Peptide substrate profiling defines fibroblast activation protein as an endopeptidase of strict Gly(2)-Pro(1)-cleaving specificity

Fibroblast activation protein (FAP) is a serine protease of undefined endopeptidase specificity implicated in tumorigenesis. To characterize FAP's P(4)-P(2)(') specificity, we synthesized intramolecularly quenched fluorescent substrate sets based on the FAP cleavage site in alpha(2)-antiplasmin (TSGP-NQ). FAP required substrates with Pro at P(1) and Gly or d-amino acids at P(2) and preferred small, uncharged amino acids at P(3), but tolerated most amino acids at P(4), P(1)(') and P(2)('). These substrate preferences allowed design of peptidyl-chloromethyl ketones that inhibited FAP, but not the related protease, dipeptidyl peptidase-4. Thus, FAP is a narrow specificity endopeptidase and this can be exploited for inhibitor design.     

A novel association of mGluR1a with the PDZ scaffold protein CAL modulates receptor activity

Metabotropic glutamate receptor subtype 1a (mGluR1a) associates with the proteins mediating its receptor activity, suggesting a complex-controlled function of mGluR1a. Here, using glutathione-S-transferase pull-down, co-immnoprecipitation and immnoflurescence assays in vitro and in vivo, we have found CFTR-associated ligand (CAL) to be a novel binding partner of mGluR1a, through its PSD95/discslarge/ZO1homology domain. Deletion of mGluR1a-carboxyl terminus (CT) or mutation of Leu to Ala in the CT of mGluR1a reduces the association, indicating the essential binding region of mGluR1a for CAL. Functionally, the interaction of mGluR1a with CAL was shown to inhibit mGluR1a-mediated ERK1/2 activation, without an apparent effect, via the C-terminal-truncated receptor. These findings might provide a novel mechanism for the regulation of mGluR1a-mediated signaling through the interaction with CAL.

Structured summary

MINT-6797987, MINT-6798009:

NHERF-2 (uniprotkb:Q15599) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by proteinarray (MI:0089)

MINT-6798026, MINT-6798048, MINT-6798066:

mGluR1a (uniprotkb:Q9R0W0) physically interacts (MI:0218) with CAL (uniprotkb:Q9HD26) by pull down (MI:0096)

MINT-6797953, MINT-6797970:

NHERF-1 (uniprotkb:O14745) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by protein array (MI:0089)

MINT-6797935:

CAL (uniprotkb:Q9HD26) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by protein array (MI:0089)

MINT-6798084:

CAL (uniprotkb:Q9HD26) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by filter binding (MI:0049)

MINT-6798134:

mGluR1a (uniprotkb:Q9R0W0) physically interacts (MI:0218) with CAL (uniprotkb:Q9HD26) by anti tag coimmunoprecipitation (MI:0007)

MINT-6798158:

CAL (uniprotkb:B4F775) physically interacts (MI:0218) with mGluR1a (uniprotkb:Q9R0W0) by anti bait coimmunoprecipitation (MI:0006)

MINT-6798233:

CAL (uniprotkb:Q9HD26) colocalizes (MI:0403) with mGluR1a (uniprotkb:Q9R0W0) by fluorescence microscopy (MI:0416)

     

Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

The (pro)renin receptor (PRR) is an important component of the renin-angiotensin system (RAS), which regulates blood pressure and cardiovascular function. The integral membrane protein PRR contains a large extracellular domain (∼310 amino acids), a single transmembrane domain (∼20 amino acids) and an intracellular domain (∼19 amino acids). Although short, the intracellular (IC) domain of the PRR has functionally important roles in a number of signal transduction pathways activated by (pro)renin binding. Meanwhile, together with the transmembrane domain and a small portion of the extracellular domain (∼30 amino acids), the IC domain is also involved in assembly of V₀ portion of the vacuolar proton-translocating ATPase (V-ATPase). To better understand structural and multifunctional roles of the PRR-IC, we report the crystal structure of the PRR-IC domain as maltose-binding protein (MBP) fusion proteins at 2.0 Å (maltose-free) and 2.15 Å (maltose-bound). In the two separate crystal forms having significantly different unit-cell dimensions and molecular packing, MBP–PRR-IC fusion protein was found to be a dimer, which is different with the natural monomer of native MBP. The PRR-IC domain appears as a relatively flexible loop and is responsible for the dimerization of MBP fusion protein. Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermonomer interactions, suggesting a role for the PRR-IC domain in protein oligomerization.     

Human heat shock protein 70 (Hsp70) as a peripheral membrane protein

While a significant fraction of heat shock protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-?SBD and Hsp70-?NBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD.     

Structure of thiocyanate hydrolase: a new nitrile hydratase family protein with a novel five-coordinate cobalt(III) center

Thiocyanate hydrolase (SCNase) of Thiobacillus thioparus THI115 is a cobalt(III)-containing enzyme catalyzing the degradation of thiocyanate to carbonyl sulfide and ammonia. We determined the crystal structures of the apo- and native SCNases at a resolution of 2.0 A. SCNases in both forms had a conserved hetero-dodecameric structure, (alphabetagamma)(4). Four alphabetagamma hetero-trimers were structurally equivalent. One alphabetagamma hetero-trimer was composed of the core domain and the betaN domain, which was located at the center of the molecule and linked the hetero-trimers with novel quaternary interfaces. In both the apo- and native SCNases, the core domain was structurally conserved between those of iron and cobalt-types of nitrile hydratase (NHase). Native SCNase possessed the post-translationally modified cysteine ligands, gammaCys131-SO(2)H and gammaCys133-SOH like NHases. However, the low-spin cobalt(III) was found to be in the distorted square-pyramidal geometry, which had not been reported before in any protein. The size as well as the electrostatic properties of the substrate-binding pocket was totally different from NHases with respect to the charge distribution and the substrate accessibility, which rationally explains the differences in the substrate preference between SCNase and NHase.     

Tyrosine kinase activity of a Ca2+/calmodulin-dependent protein kinase II catalytic fragment

A 30-kDa fragment of Ca²⁺/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active protein Ser/Thr kinase devoid of autophosphorylation activity. We have produced a chimeric enzyme of 30K-CaMKII (designated CX₄₀-30K-CaMKII), in which the N-terminal 40 amino acids of Xenopus Ca²⁺/calmodulin-dependent protein kinase I (CX₄₀) were fused to the N-terminal end of 30K-CaMKII. Although CX₄₀-30K-CaMKII exhibited essentially the same substrate specificity as 30K-CaMKII, it underwent significant autophosphorylation. Surprisingly, its autophosphorylation site was found to be Tyr-18 within the N-terminal CX₄₀ region of the fusion protein, although it did not show any Tyr kinase activity toward exogenous substrates. Several lines of evidence suggested that the autophosphorylation occurred via an intramolecular mechanism. These data suggest that even typical Ser/Thr kinases such as 30K-CaMKII can phosphorylate Tyr residues under certain conditions. The possible mechanism of the Tyr residue autophosphorylation is discussed.     

Electrostatic contributions to the kinetics and thermodynamics of protein assembly

The role of electrostatic interactions in the assembly of a native protein structure was studied using fragment complementation. Contributions of salt, pH, or surface charges to the kinetics and equilibrium of calbindin D(9k) reconstitution was measured in the presence of Ca(2+) using surface plasmon resonance and isothermal titration calorimetry. Whereas surface charge substitutions primarily affect the dissociation rate constant, the association rates are correlated with subdomain net charge in a way expected for Coulomb interactions. The affinity is reduced in all mutants, with the largest effect (260-fold) observed for the double mutant K25E+K29E. At low net charge, detailed charge distribution is important, and charges remote from the partner EF-hand have less influence than close ones. The effects of salt and pH on the reconstitution are smaller than mutational effects. The interaction between the wild-type EF-hands occurs with high affinity (K(A) = 1.3 x 10(10) M(-1); K(D) = 80 pM). The enthalpy of association is overall favorable and there appears to be a very large favorable entropic contribution from the desolvation of hydrophobic surfaces that become buried in the complex. Electrostatic interactions contribute significantly to the affinity between the subdomains, but other factors, such as hydrophobic interactions, dominate.     

Monitoring ligand-mediated internalization of G protein-coupled receptor as a novel pharmacological approach

Agonist activation of a G protein-coupled receptor (GPCR) results in the redistribution of the receptor protein away from the cell surface into internal cellular compartments through a process of endocytosis known as internalization. Visualization of receptor internalization has become experimentally practicable by using fluorescent reagents such as green fluorescent protein (GFP). In this study, we examined whether the ligand-mediated internalization of a GPCR can be exploited for pharmacological evaluations. We acquired fluorescent images of cells expressing GFP-labeled GPCRs and evaluated the ligand-mediated internalization quantitatively by image processing. Using beta2-adrenoceptor and vasopressin V1a receptor as model GPCRs that couple to Gs and Gq, respectively, we first examined whether these GFP-tagged GPCRs exhibited appropriate pharmacology. The rank order of receptor internalization potency for a variety of agonists and antagonists specific to each receptor corresponded well with that previously observed in ligand binding studies. In addition to chemical ligand-induced internalization, this cell-based fluorescence imaging system successfully monitored the internalization of the proton-sensing GPCR TDAG8, and that of the free fatty acid-sensitive GPCR GPR120. The results show that monitoring receptor internalization can be a useful approach for pharmacological characterization of GPCRs and in fishing for ligands of orphan GPCRs.     

Crystal structure of the RRM domain of poly(A)-specific ribonuclease reveals a novel m(7)G-cap-binding mode

Poly(A)-specific ribonuclease (PARN) is a processive 3′-exoribonuclease involved in the decay of eukaryotic mRNAs. Interestingly, PARN interacts not only with the 3′ end of the mRNA but also with its 5′ end as PARN contains an RRM domain that specifically binds both the poly(A) tail and the 7-methylguanosine (m⁷G) cap. The interaction of PARN with the 5′ cap of mRNAs stimulates the deadenylation activity and enhances the processivity of this reaction. We have determined the crystal structure of the PARN-RRM domain with a bound m⁷G triphosphate nucleotide, revealing a novel binding mode for the m⁷G cap. The structure of the m⁷G binding pocket is located outside of the canonical RNA-binding surface of the RRM domain and differs significantly from that of other m⁷G-cap-binding proteins. The crystal structure also shows a remarkable conformational flexibility of the RRM domain, leading to a perfect exchange of two α-helices with an adjacent protein molecule in the crystal lattice.     

Amino acids as a nitrogen source for tomato seedlings: The use of dual-labeled (C, N) glycine to test for direct uptake by tomato seedlings

Direct uptake of organic nitrogen (ON) compounds, rather than inorganic N, by plant roots has been hypothesized to constitute a significant pathway for plant nutrition. The aim of this study was to test whether tomatoes (Solanum lycopersicum cv. Huying932) can take up ON directly from the soil by using ¹⁵NH₄Cl, K¹⁵NO₃, 1, 2-¹³C₂¹⁵N-glycine labeling techniques. The ¹³C and ¹⁵N in the plants increased significantly indicating that a portion of the glycine-N was taken up in the form of intact amino acids by the tomatoes within 48 h after injection into the soil. Regression analysis of excess ¹³C against excess ¹⁵N showed that approximately 21% of the supplied glycine-N was taken up intact by the tomatoes. Atom% excesses of ¹⁵N and ¹³C in the roots were higher than in any shoots. Results also indicated rapid turnover of amino acids (e.g., glycine) by soil microorganisms, and the poor competitive ability of tomatoes in absorbing amino acids from the soil solution. This implies that tomatoes can take up ON in an intact form from the soil despite the rapid turnover of organic N usually found under such conditions. Given the influence of climatic change and N pollution, further studies investigating the functional ecological implications of ON in horticultural ecosystems are warranted.     

Site-directed fluorescence labeling of a membrane protein with BADAN: probing protein topology and local environment

The work presented here describes a new and simple method based on site-directed fluorescence labeling using the BADAN label that permits the examination of protein-lipid interactions in great detail. We applied this technique to a membrane-embedded, mainly α-helical reference protein, the M13 major coat protein. Using a high-throughput approach, 40 site-specific cysteine mutants were prepared of the 50-residues long protein. The steady-state fluorescence spectra were analyzed using a three-component spectral model that enabled the separation of Stokes shift contributions from water and internal label dynamics, and protein topology. We found that most of the fluorescence originated from BADAN labels that were hydrogen-bonded to water molecules even within the hydrophobic core of the membrane. Our spectral decomposition method revealed the embedment and topology of the labeled protein in the membrane bilayer under various conditions of headgroup charge and lipid chain length, as well as key characteristics of the membrane such as hydration level and local polarity, provided by the local dielectric constant.     

Energetics in Photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA1 or psbA3 gene

The main cofactors involved in the function of Photosystem II (PSII) are borne by the D1 and D2 proteins. In some cyanobacteria, the D1 protein is encoded by different psbA genes. In Thermosynechococcus elongatus the amino acid sequence deduced from the psbA₃ gene compared to that deduced from the psbA₁ gene points a difference of 21 residues. In this work, PSII isolated from a wild type T. elongatus strain expressing PsbA1 or from a strain in which both the psbA₁ and psbA₂ genes have been deleted were studied by a range of spectroscopies in the absence or the presence of either a urea type herbicide, DCMU, or a phenolic type herbicide, bromoxynil. Spectro-electrochemical measurements show that the redox potential of Pheo_D1 is increased by 17 mV from −522 mV in PsbA1-PSII to −505 mV in PsbA3-PSII. This increase is about half that found upon the D1-Q130E single site directed mutagenesis in Synechocystis PCC 6803. This suggests that the effects of the D1-Q130E substitution are, at least partly, compensated for by some of the additional amino-acid changes associated with the PsbA3 for PsbA1 substitution. The thermoluminescence from the S₂Q_A^−• charge recombination and the C ≡ N vibrational modes of bromoxynil detected in the non-heme iron FTIR difference spectra support two binding sites (or one site with two conformations) for bromoxynil in PsbA3-PSII instead of one in PsbA1-PSII which suggests differences in the Q_B pocket. The temperature dependences of the S₂Q_A^−• charge recombination show that the strength of the H-bond to Pheo_D1 is not the only functionally relevant difference between the PsbA3-PSII and PsbA1-PSII and that the environment of Q_A (and, as a consequence, its redox potential) is modified as well. The electron transfer rate between P₆₈₀^+• and Y_Z is found faster in PsbA3 than in PsbA1 which suggests that the redox potential of the P₆₈₀/P₆₈₀^+• couple (and hence that of ¹P₆₈₀^*/P₆₈₀^+•) is tuned as well when shifting from PsbA1 to PsbA3. In addition to D1-Q130E, the non-conservative amongst the 21 amino acid substitutions, D1-S270A and D1-S153A, are proposed to be involved in some of the observed changes.     

AHCYL2 (long-IRBIT) as a potential regulator of the electrogenic Na-HCO3 cotransporter NBCe1-B

Although AHCYL2 (long-IRBIT) is highly homologous to IRBIT, which regulates ion-transporting proteins including the electrogenic Na⁺-HCO₃⁻ cotransporter NBCe1-B, its functions are poorly understood. Here, we found that AHCYL2 interacts with NBCe1-B in bovine parotid acinar cells using yeast two-hybrid, immunofluorescence confocal microscopy and co-immunoprecipitation analyses. Whole-cell patch-clamp experiments revealed that co-expression of AHCYL2 reduces the apparent affinity for intracellular Mg²⁺ in inhibition of NBCe1-B currents specifically in a HCO₃⁻-deficient cellular condition. Our data unveil AHCYL2 as a potential regulator of NBCe1-B in mammalian cells. We propose that cytosolic ionic condition appropriate for AHCYL2 to function might be different from IRBIT.     

Membrane topology of loop 13-14 of the Na/glucose cotransporter (SGLT1): A SCAM and fluorescent labelling study

The accessibility of the hydrophilic loop between putative transmembrane segments XIII and XIV of the Na⁺/glucose cotransporter (SGLT1) was studied in Xenopus oocytes, using the substituted cysteine accessibility method (SCAM) and fluorescent labelling. Fifteen cysteine mutants between positions 565 and 664 yielded cotransport currents of similar amplitude than the wild-type SGLT1 (wtSGLT1). Extracellular, membrane-impermeant MTSES⁽⁻⁾ and MTSET⁽⁺⁾ had no effect on either cotransport or Na⁺ leak currents of wtSGLT1 but 9 mutants were affected by MTSES and/or MTSET. We also performed fluorescent labelling on SGLT1 mutants, using tetramethylrhodamine-5-maleimide and showed that positions 586, 588 and 624 were accessible. As amino acids 604 to 610 in SGLT1 have been proposed to form part of a phlorizin (Pz) binding site, we measured the K_i^Pz and K_m^αMG for wtSGLT1 and for cysteine mutants at positions 588, 605-608 and 625. Although mutants A605C, Y606C and D607C had slightly higher K_i^Pz values than wtSGLT1 with minimal changes in K_m^αMG, the effects were modest and do not support the original hypothesis. We conclude that the large, hydrophilic loop near the carboxyl terminus of SGLT1 is thus accessible to the external solution but does not appear to play a major part in the binding of phlorizin.     

Effect of poly(phosphate) anions on glyceraldehyde-3-phosphate dehydrogenase structure and thermal aggregation: comparison with influence of poly(sulfoanions)

Background

It is well documented that poly(sulfate) and poly(sulfonate) anions suppress protein thermal aggregation much more efficiently than poly(carboxylic) anions, but as a rule, they denature protein molecules. In this work, a polymer of different nature, i.e. poly(phosphate) anion (PP) was used to elucidate the influence of phosphate groups on stability and thermal aggregation of the model enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Methods

Isothermal titration calorimetry and differential scanning calorimetry were used for studying the protein–polyanion interactions and the influence of bound polyanions on the protein structure. The enzymatic activity of GAPDH and size of the complexes were measured. The aggregation level was determined from the turbidity.

Results

Highly polymerized PP chains were able to suppress the aggregation completely, but at significantly higher concentrations as compared with poly(styrenesulfonate) (PSS) or dextran sulfate chains of the same degree of polymerization. The effect of PP on the enzyme structure and activity was much gentler as opposed to the binding of dextran sulfate or, especially, PSS that denatured GAPDH molecules with the highest efficacy caused by short PSS chains. These findings agreed well with the enhanced affinity of polysulfoanions to GAPDH.

Conclusions

The revealed trends might help to illuminate the mechanism of control of proteins functionalities by insertion of charged groups of different nature through posttranslational modifications.

General significance

Practical implementation of the results could be the use of PP chains as promising tools to suppress the proteins aggregation without noticeable loss in the enzymatic activity.     

Domain movement of iron sulfur protein in cytochrome bc1 complex is facilitated by the electron transfer from cytochrome b(L) to b(H)

The key step of the "protonmotive Q-cycle" mechanism for cytochrome bc1 complex is the bifurcated oxidation of ubiquinol at the Qp site. ISP is reduced when its head domain is at the b-position and subsequent move to the c1 position, to reduce cytochrome c1, upon protein conformational changes caused by the electron transfer from cytochrome b(L) to b(H). Results of analyses of the inhibitory efficacy and the binding affinity, determined by isothermal titration calorimetry, of Pm and Pf, on different redox states of cytochrome bc1 complexes, confirm this speculation. Pm inhibitor has a higher affinity and better efficacy with the cytochrome b(H) reduced complex and Pf binds better and has a higher efficacy with the ISP reduced complex.     

Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in QB-site of Chlamydomonas reinhardtii

Tenuazonic acid (TeA) is a natural phytotoxin produced by Alternaria alternata, the causal agent of brown leaf spot disease of Eupatorium adenophorum. Results from chlorophyll fluorescence revealed TeA can block electron flow from Q_A to Q_B at photosystem II acceptor side. Based on studies with D1-mutants of Chlamydomonas reinhardtii, the No. 256 amino acid plays a key role in TeA binding to the Q_B-niche. The results of competitive replacement with [¹⁴C]atrazine combined with JIP-test and D1-mutant showed that TeA should be considered as a new type of photosystem II inhibitor because it has a different binding behavior within Q_B-niche from other known photosystem II inhibitors. Bioassay of TeA and its analogues indicated 3-acyl-5-alkyltetramic and even tetramic acid compounds may represent a new structural framework for photosynthetic inhibitors.     

Bifunctional chelates with mixed aromatic and aliphatic amine donors for labeling of biomolecules with the {Tc(CO)3} and {Re(CO)3} cores

A series of tridentate ligands consisting of mixed aromatic and aliphatic amine derivatives of single amino acid chelates and phenylpiperazine have been developed, and their reactions with [NEt₄]₂[ReBr₃(CO)₃] have been investigated. The compounds [Re(CO)₃{(NC₅H₄CH₂)NCH₃(C₂H₄)NHCH₃}]Br (4), [Re(CO)₃{(NC₅H₄CH₂)NCH₃(C₂H₄)NCH₃(CH₂)_xCOOC₂H₅}]Br (x = 1, 5; x = 4, 6) [Re(CO)₃{(NC₅H₄CH₂)NH(C₂H₄)N(CH₃)₂}]Br (7), [Re(CO)₃{(NC₅H₄CH₂)N(CH ₂COOC₂H₅)(C₂H₄)N(CH₃)₂}]Br (8) and [Re(CO)₃(NC₅H₄CH₂)(C₂H₄NH₂)N(CH₂)₃-CH₃Ophenpip]Br (9) (phenpip: phenylpiperazine, -C₆H₄-(CH₂CH₂)₂N-) were prepared and characterized by elemental analysis, NMR, IR, HSMS and X-ray crystallography. All complexes exhibit fac-{Re(CO)₃N₃} coordination geometry in the cationic molecular unit. Crystal data for C₁₃H₁₇BrN₃O₃Re (4): orthorhombic, Pbca, a = 13.4510(8) Å, b = 10.5728(6) Å, c = 22.5378(13) Å, V = 3205.2(3) ų, Z = 8; C₁₇H₂₃BrN₃O₅Re (5): orthorhombic, Pcca, a = 16.5907(7) Å,b = 14.8387(6) Å, c = 16.7075(7) Å, V = 4113.1(3) ų, Z = 8; C₁₃H₂₅BrN₃O₇Re (7 · 4H₂O): monoclinic, P2₁/n, a = 14.0698(17) Å, b = 9.6760(12) Å, c = 15.6099 (19) Å, β = 114.930(2)°, V = 1927.1(4) ų, Z = 4; C₁₇H₂₃BrN₃O₅Re (8): monoclinic, P2₁/n, a = 7.5312(5) Å, b = 16.0366(10) Å, c = 16.8741(10) Å, β = 98.9990(10)°, V = 2012.9(2) ų, Z = 4.