Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation

The proteasome is responsible for degradation of substrates of the ubiquitin pathway. 20S proteasomes are cylindrical particles with subunits arranged in a stack of four heptameric rings. The outer rings are composed of α subunits, and the inner rings are composed of β subunits. A well-characterized archaeal proteasome has a single type of each subunit, and the N-terminal threonine of the β subunit is the active-site nucleophile. Yeast proteasomes have seven different β subunits and exhibit several distinct peptidase activities, which were proposed to derive from disparate active sites. We show that mutating the N-terminal threonine in the yeast Pup1 β subunit eliminates cleavage after basic residues in peptide substrates, and mutating the corresponding threonine of Pre3 prevents cleavage after acidic residues. Surprisingly, neither mutation has a strong effect on cell growth, and they have at most minor effects on ubiquitin-dependent proteolysis. We show that Pup1 interacts with Pup3 in each β subunit ring. Our data reveal that different proteasome active sites contribute very differently to protein breakdown in vivo, that contacts between particular subunits in each β subunit ring are critical for active-site formation, and that active sites in archaea and different eukaryotes are highly similar.

Vascular endothelial growth factor: Crystal structure and functional mapping of the kinase domain receptor binding site

Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine knot family of growth factors, with limited sequence homology to platelet-derived growth factor (PDGF) and transforming growth factor β2 (TGF-β). We have determined its crystal structure at a resolution of 2.5 Å, and identified its kinase domain receptor (KDR) binding site using mutational analysis. Overall, the VEGF monomer resembles that of PDGF, but its N-terminal segment is helical rather than extended. The dimerization mode of VEGF is similar to that of PDGF and very different from that of TGF-β. Mutational analysis of VEGF reveals that symmetrical binding sites for KDR are located at each pole of the VEGF homodimer. Each site contains two functional “hot spots” composed of binding determinants presented across the subunit interface. The two most important determinants are located within the largest hot spot on a short, three-stranded sheet that is conserved in PDGF and TGF-β. Functional analysis of the binding epitopes for two receptor-blocking antibodies reveal different binding determinants near each of the KDR binding hot spots.

Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein

Protein–protein interacting surfaces are usually large and intricate, making the rational design of small mimetics of these interfaces a daunting problem. On the basis of a structural similarity between the CDR2-like loop of CD4 and the β-hairpin region of a short scorpion toxin, scyllatoxin, we transferred the side chains of nine residues of CD4, central in the binding to HIV-1 envelope glycoprotein (gp120), to a structurally homologous region of the scorpion toxin scaffold. In competition experiments, the resulting 27-amino acid miniprotein inhibited binding of CD4 to gp120 with a 40 μM IC50. Structural analysis by NMR showed that both the backbone of the chimeric β-hairpin and the introduced side chains adopted conformations similar to those of the parent CD4. Systematic single mutations suggested that most CD4 residues from the CDR2-like loop were reproduced in the miniprotein, including the critical Phe-43. The structural and functional analysis performed suggested five additional mutations that, once incorporated in the miniprotein, increased its affinity for gp120 by 100-fold to an IC50 of 0.1–1.0 μM, depending on viral strains. The resulting mini-CD4 inhibited infection of CD4+ cells by different virus isolates. Thus, core regions of large protein–protein interfaces can be reproduced in miniprotein scaffolds, offering possibilities for the development of inhibitors of protein–protein interactions that may represent useful tools in biology and in drug discovery.

Strychnine activates neuronal α7 nicotinic receptors after mutations in the leucine ring and transmitter binding site domains

Recent work has shown that strychnine, the potent and selective antagonist of glycine receptors, is also an antagonist of nicotinic acetylcholine (AcCho) receptors including neuronal homomeric α7 receptors, and that mutating Leu-247 of the α7 nicotinic AcCho receptor-channel domain (L247Tα7; mut1) converts some nicotinic antagonists into agonists. Therefore, a study was made of the effects of strychnine on Xenopus oocytes expressing the chick wild-type α7 or L247Tα7 receptors. In these oocytes, strychnine itself did not elicit appreciable membrane currents but reduced the currents elicited by AcCho in a reversible and dose-dependent manner. In sharp contrast, in oocytes expressing L247Tα7 receptors with additional mutations at Cys-189 and Cys-190, in the extracellular N-terminal domain (L247T/C189–190Sα7; mut2), micromolar concentrations of strychnine elicited inward currents that were reversibly inhibited by the nicotinic receptor blocker α-bungarotoxin. Single-channel recordings showed that strychnine gated mut2-channels with two conductance levels, 56 pS and 42 pS, and with kinetic properties similar to AcCho-activated channels. We conclude that strychnine is a modulator, as well as an activator, of some homomeric nicotinic α7 receptors. After injecting oocytes with mixtures of cDNAs encoding mut1 and mut2 subunits, the expressed hybrid receptors were activated by strychnine, similar to the mut2, and had a high affinity to AcCho like the mut1. A pentameric symmetrical model yields the striking conclusion that two identical α7 subunits may be sufficient to determine the functional properties of α7 receptors.

Cannabinoid-induced mesenteric vasodilation through an endothelial site distinct from CB1 or CB2 receptors

Cannabinoids, including the endogenous ligand arachidonyl ethanolamide (anandamide), elicit not only neurobehavioral but also cardiovascular effects. Two cannabinoid receptors, CB1 and CB2, have been cloned, and studies with the selective CB1 receptor antagonist SR141716A have implicated peripherally located CB1 receptors in the hypotensive action of cannabinoids. In rat mesenteric arteries, anandamide-induced vasodilation is inhibited by SR141716A, but other potent CB1 receptor agonists, such as HU-210, do not cause vasodilation, which implicates an as-yet-unidentified receptor in this effect. Here we show that “abnormal cannabidiol” (Abn-cbd) is a neurobehaviorally inactive cannabinoid that does not bind to CB1 receptors, yet causes SR141716A-sensitive hypotension and mesenteric vasodilation in wild-type mice and in mice lacking CB1 receptors or both CB1 and CB2 receptors. Hypotension by Abn-cbd is also inhibited by cannabidiol (20 μg/g), which does not influence anandamide- or HU-210-induced hypotension. In the rat mesenteric arterial bed, Abn-cbd-induced vasodilation is unaffected by blockade of endothelial NO synthase, cyclooxygenase, or capsaicin receptors, but it is abolished by endothelial denudation. Mesenteric vasodilation by Abn-cbd, but not by acetylcholine, sodium nitroprusside, or capsaicine, is blocked by SR141716A (1 μM) or by cannabidiol (10 μM). Abn-cbd-induced vasodilation is also blocked in the presence of charybdotoxin (100 nM) plus apamin (100 nM), a combination of K+-channel toxins reported to block the release of an endothelium-derived hyperpolarizing factor (EDHF). These findings suggest that Abn-cbd and cannabidiol are a selective agonist and antagonist, respectively, of an as-yet-unidentified endothelial receptor for anandamide, activation of which elicits NO-independent mesenteric vasodilation, possibly by means of the release of EDHF.

Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding

tRNA binding to the ribosomal P site is dependent not only on correct codon–anticodon interaction but also involves identification of structural elements of tRNA by the ribosome. By using a phosphorothioate substitution–interference approach, we identified specific nonbridging Rp-phosphate oxygens in the anticodon loop of tRNAPhe from Escherichia coli which are required for P-site binding. Stereo-specific involvement of phosphate oxygens at these positions was confirmed by using synthetic anticodon arm analogues at which single Rp- or Sp-phosphorothioates were incorporated. Identical interference results with yeast tRNAPhe and E. coli tRNAfMet indicate a common backbone conformation or common recognition elements in the anticodon loop of tRNAs. N-ethyl-N-nitrosourea modification–interference experiments with natural tRNAs point to the importance of the same phosphates in the loop. Guided by the crystal structure of tRNAPhe, we propose that specific Rp-phosphate oxygens are required for anticodon loop (“U-turn”) stabilization or are involved in interactions with the ribosome on correct tRNA–mRNA complex formation.

TnTIN and TnTAP: Mini-transposons for site-specific proteolysis in vivo

Tobacco etch virus (TEV) protease recognizes a 7-aa consensus sequence, Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser, where Xaa can be almost any amino acyl residue. Cleavage occurs between the conserved Gln and Ser residues. Because of its distinct specificity, TEV protease can be expressed in the cytoplasm without interfering with viability. Polypeptides that are not natural substrates of TEV protease are proteolyzed if they carry the appropriate cleavage site. Thus, this protease can be used to study target proteins in their natural environment in vivo, as well as in vitro. We describe two Tn5-based mini-transposons that insert TEV protease cleavage sites at random into target proteins. TnTIN introduces TEV cleavage sites into cytoplasmic proteins. TnTAP facilitates the same operation for proteins localized to the bacterial cell envelope. By using two different target proteins, SecA and TolC, we show that such modified proteins can be cleaved in vivo and in vitro by TEV protease. Possible applications of the site-specific proteolysis approach are topological studies of soluble as well as of inner and outer membrane proteins, protein inactivation, insertion mutagenesis experiments, and protein tagging.

Insecticide resistance resulting from an absence of target-site gene product

Genetic changes in insects that lead to insecticide resistance include point mutations and up-regulation/amplification of detoxification genes. Here, we report a third mechanism, resistance caused by an absence of gene product. Mutations of the Methoprene-tolerant (Met) gene of Drosophila melanogaster result in resistance to both methoprene, a juvenile hormone (JH) agonist insecticide, and JH. Previous results have demonstrated a mechanism of resistance involving an intracellular JH binding protein that has reduced ligand affinity in Met flies. We show that a γ-ray induced allele, Met27, completely lacks Met transcript during the insecticide-sensitive period in development. Although Met27 homozygotes have reduced oogenesis, they are viable, demonstrating that Met is not a vital gene. Most target-site resistance genes encode vital proteins and thus have few mutational changes that permit both resistance and viability. In contrast, resistance genes such as Met that encode nonvital insecticide target proteins can have a variety of mutational changes that result in an absence of functional gene product and thus should show higher rates of resistance evolution.

A transmembrane form of annexin XII detected by site-directed spin labeling

Previous studies of the annexin family of Ca2+ binding proteins identified a soluble monomer in the absence of Ca2+ and a trimer adsorbed on the membrane surface in the presence of Ca2+. On the basis of site-directed spin-labeling studies of annexin XII at low pH, we now report a membrane-inserted form of the protein with a dramatically different structure. The data suggest that upon insertion a continuous transmembrane α-helix is reversibly formed from a helix–loop–helix motif in the solution structure. Other regions with similar membrane-insertion potential were identified in the amino acid sequence, and we propose that the corresponding helices come together to form an aqueous pore that mediates the ion channel activity reported for several annexins.

Expression of myogenin during embryogenesis is controlled by Six/sine oculis homeoproteins through a conserved MEF3 binding site

Myogenin, one of the MyoD family of proteins, is expressed early during somitogenesis and is required for myoblast fusion in vivo. Previous studies in transgenic mice have shown that a 184-bp myogenin promoter fragment is sufficient to correctly drive expression of a β-galactosidase transgene during embryogenesis. We show here that mutation of one of the DNA motifs present in this region, the MEF3 motif, abolished correct expression of this β-galactosidase transgene. We have found that the proteins that bind to the MEF3 site are homeoproteins of the Six/sine oculis family. Antibodies directed specifically against Six1 or Six4 proteins reveal that each of these proteins is present in the embryo when myogenin is activated and constitutes a muscle-specific MEF3-binding activity in adult muscle nuclear extracts. Both of these proteins accumulate in the nucleus of C2C12 myogenic cells, and transient transfection experiments confirm that Six1 and Six4 are able to transactivate a reporter gene containing MEF3 sites. Altogether these results establish Six homeoproteins as a family of transcription factors controlling muscle formation through activation of one of its key regulators, myogenin.

Evidence for a voltage-dependent enhancement of neurotransmitter release mediated via the synaptic protein interaction site of N-type Ca2+ channels

Secretion of neurotransmitters is initiated by voltage-gated calcium influx through presynaptic, voltage-gated N-type calcium channels. These channels interact with the SNARE proteins, which are core components of the exocytosis process, via the synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their α1B subunit. Interruption of this interaction by competing synprint peptides inhibits fast, synchronous transmitter release. Here we identify a voltage-dependent, but calcium-independent, enhancement of transmitter release that is elicited by trains of action potentials in the presence of a hyperosmotic extracellular concentration of sucrose. This enhancement of transmitter release requires interaction of SNARE proteins with the synprint site. Our results provide evidence for a voltage-dependent signal that is transmitted by protein–protein interactions from the N-type calcium channel to the SNARE proteins and enhances neurotransmitter release by altering SNARE protein function.

Rat heart: A site of oxytocin production and action

We report here that the rat heart is a site of oxytocin (OT) synthesis and release. Oxytocin was detected in all four chambers of the heart. The highest OT concentration was in the right atrium (2128 ± 114 pg/mg protein), which was 19-fold higher than in rat uterus but 3.3-fold lower than in the hypothalamus. OT concentrations were significantly greater in the right and left atria than in the corresponding ventricles. Furthermore, OT was released into the effluent of isolated, perfused rat heart (34.5 ± 4.7 pg/min) and into the medium of cultured atrial myocytes. Reverse-phase HPLC purification of the heart extracts and heart perfusates revealed a main peak identical with the retention time of synthetic OT. Southern blots of reverse transcription–PCR products from rat heart revealed gene expression of specific OT mRNA. OT immunostaining likewise was found in atrial myocytes and fibroblasts, and the intensity of positive stains from OT receptors paralleled the atrial natriuretic peptide stores. Our findings suggest that heart OT is structurally identical, and therefore derived from, the same gene as the OT that is primarily found in the hypothalamus. Thus, the heart synthesizes and processes a biologically active form of OT. The presence of OT and OT receptor in all of the heart’s chambers suggests an autocrine and/or paracrine role for the peptide. Our finding of abundant OT receptor in atrial myocytes supports our hypothesis that OT, directly and/or via atrial natriuretic peptide release, can regulate the force of cardiac contraction.

The small ribosomal subunit from Thermus thermophilus at 4.5 Å resolution: Pattern fittings and the identification of a functional site

The electron density map of the small ribosomal subunit from Thermus thermophilus, constructed at 4.5 Å resolution, shows the recognizable morphology of this particle, as well as structural features that were interpreted as ribosomal RNA and proteins. Unbiased assignments, carried out by quantitative covalent binding of heavy atom compounds at predetermined sites, led to the localization of the surface of the ribosomal protein S13 at a position compatible with previous assignments, whereas the surface of S11 was localized at a distance of about twice its diameter from the site suggested for its center by neutron scattering. Proteins S5 and S7, whose structures have been determined crystallographically, were visually placed in the map with no alterations in their conformations. Regions suitable to host the fold of protein S15 were detected in several positions, all at a significant distance from the location of this protein in the neutron scattering map. Targeting the 16S RNA region, where mRNA docks to allow the formation of the initiation complex by a mercurated mRNA analog, led to the characterization of its vicinity.

Yeast chorismate mutase in the R state: Simulations of the active site

The isomerization of chorismate to prephenate by chorismate mutase in the biosynthetic pathway that forms Tyr and Phe involves C5—O (ether) bond cleavage and C1—C9 bond formation in a Claisen rearrangement. Development of negative charge on the ether oxygen, stabilized by Lys-168 and Glu-246, is inferred from the structure of a complex with a transition state analogue (TSA) and from the pH-rate profile of the enzyme and the E246Q mutant. These studies imply a protonated Glu-246 well above pH 7. Here, several 500-ps molecular dynamics simulations test the stability of enzyme–TSA complexes by using a solvated system with stochastic boundary conditions. The simulated systems are (i) protonated Glu-246 (stable), (ii) deprotonated Glu-246 (unstable), (iii) deprotonated Glu-246 plus one H2O between Glu-246 and the ether oxygen (unstable), (iv) the E246Q mutant (stable), and (v) addition of OH− between protonated Glu-246 and the ether oxygen. In (v), a local conformational change of Lys-168 displaced the OH− into the solvent region, suggesting a possible rate-determining step that precedes the catalytic step. In a 500-ps simulation of the enzyme complexed with the reactant chorismate or the product prephenate, no water molecule remained near the oxygen of the ligand. Calculations using the linearized Poisson–Boltzmann equation show that the effective pKa of Glu-246 is shifted from 5.8 to 8.1 as the negative charge on the ether oxygen of the TSA is changed from −0.56 electron to −0.9 electron. Altogether, these results support retention of a proton on Glu-246 to high pH and the absence of a water molecule in the catalytic steps.

Crystal structure of glutamate-1-semialdehyde aminomutase: An α2-dimeric vitamin B6-dependent enzyme with asymmetry in structure and active site reactivity

The three-dimensional structure of glutamate-1-semialdehyde aminomutase (EC 5.4.3.8), an α2-dimeric enzyme from Synechococcus, has been determined by x-ray crystallography using heavy atom derivative phasing. The structure, refined at 2.4-Å resolution to an R-factor of 18.7% and good stereochemistry, explains many of the enzyme’s unusual specificity and functional properties. The overall fold is that of aspartate aminotransferase and related B6 enzymes, but it also has specific features. The structure of the complex with gabaculine, a substrate analogue, shows unexpectedly that the substrate binding site involves residues from the N-terminal domain of the molecule, notably Arg-32. Glu-406 is suitably positioned to repel α-carboxylic acids, thereby suggesting a basis for the enzyme’s reaction specificity. The subunits show asymmetry in cofactor binding and in the mobilities of the residues 153–181. In the unliganded enzyme, one subunit has the cofactor bound as an aldimine of pyridoxal phosphate with Lys-273 and, in this subunit, residues 153–181 are disordered. In the other subunit in which the cofactor is not covalently bound, residues 153–181 are well defined. Consistent with the crystallographically demonstrated asymmetry, a form of the enzyme in which both subunits have pyridoxal phosphate bound to Lys-273 through a Schiff base showed biphasic reduction by borohydride in solution. Analysis of absorption spectra during reduction provided evidence of communication between the subunits. The crystal structure of the reduced form of the enzyme shows that, despite identical cofactor binding in each monomer, the structural asymmetry at residues 153–181 remains.