusable for assembly as studied using fluorescence microscopy.

Crystal Structures of the Scaffolding Protein LGN Reveal the General Mechanism by Which GoLoco Binding Motifs Inhibit the Release of GDP from Gαi

GoLoco (GL) motif-containing proteins regulate G protein signaling by binding to Gα subunit and acting as guanine nucleotide dissociation inhibitors. GLs of LGN are also known to bind the GDP form of Gα(i/o) during asymmetric cell division. Here, we show that the C-terminal GL domain of LGN binds four molecules of Gα(i)·GDP. The crystal structures of Gα(i)·GDP in complex with LGN GL3 and GL4, respectively, reveal distinct GL/Gα(i) interaction features when compared with the only high resolution structure known with GL/Gα(i) interaction between RGS14 and Gα(i1.) Only a few residues C-terminal to the conserved GL sequence are required for LGN GLs to bind to Gα(i)·GDP. A highly conserved "double Arg finger" sequence (RΨ(D/E)(D/E)QR) is responsible for LGN GL to bind to GDP bound to Gα(i). Together with the sequence alignment, we suggest that the LGN GL/Gα(i) interaction represents a general binding mode between GL motifs and Gα(i). We also show that LGN GLs are potent guanine nucleotide dissociation inhibitors.

Calcyon Forms a Novel Ternary Complex with Dopamine D1 Receptor through PSD-95 Protein and Plays a Role in Dopamine Receptor Internalization

Calcyon, once known for interacting directly with the dopamine D(1) receptor (D(1)DR), is implicated in various neuropsychiatric disorders including schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder. Although its direct interaction with D(1)DR has been shown to be misinterpreted, it still plays important roles in D(1)DR signaling. Here, we found that calcyon interacts with the PSD-95 and subsequently forms a ternary complex with D(1)DR through PSD-95. Calcyon is phosphorylated on Ser-169 by the PKC activator phorbol 12-myristate 13-acetate or by the D(1)DR agonist SKF-81297, and its phosphorylation increases its association with PSD-95 and recruitment to the cell surface. Interestingly, the internalization of D(1)DR at the cell surface was enhanced by phorbol 12-myristate 13-acetate and SKF-81297 in the presence of calcyon, but not in the presence of its S169A phospho-deficient mutant, suggesting that the phosphorylation of calcyon and the internalization of the surface D(1)DR are tightly correlated. Our results suggest that calcyon regulates D(1)DR trafficking by forming a ternary complex with D(1)DR through PSD-95 and thus possibly linking glutamatergic and dopamine receptor signalings. This also raises the possibility that a novel ternary complex could represent a potential therapeutic target for the modulation of related neuropsychiatric disorders.

Structure of an Asymmetric Ternary Protein Complex Provides Insight for Membrane Interaction

Plasma membrane repair involves the coordinated effort of proteins and the inner phospholipid surface to mend the rupture and return the cell back to homeostasis. Here, we present the three-dimensional structure of a multiprotein complex that includes S100A10, annexin A2, and AHNAK, which along with dysferlin, functions in muscle and cardiac tissue repair. The 3.5Å resolution X-ray structure shows that a single region from the AHNAK Cterminus is recruited by an S100A10-annexin A2 heterotetramer, forming an asymmetric ternary complex. The AHNAK peptide adopts a coil conformation that arches across the heterotetramer contacting both annexin A2 and S100A10 protomers with tight affinity (∼30nM) and establishing a structural rationale whereby both S100A10 and annexin proteins are needed in AHNAK recruitment. The structure evokes a model whereby AHNAK is targeted to the membrane surface through sandwiching of the binding region between the S100A10/annexin A2 complex and the phospholipid membrane.

Scaffoldin Conformation and Dynamics Revealed by a Ternary Complex from the Clostridium thermocellum Cellulosome

Cellulosomes are multienzyme complexes responsible for efficient degradation of plant cell wall polysaccharides. The nonenzymatic scaffoldin subunit provides a platform for cellulolytic enzyme binding that enhances the overall activity of the bound enzymes. Understanding the unique quaternary structural elements responsible for the enzymatic synergy of the cellulosome is hindered by the large size and inherent flexibility of these multiprotein complexes. Herein, we have used x-ray crystallography and small angle x-ray scattering to structurally characterize a ternary protein complex from the Clostridium thermocellum cellulosome that comprises a C-terminal trimodular fragment of the CipA scaffoldin bound to the SdbA type II cohesin module and the type I dockerin module from the Cel9D glycoside hydrolase. This complex represents the largest fragment of the cellulosome solved by x-ray crystallography to date and reveals two rigid domains formed by the type I cohesin·dockerin complex and by the X module-type II cohesin·dockerin complex, which are separated by a 13-residue linker in an extended conformation. The type I dockerin modules of the four structural models found in the asymmetric unit are in an alternate orientation to that previously observed that provides further direct support for the dual mode of binding. Conserved intermolecular contacts between symmetry-related complexes were also observed and may play a role in higher order cellulosome structure. SAXS analysis of the ternary complex revealed that the 13-residue intermodular linker of the scaffoldin subunit is highly dynamic in solution. These studies provide fundamental insights into modular positioning, linker flexibility, and higher order organization of the cellulosome.

Augmentation of Kv4.2-encoded Currents by Accessory Dipeptidyl Peptidase 6 and 10 Subunits Reflects Selective Cell Surface Kv4.2 Protein Stabilization

Rapidly activating and inactivating somatodendritic voltage-gated K(+) (Kv) currents, I(A), play critical roles in the regulation of neuronal excitability. Considerable evidence suggests that native neuronal I(A) channels function in macromolecular protein complexes comprising pore-forming (α) subunits of the Kv4 subfamily together with cytosolic, K(+) channel interacting proteins (KChIPs) and transmembrane, dipeptidyl peptidase 6 and 10 (DPP6/10) accessory subunits, as well as other accessory and regulatory proteins. Several recent studies have demonstrated a critical role for the KChIP subunits in the generation of native Kv4.2-encoded channels and that Kv4.2-KChIP complex formation results in mutual (Kv4.2-KChIP) protein stabilization. The results of the experiments here, however, demonstrate that expression of DPP6 in the mouse cortex is unaffected by the targeted deletion of Kv4.2 and/or Kv4.3. Further experiments revealed that heterologously expressed DPP6 and DPP10 localize to the cell surface in the absence of Kv4.2, and that co-expression with Kv4.2 does not affect total or cell surface DPP6 or DPP10 protein levels. In the presence of DPP6 or DPP10, however, cell surface Kv4.2 protein expression is selectively increased. Further addition of KChIP3 in the presence of DPP10 markedly increases total and cell surface Kv4.2 protein levels, compared with cells expressing only Kv4.2 and DPP10. Taken together, the results presented here demonstrate that the expression and localization of the DPP accessory subunits are independent of Kv4 α subunits and further that the DPP6/10 and KChIP accessory subunits independently stabilize the surface expression of Kv4.2.

A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4(IETDV)(2) (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.

Dynamic Assembly of the Receptor-G-Protein Signaling Complex

The recent crystal structure1 of the β2 adrenergic receptor-Gs protein complex (β2AR-Gs) marks an important milestone in a two decades long race to decipher one of the most important and widely utilized signal-transduction mechanisms in biology. However, it is unclear how the relatively drastic and unexpected conformational changes evolve from the initial encounter complex. The number of compositional and conformational microstates that evolve along the reaction pathway from the initial encounter of agonist-activated receptor with the GDP-bound heterotrimeric G-protein to the nucleotide-free agonist-receptor-G protein ternary complex is potentially very large.2 Identification of these pathways is out of reach for crystallographic experiments, and we follow a complementary approach with large-scale molecular dynamics (MD) simulations of membrane-embedded, fully solvated metarhodopsin II alone and in complex with its cognate G-protein, transducin, in GDP-bound form. Here, we compare and contrast the relaxation of two different encounter complexes, one with an orientation similar to β2AR-Gs, and the other 120° rotated. Interestingly, the two different pre-aligned encounter complexes yield induced-fit complexes that share a signature set of conformational changes involving highly conserved residues in the three allosterically coupled domains, the agonist-binding, G-protein-binding, and nucleotide-binding sites. How are these canonical and non-canonical binding modes possible for such a high fidelity signal-transduction system? Is it possible that Nature utilizes multiple, energetically similar states to relay the information encoded in the relatively small free energy of agonist binding across about ten nanometers distance to the nucleotide-binding pocket? It is tempting to speculate about the role of these energetically near-equivalent microstates when discussing the dominating entropic contribution to the binding free energy of agonists at the β adrenergic receptors.1Rasmussen, et al. (2011) Nature 477, 549.2Huber and Sakmar (2011) Trends. Pharmacol. Sci. 32, 410.

Formation of Ternary Complex of Human Biliverdin Reductase-Protein Kinase Cδ-ERK2 Protein Is Essential for ERK2-mediated Activation of Elk1 Protein, Nuclear Factor-κB, and Inducible Nitric-oxidase Synthase (iNOS)

Growth factors, insulin, oxidative stress, and cytokines activate ERK1/2 by PKCδ and MEK1/2. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase and intracellular scaffold/bridge/anchor, is a nuclear transporter of MEK1/2-stimulated ERK1/2 (Lerner-Marmarosh, N., Miralem, T., Gibbs, P. E., and Maines, M. D. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 6870-6875). hBVR, PKCδ, and MEK1/2 overlap in their tissue expression profile and type of activators. Presently, we report on formation of an hBVR-PKCδ-ERK2 ternary complex that is essential for ERK2 signal transduction and activation of genes linked to cell proliferation and cancer. MEK1/2 and the protein phosphatase PP2A were also present in the complex. When cells were stimulated with insulin-like growth factor-1 (IGF-1), an increased interaction between hBVR and PKCδ was detected by FRET-fluorescence lifetime imaging microscopy. hBVR and ERK2 were phosphorylated by PKCδ; however, the PKC was not a substrate for either ERK2 or hBVR. IGF-1 and phorbol ester increased hBVR/PKCδ binding; hBVR was required for the activation of PKCδ and its interaction with ERK2. The C-terminal phenylalanine residues of PKCδ (Phe(660), Phe(663), and Phe(665)) were necessary for binding to ERK2 but not for hBVR binding. Formation of the hBVR-PKCδ-ERK2 complex required the hBVR docking site for ERK, FXFP (DEF, C-box) and D(δ)-box (ILXXLXL) motifs. The hBVR-based peptide KKRILHCLGLA inhibited PKC activation and PKCδ/ERK2 interaction. Phorbol ester- and TNF-α-dependent activation of the ERK-regulated transcription factors Elk1 and NF-κB and expression of the iNOS gene were suppressed by hBVR siRNA; those activities were rescued by hBVR. The findings reveal the direct input of hBVR in PKCδ/ERK signaling and identify hBVR-based peptide regulators of ERK-mediated gene activation.