Conformation and stability of alpha-helical membrane proteins. 1. Influence of salts on conformational equilibria between active and Inactive states of rhodopsin

We studied the influence of salts on the pH-dependent conformational equilibria between the active and the inactive photoproduct states of rhodopsin, Meta II and Meta I, respectively, and between the active and inactive conformations of the apoprotein opsin. In both equilibria, the active species is favored in the presence of medium to high concentration of salt. The ion selectivity for the Meta I/Meta II equilibrium is particularly pronounced for the anions and follows the series trichloroacetate > thiocyanate > iodide > bromide > sulfate > chloride > acetate. The Hill coefficient of this salt-induced transition is close to 2.0. Both ion selectivity and Hill coefficient suggest that the transition is mainly regulated by ion binding to two specific charged binding sites in the protein with smaller contributions being due to the Hofmeister effect. We propose that these putative ion binding sites are identical to those sites that are titrated in the corresponding pH-dependent conformational transition. They presumably function as ionic locks, which keep the receptor in an inactive conformation, and which may be disrupted either by pH-dependent protonation or by salt-dependent ion binding.     

CheW binding interactions with CheA and Tar. Importance for chemotaxis signaling in Escherichia coli

The initial signaling events underlying the chemotactic response of Escherichia coli to aspartic acid occur within a ternary complex that includes Tar (an aspartate receptor), CheA (a protein kinase), and CheW. Because CheW can bind to CheA and to Tar, it is thought to serve as an adapter protein in this complex. The functional importance of CheW binding interactions, however, has not been investigated. To better define the role of CheW and its binding interactions, we performed biochemical characterization of six mutant variants of CheW. We examined the ability of the purified mutant CheW proteins to bind to CheA and Tar, to promote formation of active ternary complexes, and to support chemotaxis in vivo. Our results indicate that mutations which eliminate CheW binding to Tar (V36M) or to CheA (G57D) result in a complete inability to form active ternary complexes in vitro and render the CheW protein incapable of mediating chemotaxis in vivo. The in vivo signaling pathway can, however, tolerate moderate changes in CheW-Tar and CheW-CheA affinities observed with several of the mutants (G133E, G41D, and 154ocr). One mutant (R62H) provided surprising results that may indicate a role for CheW in addition to binding CheA/receptors and promoting ternary complex formation.     

Altering a histone H3K4 methylation pathway in glomerular podocytes promotes a chronic disease phenotype

Methylation of specific lysine residues in core histone proteins is essential for embryonic development and can impart active and inactive epigenetic marks on chromatin domains. The ubiquitous nuclear protein PTIP is encoded by the Paxip1 gene and is an essential component of a histone H3 lysine 4 (H3K4) methyltransferase complex conserved in metazoans. In order to determine if PTIP and its associated complexes are necessary for maintaining stable gene expression patterns in a terminally differentiated, non-dividing cell, we conditionally deleted PTIP in glomerular podocytes in mice. Renal development and function were not impaired in young mice. However, older animals progressively exhibited proteinuria and podocyte ultra structural defects similar to chronic glomerular disease. Loss of PTIP resulted in subtle changes in gene expression patterns prior to the onset of a renal disease phenotype. Chromatin immunoprecipitation showed a loss of PTIP binding and lower H3K4 methylation at the Ntrk3 (neurotrophic tyrosine kinase receptor, type 3) locus, whose expression was significantly reduced and whose function may be essential for podocyte foot process patterning. These data demonstrate that alterations or mutations in an epigenetic regulatory pathway can alter the phenotypes of differentiated cells and lead to a chronic disease state.     

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRP-receptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo. Using both biochemical and genetic experiments, we show here that SRP RNA enhances GTPase activity of the SRP-receptor complex above a critical threshold required for cell viability. Furthermore, this stimulation is a property of the SRP RNA tetraloop. SRP RNA tetraloop mutants that confer defective growth phenotypes can assemble into SRP-receptor complexes, but fail to stimulate GTP hydrolysis in these complexes in vitro. Tethered hydroxyl radical probing data reveal that specific positioning of the RNA tetraloop within the SRP-receptor complex is required to stimulate GTPase activity to a level sufficient to support cell growth. These results explain why no external GAP is needed and why the phylogenetically conserved SRP RNA tetraloop is required in vivo.     

Identification of determinants involved in binding of tissue-type plasminogen activator-plasminogen activator inhibitor type 1 complexes to HepG2 cells

Complexes between tissue-type plasminogen activator (t-PA) and its rapidly acting inhibitor plasminogen activator inhibitor type 1 (PAI-1) are bound, internalized, and degraded by HepG2 cells. The mechanism involves endocytosis mediated by a specific high-affinity receptor. However, the particular domains of the complex that are recognized by the receptor have not been elucidated. To identify the determinants involved in ligand binding to the receptor, several variants of t-PA were assessed for their ability to form complexes with PAI-1 and thereby to inhibit specific cellular binding of complexes between structurally unmodified 125I-t-PA and PAI-1. Catalytically active variants lacking selected structural domains form complexes with PAI-1 and inhibit 125I-t-PA.PAI-1 binding to HepG2 cells. In addition, several forms of the plasminogen activator urokinase (u-PA), which shares partial structural homology with t-PA, were evaluated as competitors of cellular binding. The catalytically active two-chain forms of u-PA, but not the inactive proenzyme single-chain form, complex with PAI-1 and inhibit specific binding of 125I-t-PA.PAI-1, suggesting that the serine protease domain, rather than other domains, may confer the determinants required for cellular binding. However, a mutant t-PA with markedly reduced catalytic activity, resulting from replacement of the active site serine with threonine, not only forms complexes with PAI-1 but also inhibits specific cellular binding of unmodified 125I-t-PA.PAI-1. These data indicate that specific binding of t-PA.PAI-1 to HepG2 cells does not require a serine-containing catalytic site in the protease domain. To determine whether binding of the complex is mediated through other components of t-PA or through structural elements of PAI-1, both t-PA and PAI-1 were examined separately for capacity to bind directly to HepG2 cells. To exclude potential interactions with components of the extracellular matrix which contains binding sites for PAI-1, ligand binding to HepG2 cells in suspension was assessed. Although neither t-PA nor PAI-1 alone binds specifically to HepG2 cells, the preformed t-PA.PAI-1 complexes do. These findings suggest that specific binding of t-PA.PAI-1 requires elements of the PAI-1 moiety and/or parts of the protease domain of t-PA.     

Probing the solution structure of tumor necrosis factor-α homotrimer and heterotrimer after complex perturbation using electrospray ionization mass spectrometry

There are a number of proteins whose active forms are non-covalent multichain complexes. Therapeutic intervention involving such complexes has been proposed through the use of muteins to form heterostructures. These resulting structures would either not be recognized by receptors or would be inactive competitive inhibitors to wild-type (wt) proteins. We have used tumor necrosis factor-α (TNF-α) to establish that it is possible to use mass spectrometry to monitor the non-covalent solution structure of therapeutically relevant proteins and correlate the results with binding data. Mass spectrometry is shown to be able to directly monitor the state of the solution complexes to within 5 Da errors mass accuracy of theoretical mass at 50 kDa, as well as to resolve homocomplex from heterocomplex. Furthermore, it was determined that perturbation of the TNF-α complex, at or below pH 4.0, results in monomers that cannot reform into the multimeric complex, and the resulting protein solution can no longer bind to an anti-TNF-α antibody. Dissociation and re-association of the trimer was possible with the use of dimethyl sulfoxide at pH 5.5 and allowed for the resulting detection of both homotrimer and heterotrimer in solution with no impact on antibody binding. This work demonstrates that mass spectrometric techniques offer a means to monitor native solution interactions of non-covalent complexes and to differentiate multiple complexes from each other in solution. This method has applicability in the biopharmaceutical arena for monitoring engineering non-covalent drug complexes for the purpose of altering biological activity.     

RNase A effects on sedimentation and DNA binding properties of dexamethasone-receptor complexes from HeLa cell cytosol

Dexamethasone-receptor complexes from HeLa cell cytosol sediment at 7.4S in low salt sucrose gradients, and at 3.8S in high salt gradients. If cytosol is heated at 25 degrees C, receptor complexes sediment at 6.9S in low salt, and at 3.6S in high salt gradients. RNase A treatment at 25 degrees C, instead, results in receptor complexes which sediment in low salt gradients as two major forms at 6.5 and 4.8S. Receptor complexes from RNase A-treated cytosols sediment as their counterparts from untreated cytosols in high salt gradients. Although the shift in sedimentation properties of receptor complexes at 2 degrees C is induced by RNase A, and not by other low molecular weight basic proteins or RNase T1, the effect can be also obtained by inactive RNase A. The catalytically active enzyme, however, is required to observe 6.5 and 4.8S complexes after cytosol incubations at 25 degrees C. Placental ribonuclease inhibitor prevents the appearance of RNase A-induced receptor forms at 25 degrees C, but not at 2 degrees C. Moreover, this inhibitor can prevent the 7.4 to 6.9S shift in sedimentation coefficient of receptor complexes caused by cytosol heating. Dexamethasone-receptor complexes from HeLa cell cytosol show low levels of binding to DNA-cellulose, and heating at 25 degrees C is required to observe a six-fold increase in DNA binding levels. RNase A treatment of cytosols at 2 degrees C does not result in significant enhancement in receptor complex binding to DNA. If RNase A treatment is carried out at 25 degrees C, however, DNA binding levels of receptor complexes increased by 25% over the values observed with control heated cytosol. This effect cannot be observed if RNase T1 substitutes for RNase A. Placental ribonuclease inhibitor can prevent the temperature-dependent increase in DNA binding properties of dexamethasone-receptor complexes either in the presence or absence of exogenous RNase A. These findings indicate that exogenous RNases can perturb the structure of dexamethasone-receptor complexes without being involved in the transformation process.     

Toward the active conformations of rhodopsin and the beta2-adrenergic receptor

Using sets of experimental distance restraints, which characterize active or inactive receptor conformations, and the X-ray crystal structure of the inactive form of bovine rhodopsin as a starting point, we have constructed models of both the active and inactive forms of rhodopsin and the beta2-adrenergic G-protein coupled receptors (GPCRs). The distance restraints were obtained from published data for site-directed crosslinking, engineered zinc binding, site-directed spin-labeling, IR spectroscopy, and cysteine accessibility studies conducted on class A GPCRs. Molecular dynamics simulations in the presence of either "active" or "inactive" restraints were used to generate two distinguishable receptor models. The process for generating the inactive and active models was validated by the hit rates, yields, and enrichment factors determined for the selection of antagonists in the inactive model and for the selection of agonists in the active model from a set of nonadrenergic GPCR drug-like ligands in a virtual screen using ligand docking software. The simulation results provide new insights into the relationships observed between selected biochemical data, the crystal structure of rhodopsin, and the structural rearrangements that occur during activation.     

A Monte Carlo study of the dynamics of G-protein activation.

To link quantitatively the cell surface binding of ligand to receptor with the production of cellular responses, it may be necessary to explore early events in signal transduction such as G-protein activation. Two different model frameworks relating receptor/ligand binding to G-protein activation are examined. In the first framework, a simple ordinary differential equation model is used to describe receptor/ligand binding and G-protein activation. In the second framework, the events leading to G-protein activation are simulated using a dynamic Monte Carlo model. In both models, reactions between ligand-bound receptors and G-proteins are assumed to be diffusion-limited. The Monte Carlo model predicts two regimes of G-protein activation, depending upon whether the lifetime of a receptor/ligand complex is long or short compared with the time needed for diffusional encounters of complexes and G-proteins. When the lifetime of a complex is relatively short compared with the diffusion time, the movement of ligand among free receptors by binding and unbinding ("switching") significantly enhances G-protein activation. Receptor antagonists dramatically reduce G-protein activation and, thus, signal transduction in this case, and significant clustering of active G-proteins near receptor/ligand complexes results. The simple ordinary differential equation model poorly predicts G-protein activation for this situation. In the alternative case, when diffusion is relatively fast, ligand movement among receptors is less important and the simple ordinary differential equation model and Monte Carlo model results are similar. In this case, there is little clustering of active G-proteins near receptor/ligand complexes. Results also indicate that as the GTPase activity of the alpha-subunit decreases, the steady-state level of alpha-GTP increases, although temporal sensitivity is compromised.     

Monocyte Chemoattractant Activity of Ser → Ala Active Site Mutant Recombinant α-Thrombin

α-Thrombin is chemotactic for human monocytes with optimal activity between 10-100 nM. The mechanism by which this response is mediated remains a point of controversy. The purpose of this study was to compare the chemotactic activity of proteolytically inactive thrombin (active site Ser¹⁹⁵ → Ala mutant or Phe-Pro-Arg-chloromethyl ketone-inactivated thrombin) to thrombin and the "tethered ligand" thrombin receptor agonist peptide SFLLRN (single-letter amino acid code). Monocyte chemotaxis was compared to an optimal concentration (10 nM, considered to be 100%) of formyl-Met-Leu-Phe (fMLP). Proteolytically inactive thrombin (38% of fMLP) had similar chemotactic activity to active thrombin (46% of fMLP) at a concentration of 100 nM. Chemotaxis to SFLLRN was comparable to that of a control hexapeptide (FSLNLR) which is not an agonist for the tethered ligand thrombin receptor. Cross-desensitization experiments showed that pretreatment of monocytes with either mutant or active thrombin reduced subsequent chemotaxis to both thrombin chemotaxins. Pretreatment with SFLLRN did not decrease subsequent chemotaxis to either form of thrombin. Calcium flux measurements showed that both active thrombin and SFLLRN induced a rapid increase in monocyte and platelet intracellular calcium concentration. However, there was no intracellular calcium change in response to mutant thrombin or FSLNLR. Likewise, active thrombin and SFLLRN induced a rapid net increase in polymerized actin, but mutant thrombin and FSLNLR did not. By contrast, both active and mutant thrombin induced a polarization of monotocyte morphology and actin distribution. This polarization has been associated with directed migration in many cell types. SFLLRN, however, induced a symmetrical increase in polymerized actin. These results suggest that measurements of intracellular calcium and polymerized actin are not perfect surrogate tests for true chemotactic activity. These results show that thrombin proteolysis is not required for monocyte chemotaxis and may be mediated by interaction with a binding site other than the tethered ligand thrombin receptor.     

Statistical analyses of protein sequence alignments identify structures and mechanisms in signal activation of sensor histidine kinases

Statistical analyses of genome sequence‐derived protein sequence data can identify amino acid residues that interact between proteins or between domains of a protein. These statistical methods are based on evolution‐directed amino acid variation responding to structural and functional constraints in proteins. The identified residues form a basis for determining structure and folding of proteins as well as inferring mechanisms of protein function. When applied to two‐component systems, several research groups have shown they can be used to identify the amino acid interactions between response regulators and histidine kinases and the specificity therein. Recently, statistical studies between the HisKA and HATPase‐ATP‐binding domains of histidine kinases identified amino acid interactions for both the inactive and the active catalytic states of such kinases. The identified interactions generated a model structure for the domain conformation of the active state. This conformation requires an unwinding of a portion of the C‐terminal helix of the HisKA domain that destroys the inactive state residue contacts and suggests how signal‐binding determines the equilibrium between the inactive and active states of histidine kinases. The rapidly accumulating protein sequence databases from genome, metagenome and microbiome studies are an important resource for functional and structural understanding of proteins and protein complexes in microbes.     

Stoichiometry for binding and transport by the twin arginine translocation system

Twin arginine translocation (Tat) systems transport large folded proteins across sealed membranes. Tat systems accomplish this feat with three membrane components organized in two complexes. In thylakoid membranes, cpTatC and Hcf106 comprise a large receptor complex containing an estimated eight cpTatC-Hcf106 pairs. Protein transport occurs when Tha4 joins the receptor complex as an oligomer of uncertain size that is thought to form the protein-conducting structure. Here, binding analyses with intact membranes or purified complexes indicate that each receptor complex could bind eight precursor proteins. Kinetic analysis of translocation showed that each precursor-bound site was independently functional for transport, and, with sufficient Tha4, all sites were concurrently active for transport. Tha4 titration determined that ～26 Tha4 protomers were required for transport of each OE17 (oxygen-evolving complex subunit of 17 kD) precursor protein. Our results suggest that, when fully saturated with precursor proteins and Tha4, the Tat translocase is an ～2.2-megadalton complex that can individually transport eight precursor proteins or cooperatively transport multimeric precursors.     

Prodomains of transforming growth factor beta (TGFbeta) superfamily members specify different functions: extracellular matrix interactions and growth factor bioavailability

The specific functions of the prodomains of TGFβ superfamily members are largely unknown. Interactions are known between prodomains of TGFβ-1-3 and latent TGFβ-binding proteins and between prodomains of BMP-2, -4, -7, and -10 and GDF-5 and fibrillins, raising the possibility that latent TGFβ-binding proteins and fibrillins may mediate interactions with all other prodomains of this superfamily. This possibility is tested in this study. Results show that the prodomain of BMP-5 interacts with the N-terminal regions of fibrillin-1 and -2 in a site similar to the binding sites for other bone morphogenetic proteins. However, in contrast, the prodomain of GDF-8 (myostatin) interacts with the glycosaminoglycan side chains of perlecan. The binding site for the GDF-8 prodomain is likely the heparan sulfate chain present on perlecan domain V. These results support and extend the emerging concept that TGFβ superfamily prodomains target their growth factor dimers to extracellular matrix macromolecules. In addition, biochemical studies of prodomain·growth factor complexes were performed to identify inactive complexes. For some members of the superfamily, the prodomain is noncovalently associated with its growth factor dimer in an inactive complex; for others, the prodomain·growth factor complex is active, even though the prodomain is noncovalently associated with its growth factor dimer. Results show that the BMP-10 prodomain, in contrast to BMP-4, -5, and -7 prodomains, can inhibit the bioactivity of the BMP-10 growth factor and suggest that the BMP-10 complex is like TGFβ and GDF-8 complexes, which can be activated by cleavage of the associated prodomain.     

Efficient and specific targeting of Polycomb group proteins requires cooperative interaction between Grainyhead and Pleiohomeotic

Specific targeting of the protein complexes formed by the Polycomb group of proteins is critically required to maintain the inactive state of a group of developmentally regulated genes. Although the role of DNA binding proteins in this process has been well established, it is still not understood how these proteins target the Polycomb complexes specifically to their response elements. Here we show that the grainyhead gene, which encodes a DNA binding protein, interacts with one such Polycomb response element of the bithorax complex. Grainyhead binds to this element in vitro. Moreover, grainyhead interacts genetically with pleiohomeotic in a transgene-based, pairing-dependent silencing assay. Grainyhead also interacts with Pleiohomeotic in vitro, which facilitates the binding of both proteins to their respective target DNAs. Such interactions between two DNA binding proteins could provide the basis for the cooperative assembly of a nucleoprotein complex formed in vitro. Based on these results and the available data, we propose that the role of DNA binding proteins in Polycomb group-dependent silencing could be described by a model very similar to that of an enhanceosome, wherein the unique arrangement of protein-protein interaction modules exposed by the cooperatively interacting DNA binding proteins provides targeting specificity.     

Allosteric activation of metabotropic glutamate receptor 5

Abstract

Metabotropic glutamate receptor 5 (mGluR5) is a class C G protein-coupled receptor (GPCR) with both an extracellular ligand binding site and an allosteric intrahelical chamber located similarly to the orthosteric ligand binding site of Class A GPCRs. Ligands binding to this ancestral site of mGluR5 can act as positive (PAM), negative (NAM) or silent (SAM) allosteric modulators, and their medicinal chemistry optimization is notoriously difficult, as subtle structural changes may cause significant variation in activity and switch in the functional response. Here we present all atom molecular dynamics simulations of NAM, SAM and PAM complexes formed by closely related ligands and analyse the structural differences of the complexes. Several residues involved in the activation are identified and the formation of a continuous water channel in the active complex but not in the inactive ones is recognized. Our results suggest that the mechanism of mGluR5 activation is similar to that of class A GPCRs.

Communicated by Ramaswamy H. Sarma     

Selective interactions between G protein subunits and RGS4 with the C-terminal domains of the mu- and delta-opioid receptors regulate opioid receptor signaling

To define receptor subdomains important for protein interaction and identify components of novel signal transduction complexes for the mu- and delta-opioid receptors (mu-OR, delta-OR), we generated glutathione S-transferase fusion proteins of the carboxyl-termini of the mu-opioid receptor (mu-CT), the delta- (delta-CT), and the third intracellular loop of the delta-opioid receptor (delta-i3L) to search for interactive proteins. Results from pull down experiments have demonstrated for the first time that Gbetagamma complexes, derived from the heterotrimeric Galphatbeta1gamma1, purified Gbeta1gamma1, or Gbeta endogenously present in cell lysates and rat striatal extracts, interact with all mu- and delta-opioid receptor subdomains. On the other hand, the C-terminal peptides of the delta- and the mu-ORs exhibit differential profiles for Galpha subunit binding. Indeed, while mu-CT was unable to bind any form of Galpha, both the delta-CT and the delta-i3L displayed interactive regions for heterotrimeric Galphatbeta1gamma1, inactive Galpha(GDP) and active Galpha(GTPgammaS). Regulators of G protein signaling (RGS) proteins are another class of proteins that can modulate G protein signaling events. We demonstrate for the first time that RGS4 directly interacts with the mu-CT, the delta-CT as well as delta-i3L in a dose dependent manner. Moreover, RGS4 modulates mu-OR signaling and can form stable heterotrimeric complexes with the activated Galpha. Collectively, our data demonstrate that the C-termini of the mu- and delta-ORs provide direct physical scaffolding in which G protein subunits and RGS4 protein can be bound.     

Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action.

ATP-dependent DNA ligases are essential enzymes in both DNA replication and DNA repair processes. Here we report a functional characterization of the T4 DNA ligase. One N-terminal and two C-terminal deletion mutants were expressed in Escherichia coli as histidine- tagged proteins. An additional mutant bore a substitution of Lys159 in the active site that abolished ATP binding. All the proteins were tested in biochemical assays for ATP-dependent self-adenylation, DNA binding, nick joining, blunt-end ligation and AMP- dependent DNA relaxation. From this analysis we conclude that binding to DNA is mediated by sequences at both protein ends and plays a key role in the reaction. The enzyme establishes two different complexes with DNA: (i) a transient complex (T.complex) involving the adenylated enzyme; (ii) a stable complex (S.complex) requiring the deadenylated T4 DNA ligase. The formation of an S. complex seems to be relevant during both blunt-end ligation and DNA relaxation. Moreover the inactive His-K159L substitution mutant, although unable to self-adenylate, still possesses AMP-dependent DNA nicking activity.