Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its gamma subunit and transducin

The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (T alpha-GTP) is a key step in visual excitation. The finding that trypsin activates PDE (alpha beta gamma) by degrading its gamma subunit and the reversal of this activation by gamma led to the proposal that T alpha-GTP activates PDE by relieving an inhibitory constraint imposed by gamma (Hurley and Stryer: J. Biol. Chem. 257:11094-11099, 1982). We report here studies showing that the addition of gamma subunit also reverses the activation of PDE by T alpha-GTP-gamma S. A procedure for preparing gamma in high yield (50-80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitory activity resides in the gamma subunit. Nanomolar gamma blocks the activation of PDE by micromolar T alpha-GTP gamma S. The degree of activation of PDE depends reciprocally on the concentrations of gamma and T alpha-GTP gamma S. gamma remains bound to the disk membrane during the activation of PDE by transducin. The binding of gamma to the alpha beta subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of T alpha-GTP.     

Assembly and function of the Photosystem II manganese stabilizing protein: lessons from its natively unfolded behavior

The Photosystem II (PS II) manganese stabilizing protein (MSP) possesses characteristics, including thermostability, ascribed to the natively unfolded class of proteins (Lydakis-Simantiris et al. (1999) Biochemistry 38: 404–414). A site-directed mutant of MSP, C28A, C51A, which lacks the -S–S- bridge, also binds to PS II at wild-type levels and reconstitutes oxygen evolution activity [Betts et al. (1996) Biochim Biophys Acta 1274: 135–142], although the mutant protein is even more disordered in solution. Both WT and C28A, C51A MSP aggregate upon heating, but an examination of the effects of protein concentration and pH on heat-induced aggregation showed that each MSP species exhibited greater resistance to aggregation at a pH near their pI (5.2) than do either bovine serum albumin (BSA) or carbonic anhydrase, which were used as model water soluble proteins. Increases in pH above the pI of the MSPs and BSA enhanced their aggregation resistance, a behavior which can be predicted from their charge (MSP) or a combination of charge and stabilization by -S–S- bonds (BSA). In the case of aggregation resistance by MSP, this is likely to be an important factor in its ability to avoid unproductive self-association reactions in favor of formation of the protein–protein interactions that lead to formation of the functional oxygen evolving complex.     

MARCKS is a natively unfolded protein with an inaccessible actin-binding site: evidence for long-range intramolecular interactions

Myristoylated alanine-rich C kinase substrate (MARCKS) is an unfolded protein that contains well characterized actin-binding sites within the phosphorylation site domain (PSD), yet paradoxically, we now find that intact MARCKS does not bind to actin. Intact MARCKS also does not bind as well to calmodulin as does the PSD alone. Myristoylation at the N terminus alters how calmodulin binds to MARCKS, implying that, despite its unfolded state, the distant N terminus influences binding events at the PSD. We show that the free PSD binds with site specificity to MARCKS, suggesting that long-range intramolecular interactions within MARCKS are also possible. Because of the unusual primary sequence of MARCKS with an overall isoelectric point of 4.2 yet a very basic PSD (overall charge of +13), we speculated that ionic interactions between oppositely charged domains of MARCKS were responsible for long-range interactions within MARCKS that sterically influence binding events at the PSD and that explain the observed differences between properties of the PSD and MARCKS. Consistent with this hypothesis, chemical modifications of MARCKS that neutralize negatively charged residues outside of the PSD allow the PSD to bind to actin and increase the affinity of MARCKS for calmodulin. Similarly, both myristoylation of MARCKS and cleavage of MARCKS by calpain are shown to increase the availability of the PSD so as to activate its actin-binding activity. Because abundant evidence supports the conclusion that MARCKS is an important protein in regulating actin dynamics, our data imply that post-translational modifications of MARCKS are necessary and sufficient to regulate actin-binding activity.     

The inactivating factor of glutamine synthetase,IF7, is a "natively unfolded" protein

Glutamine synthetase (GS) is the key enzyme responsible for the primary assimilation of ammonium in all living organisms, and it catalyses the synthesis of glutamine from glutamic acid, ATP, and ammonium. One of the recently discovered mechanisms of GS regulation involves protein-protein interactions with a small 65-residue-long protein named IF7. Here, we study the structure and stability of IF7 and its binding properties to GS, by using several biophysical techniques (fluorescence, circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopies, and gel filtration chromatography) which provide complementary structural information. The findings show that IF7 has a small amount of residual secondary structure, but lacks a well defined tertiary structure, and is not compact. Thus, all of the studies indicate that IF7 is a "natively unfolded" protein. The binding of IF7 to GS, its natural binding partner, occurs with an apparent dissociation constant of K(D) = 0.3 +/- 0.1 microM, as measured by fluorescence. We discuss the implications for the GS regulation mechanisms of IF7 being unfolded.     

Evidences of a natively unfolded state for the human topoisomerase IB N-terminal domain

The N-terminal domain of human topoisomerase IB has been expressed, purified and characterized by spectroscopic techniques. CD spectra as a function of concentration and pH indicate that the domain does not possess any defined secondary structure. The protein is probably in a natively unfolded state since its denaturation curve is indicative of a non-cooperative transition. Evidence of a partially folded structure comes from the fluorescence spectrum of ANS, whose intensity increases in presence of the domain. Indication of a partial structural arrangement of the domain comes also from the endogenous fluorescence of tryptophans that is centred at 350 nm in the native and shifts to 354 nm in the fully denaturated protein. Interestingly despite the poor structural degree, as also confirmed by a predictive approach, the domain efficiently binds DNA, suggesting that the absence of a defined 3D structure has a functional meaning that permits the domain to be available for the interaction with different molecular partners.     

Close packing of Listeria monocytogenes ActA, a natively unfolded protein, enhances F-actin assembly without dimerization

Studies of the biochemistry of Listeria monocytogenes virulence protein ActA have typically focused on the behavior of bacteria in complex systems or on the characterization of the protein after expression and purification. Although prior in vivo work has proposed that ActA forms dimers on the surface of L. monocytogenes, dimerization has not been demonstrated in vitro, and little consideration has been given to the surface environment where ActA performs its pivotal role in bacterial actin-based motility. We have synthesized and characterized an ActA dimer and provide evidence that the two ActA molecules do not interact with each other even when tethered together. However, we also demonstrate that artificial dimers provide superior activation of actin nucleation by the Arp2/3 complex compared with monomers and that increased activation of the Arp2/3 complex by dimers may be a general property of Arp2/3 activators. It appears that the close packing ( approximately 19 nm) of ActA molecules on the surface of L. monocytogenes is so dense that the kinetics of actin nucleation mimic that of synthetic ActA dimers. We also present observations indicating that ActA is a natively unfolded protein, largely random coil that is responsible for many of the unique physical properties of ActA including its extended structure, aberrant mobility during SDS-PAGE, and ability to resist irreversible denaturation upon heating.     

The intracellular domain of the Drosophila cholinesterase-like neural adhesion protein, gliotactin, is natively unfolded

Drosophila gliotactin (Gli) is a 109-kDa transmembrane, cholinesterase-like adhesion molecule (CLAM), expressed in peripheral glia, that is crucial for formation of the blood-nerve barrier. The intracellular portion (Gli-cyt) was cloned and expressed in the cytosolic fraction of Escherichia coli BLR(DE3) at 45 mg/L and purified by Ni-NTA (nitrilotriacetic acid) chromatography. Although migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), under denaturing conditions, was unusually slow, molecular weight determination by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) confirmed that the product was consistent with its theoretical size. Gel filtration chromatography yielded an anomalously large Stokes radius, suggesting a fully unfolded conformation. Circular dichroism (CD) spectroscopy demonstrated that Gli-cyt was >50% unfolded, further suggesting a nonglobular conformation. Finally, 1D-(1)H NMR conclusively demonstrated that Gli-cyt possesses an extended unfolded structure. In addition, Gli-cyt was shown to possess charge and hydrophobic properties characteristic of natively unfolded proteins (i.e., proteins that, when purified, are intrinsically disordered under physiologic conditions in vitro).     

G protein-effector coupling: interactions of recombinant inhibitory gamma subunit with transducin and phosphodiesterase

A bacterial expression vector for the inhibitory gamma subunit of retinal rod phosphodiesterase has been constructed by inserting a mouse gamma cDNA into pUC19. Escherichia coli 222 transformed with this plasmid produces a 12-kDa recombinant protein consisting of 18 additional amino acids attached to the amino terminus of gamma. The fusion protein, designated beta-gal-gamma, has been refolded into an active form in formic acid and partially purified by gel filtration chromatography. Despite a large extended sequence at the amino terminus, beta-gal-gamma is able to inhibit the activity of trypsin-activated phosphodiesterase, bind tightly to the catalytic alpha beta subunits, and interact with the alpha subunit of transducin in a nucleotide-dependent manner. The availability of large quantities of active bacterial gamma, together with the ability to change its primary structure by site-directed mutagenesis, promises to provide considerable new information on the interaction between transducin and phosphodiesterase, as well as insights into the molecular mechanism of G protein-effector coupling.     

Limited proteolysis of natively unfolded protein 4E‐BP1 in the presence of trifluoroethanol

Natively unfolded (intrinsically disordered (ID) proteins) have been attracting an increasing attention due to their involvement in many regulatory processes. Natively unfolded proteins can fold upon binding to their metabolic partners. Coupled folding and binding events usually involve only relatively short motifs (binding motifs). These binding motifs which are able to fold should have an increased propensity to form a secondary structure. The aim of the present work was to probe the conformation of the intrinsically disordered protein 4E‐BP1 in the native and partly folded states by limited proteolysis and to reveal regions with a high propensity to form an ordered structure. Trifuoroethanol (TFE) in low concentrations (up to 15 vol%) was applied to increase the helical population of protein regions with a high intrinsic propensity to fold. When forming helical structures, these regions lose mobility and become more protected from proteases than random/unfolded protein regions. Limited proteolysis followed by mass spectrometry analysis allows identification of the regions with decreased mobility in TFE solutions. Trypsin and V8 proteases were used to perform limited proteolysis of the 4E‐BP1 protein in buffer and in solutions with low TFE concentrations at 37°C and at elevated temperatures (42 and 50°C). Comparison of the results obtained with the previously established 4E‐BP1 structure and the binding motif illustrates the ability of limited proteolysis in the presence of a folding assistant (TFE) to map the regions with high and low propensities to form a secondary structure revealing potential binding motifs inside the intrinsically disordered protein. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 591–602, 2014.     

Regulation of AMP-activated protein kinase by a pseudosubstrate sequence on the gamma subunit

The AMP-activated protein kinase (AMPK) system monitors cellular energy status by sensing AMP and ATP, and is a key regulator of energy balance at the cellular and whole-body levels. AMPK exists as heterotrimeric alphabetagamma complexes, and the gamma subunits contain two tandem domains that bind the regulatory nucleotides. There is a sequence in the first of these domains that is conserved in gamma subunit homologues in all eukaryotes, and which resembles the sequence around sites phosphorylated on target proteins of AMPK, except that it has a non-phosphorylatable residue in place of serine. We propose that in the absence of AMP this pseudosubstrate sequence binds to the active site groove on the alpha subunit, preventing phosphorylation by the upstream kinase, LKB1, and access to downstream targets. Binding of AMP causes a conformational change that prevents this interaction and relieves the inhibition. We present several lines of evidence supporting this hypothesis.     

The YefM antitoxin defines a family of natively unfolded proteins: implications as a novel antibacterial target

Although natively unfolded proteins are being observed increasingly, their physiological role is not well understood. Here, we demonstrate that the Escherichia coli YefM protein is a natively unfolded antitoxin, lacking secondary structure even at low temperature or in the presence of a stabilizing agent. This conformation of the protein is suggested to have a key role in its physiological regulatory activity. Because of the unfolded state of the protein, a linear determinant rather than a conformational one is presumably being recognized by its toxin partner, YoeB. A peptide array technology allowed the identification and validation of such a determinant. This recognition element may provide a novel antibacterial target. Indeed, a pair-constrained bioinformatic analysis facilitated the definite determination of novel YefM-YoeB toxin-antitoxin systems in a large number of bacteria including major pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, and Mycobacterium tuberculosis. Taken together, the YefM protein defines a new family of natively unfolded proteins. The existence of a large and conserved group of proteins with a clear physiologically relevant unfolded state serves as a paradigm to understand the structural basis of this state.     

Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. II. Its role in the turnoff of phosphodiesterase in vivo

Retinal cGMP phosphodiesterase (PDE) is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In the accompanying paper (Matsuura, I., Bondarenko, V. A., Maeda, T., Kachi, S., Yamazaki, M., Usukura, J., Hayashi, F., and Yamazaki, A. (2000) J. Biol. Chem. 275, 32950-32957), we have shown that all known Pgammas contain a specific phosphorylation motif for cyclin-dependent protein kinase 5 (Cdk5) and that the unknown kinase is Cdk5 complexed with its activator. Here, using frog rod photoreceptor outer segments (ROS) isolated by a new method, we show that Cdk5 is involved in light-dependent Pgamma phosphorylation in vivo. Under dark conditions only negligible amounts of Pgamma were phosphorylated. However, under illumination that bleached less than 0.3% of the rhodopsin, approximately 4% of the total Pgamma was phosphorylated in less than 10 s. Pgamma dephosphorylation occurred in less than 1 s after the light was turned off. Analysis of the phosphorylated amino acid, inhibition of Pgamma phosphorylation by Cdk inhibitors in vivo and in vitro, and two-dimensional peptide map analysis of Pgamma phosphorylated in vivo and in vitro indicate that Cdk5 phosphorylates a Pgamma threonine in the same manner in vivo and in vitro. These observations, together with immunological data showing the presence of Cdk5 in ROS, suggest that Cdk5 is involved in light-dependent Pgamma phosphorylation in ROS and that the phosphorylation is significant and reversible. In an homogenate of frog ROS, PDE activated by light/guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) was inhibited by Pgamma alone, but not by Pgamma complexed with GDP/Talpha or GTPgammaS/Talpha. Under these conditions, Pgamma phosphorylated by Cdk5 inhibited the light/GTPgammaS-activated PDE even in the presence of GTPgammaS/Talpha. These observations suggest that phosphorylated Pgamma interacts with and inhibits light/GTPgammaS-activated PDE, but does not interact with GTPgammaS/Talpha in the homogenate. Together, our results strongly suggest that after activation of PDE by light/GTP, Pgamma is phosphorylated by Cdk5 and the phosphorylated Pgamma inhibits GTP/Talpha-activated PDE, even in the presence of GTP/Talpha in ROS.     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

Characterization of the cDNA and genomic sequence of a G protein gamma subunit (gamma 5).

A cDNA from human placenta and liver tissues that contained both sequence for the lysosomal glycosidase di-N-acetylchitobiase and sequence homologous to the gamma subunit of GTP-binding proteins was previously isolated. Here we have shown that the gamma-subunit-homologous portion of this unusual cDNA is derived from a member of the gamma-subunit multigene family. The partial human gamma-subunit sequence was used to isolate the corresponding full-length cDNA clones from bovine and rat livers. The two cDNAs encode identical 68-amino-acid proteins (7.3 kDa) homologous to previously cloned G protein gamma subunits. The bovine gene sequence encoding this new gamma-subunit isoform (gamma 5) was determined and found to have an intron-exon structure consistent with the original human chitobiase-gamma 5-subunit hybrid mRNA being a product of alternative splicing. Genomic cloning also resulted in the isolation of a human gamma 5 pseudogene.     

Characterization of the structure and function of W --> F WW domain variants: identification of a natively unfolded protein that folds upon ligand binding

The WW domain adopts a compact, three-stranded, antiparallel beta-sheet structure that mediates protein-protein interactions by binding to xPPxY-based protein ligands, such as the PY-ligand (EYPPYPPPPYPSG) derived from p53 binding protein-2. The conserved Trp residues, after which this domain was named, were replaced with Phe so their importance in structural integrity and for ligand binding could be evaluated. A biophysical approach was employed to compare the W17F, W39F, and W17F/W39F WW domains to the wild-type protein. The data demonstrate that replacement of Trp39 with Phe (W39F) does not disrupt the structure of the WW domain variant, but does abolish ligand binding. In contrast, the W17F WW domain variant is largely if not completely unfolded; however, this variant undergoes a PY-ligand induced disorder to order (folding) transition. The dissociation constant for the W17F WW domain-PY-ligand interaction is 15.1 +/- 1.2 microM, only slightly higher than that observed for the wild-type WW domain interaction (5.9 +/- 0.33 microM). The W17F WW domain is a natively unfolded protein which adopts a native conformation upon PY-ligand binding.     

Intramolecular cohesion of coils mediated by phenylalanine--glycine motifs in the natively unfolded domain of a nucleoporin

The nuclear pore complex (NPC) provides the sole aqueous conduit for macromolecular exchange between the nucleus and the cytoplasm of cells. Its diffusion conduit contains a size-selective gate formed by a family of NPC proteins that feature large, natively unfolded domains with phenylalanine-glycine repeats (FG domains). These domains of nucleoporins play key roles in establishing the NPC permeability barrier, but little is known about their dynamic structure. Here we used molecular modeling and biophysical techniques to characterize the dynamic ensemble of structures of a representative FG domain from the yeast nucleoporin Nup116. The results showed that its FG motifs function as intramolecular cohesion elements that impart order to the FG domain and compact its ensemble of structures into native premolten globular configurations. At the NPC, the FG motifs of nucleoporins may exert this cohesive effect intermolecularly as well as intramolecularly to form a malleable yet cohesive quaternary structure composed of highly flexible polypeptide chains. Dynamic shifts in the equilibrium or competition between intra- and intermolecular FG motif interactions could facilitate the rapid and reversible structural transitions at the NPC conduit needed to accommodate passing karyopherin-cargo complexes of various shapes and sizes while simultaneously maintaining a size-selective gate against protein diffusion.     

The N-terminal half of the peroxisomal cycling receptor Pex5p is a natively unfolded domain

Targeting of most newly synthesised peroxisomal matrix proteins to the organelle requires Pex5p, the so-called PTS1 receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with these proteins in the cytosol, transports them to the peroxisomal membrane and catalyses their translocation across the membrane. Presently, our knowledge on the structural details behind the interaction of Pex5p with the cargo proteins is reasonably complete. In contrast, information regarding the structure of the Pex5p N-terminal half (a region containing its peroxisomal targeting domain) is still limited. We have recently observed that the Stokes radius of this Pex5p domain is anomalously large, suggesting that this portion of the protein is either a structured elongated domain or that it adopts a low compactness conformation. Here, we address this issue using a combination of biophysical and biochemical approaches. Our results indicate that the N-terminal half of Pex5p is best described as a natively unfolded pre-molten globule-like domain. The implications of these findings on the mechanism of protein import into the peroxisome are discussed.