Mapping sequence differences between thimet oligopeptidase and neurolysin implicates key residues in substrate recognition

The highly homologous endopeptidases thimet oligopeptidase and neurolysin are both restricted to short peptide substrates and share many of the same cleavage sites on bioactive and synthetic peptides. They sometimes target different sites on the same peptide, however, and defining the determinants of differential recognition will help us to understand how both enzymes specifically target a wide variety of cleavage site sequences. We have mapped the positions of the 224 surface residues that differ in sequence between the two enzymes onto the surface of the neurolysin crystal structure. Although the deep active site channel accounts for about one quarter of the total surface area, only 11% of the residue differences map to this region. Four isolated sequence changes (R470/E469, R491/M490, N496/H495, and T499/R498; neurolysin residues given first) are well positioned to affect recognition of substrate peptides, and differences in cleavage site specificity can be largely rationalized on the basis of these changes. We also mapped the positions of three cysteine residues believed to be responsible for multimerization of thimet oligopeptidase, a process that inactivates the enzyme. These residues are clustered on the outside of one channel wall, where multimerization via disulfide formation is unlikely to block the substrate-binding site. Finally, we mapped the regulatory phosphorylation site in thimet oligopeptidase to a location on the outside of the molecule well away from the active site, which indicates this modification has an indirect effect on activity.     

Substrate specificity characterization of recombinant metallo oligo-peptidases thimet oligopeptidase and neurolysin

We report a systematic and detailed analysis of recombinant neurolysin (EC 3.4.24.16) specificity in parallel with thimet oligopeptidase (TOP, EC 3.4.24.15) using Bk sequence and its C- and N-terminal extensions as in human kininogen as motif for synthesis of internally quenched fluorescent substrates. The influence of the substrate size was investigated, and the longest peptide susceptible to TOP and neurolysin contains 17 amino acids. The specificities of both oligopeptidases to substrate sites P(4) to P(3)' were also characterized in great detail using seven series of peptides based on Abz-GFSPFRQ-EDDnp taken as reference substrate. Most of the peptides were hydrolyzed at the bond corresponding to P(4)-F(5) in the reference substrate and some of them were hydrolyzed at this bond or at F(2)-S(3) bond. No restricted specificity was found for P(1)' as found in thermolysin as well for P(1) substrate position, however the modifications at this position (P(1)) showed to have large influence on the catalytic constant and the best substrates for TOP contained at P(1), Phe, Ala, or Arg and for neurolysin Asn or Arg. Some amino acid residues have large influence on the K(m) constants independently of its position. On the basis of these results, we are hypothesizing that some amino acids of the substrates can bind to different sub-sites of the enzyme fitting P-F or F-S bond, which requires rapid interchange for the different forms of interaction and convenient conformations of the substrate in order to expose and fit the cleavage bonds in correct position for an efficient hydrolysis. Finally, this plasticity of interaction with the substrates can be an essential property for a class of cytosolic oligopeptidases that are candidates to participate in the selection of the peptides to be presented by the MHC class I.     

Swapping the substrate specificities of the neuropeptidases neurolysin and thimet oligopeptidase

Thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related zinc-dependent metallopeptidases that metabolize small bioactive peptides. They cleave many substrates at the same sites, but they recognize different positions on others, including neurotensin, a 13-residue peptide involved in modulation of dopaminergic circuits, pain perception, and thermoregulation. On the basis of crystal structures and previous mapping studies, four sites (Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499; thimet oligopeptidase residues listed first) in their substrate-binding channels appear positioned to account for differences in specificity. Thimet oligopeptidase mutated so that neurolysin residues are at all four positions cleaves neurotensin at the neurolysin site, and the reverse mutations in neurolysin switch hydrolysis to the thimet oligopeptidase site. Using a series of constructs mutated at just three of the sites, it was determined that mutations at only two (Glu-469/Arg-470 and Arg-498/Thr-499) are required to swap specificity, a result that was confirmed by testing the two-mutant constructs. If only either one of the two sites is mutated in thimet oligopeptidase, then the enzyme cleaves almost equally at the two hydrolysis positions. Crystal structures of both two-mutant constructs show that the mutations do not perturb local structure, but side chain conformations at the Arg-498/Thr-499 position differ from those of the mimicked enzyme. A model for differential recognition of neurotensin based on differences in surface charge distribution in the substrate binding sites is proposed. The model is supported by the finding that reducing the positive charge on the peptide results in cleavage at both hydrolysis sites.     

Selective neurotensin-derived internally quenched fluorogenic substrates for neurolysin (EC 3.4.24.16): comparison with thimet oligopeptidase (EC 3.4.24.15) and neprilysin (EC 3.4.24.11)

Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at P--Y or Y--I or R--R bonds depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S'(1) specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for neurolysin (k(cat) = 7.0 s(-1), K(m) = 1.19 microM and k(cat)/K(m) = 5882 mM(-1). s(-1)), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26). Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by neurolysin with k(cat) = 14.03 s(-1), K(m) = 0.82 microM, and k(cat)/K(m) = 17,110 mM(-1). s(-1), being the best substrate so far described for this peptidase.     

Profiling thimet oligopeptidase-mediated proteolysis in Arabidopsis thaliana

Protein homeostasis (proteostasis) is crucial for proper cellular function, including the production of peptides with biological functions through controlled proteolysis. Proteostasis has roles in maintenance of cellular functions and plant interactions with the environment under physiological conditions. Plant stress continues to reduce agricultural yields causing substantial economic losses; thus, it is critical to understand how plants perceive stress signals to elicit responses for survival. As previously shown in Arabidopsis thaliana, thimet oligopeptidases (TOPs) TOP1 (also referred to as organellar oligopeptidase) and TOP2 (also referred to as cytosolic oligopeptidase) are essential components in plant response to pathogens, but further characterization of TOPs and their peptide substrates is required to understand their contributions to stress perception and defense signaling. Herein, label-free peptidomics via liquid chromatography-tandem mass spectrometry was used to differentially quantify 1111 peptides, originating from 369 proteins, between the Arabidopsis Col-0 wild type and top1top2 knock-out mutant. This revealed 350 peptides as significantly more abundant in the mutant, representing accumulation of these potential TOP substrates. Ten direct substrates were validated using in vitro enzyme assays with recombinant TOPs and synthetic candidate peptides. These TOP substrates are derived from proteins involved in photosynthesis, glycolysis, protein folding, biogenesis, and antioxidant defense, implicating TOP involvement in processes aside from defense signaling. Sequence motif analysis revealed TOP cleavage preference for non-polar residues in the positions surrounding the cleavage site. Identification of these substrates provides a framework for TOP signaling networks, through which the interplay between proteolytic pathways and defense signaling can be further characterized.     

Anion activation site of insulin-degrading enzyme

Insulin-degrading enzyme (IDE) (insulysin) is a zinc metallopeptidase that metabolizes several bioactive peptides, including insulin and the amyloid β peptide. IDE is an unusual metallopeptidase in that it is allosterically activated by both small peptides and anions, such as ATP. Here, we report that the ATP-binding site is located on a portion of the substrate binding chamber wall arising largely from domain 4 of the four-domain IDE. Two variants having residues in this site mutated, IDEK898A,K899A,S901A and IDER429S, both show greatly decreased activation by the polyphosphate anions ATP and PPPi. IDEK898A,K899A,S901A is also deficient in activation by small peptides, suggesting a possible mechanistic link between the two types of allosteric activation. Sodium chloride at high concentrations can also activate IDE. There are no observable differences in average conformation between the IDE-ATP complex and unliganded IDE, but regions of the active site and C-terminal domain do show increased crystallographic thermal factors in the complex, suggesting an effect on dynamics. Activation by ATP is shown to be independent of the ATP hydrolysis activity reported for the enzyme. We also report that IDEK898A,K899A,S901A has reduced intracellular function relative to unmodified IDE, consistent with a possible role for anion activation of IDE activity in vivo. Together, the data suggest a model in which the binding of anions activates by reducing the electrostatic attraction between the two halves of the enzyme, shifting the partitioning between open and closed conformations of IDE toward the open form.     

The role of Tyr605 and Ala607 of thimet oligopeptidase and Tyr606 and Gly608 of neurolysin in substrate hydrolysis and inhibitor binding

The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series Abz-GFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X=Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr605 and Ala607 in TOP and at Tyr606 and Gly608 in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. The kinetic parameters for the hydrolysis of substrates with systematic variations at position P1 showed that Tyr605 and Tyr606 of TOP and NEL respectively, played a role in subsite S1. Ala607 of TOP and Gly608 of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Tyr605 was an important anchor for its interaction with wild-type TOP. The hydroxy groups of Tyr605 and Tyr606 did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-kcat/Km dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.     

Molecular dynamics, crystallography and mutagenesis studies on the substrate gating mechanism of prolyl oligopeptidase

Altered prolyl oligopeptidase (PREP) activity is found in many common neurological and other genetic disorders, and in some cases PREP inhibition may be a promising treatment. The active site of PREP resides in an internal cavity; in addition to the direct interaction between active site and substrate or inhibitor, the pathway to reach the active site (the gating mechanism) must be understood for more rational inhibitor design and understanding PREP function. The gating mechanism of PREP has been investigated through molecular dynamics (MD) simulation combined with crystallographic and mutagenesis studies. The MD results indicate the inter-domain loop structure, comprised of 3 loops at residues, 189-209 (loop A), 577-608 (loop B), and 636-646 (loop C) (porcine PREP numbering), are important components of the gating mechanism. The results from enzyme kinetics of PREP variants also support this hypothesis: When loop A is (1) locked to loop B through a disulphide bridge, all enzyme activity is halted, (2) nicked, enzyme activity is increased, and (3) removed, enzyme activity is only reduced. Limited proteolysis study also supports the hypothesis of a loop A driven gating mechanism. The MD results show a stable network of H-bonds that hold the two protein domains together. Crystallographic study indicates that a set of known PREP inhibitors inhabit a common binding conformation, and this H-bond network is not significantly altered. Thus the domain separation, seen to occur in lower taxa, is not involved in the gating mechanism for mammalian PREP. In two of the MD simulations we observed a conformational change that involved the breaking of the H-bond network holding loops A and B together. We also found that this network was more stable when the active site was occupied, thus decreasing the likelihood of this transition.     

Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase.

We report the recombinant neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R-R or at R-X bonds, and TOP hydrolyzed at R-R or L-R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05-0.2 m for NaCl, KCl, NH4Cl and NaI, and 0.025-0.1 m for Na2SO4. Concentration higher than 0.2 N NH4Cl and NaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values of enthalpy (DeltaH*) and entropy (DeltaS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.     

Identification of essential residues within Lit, a cell death peptidase of Escherichia coli K-12

Bacteriophage exclusion is a suicide response to viral infection. In strains of Escherichia coli K-12 infected with T4 phage this process is mediated by the host-encoded Lit peptidase. Lit is activated by a unique sequence in the major head protein of the T4 phage (the Gol sequence) which then cleaves site-specifically the host translation factor EF-Tu, ultimately leading to cell death. Lit has very low sequence identity with other peptidases, with only a putative metallopeptidase motif, H(160)EXXH, giving an indication of its catalytic activity. The aim of the present study was to ascertain if Lit is a metallopeptidase, identify residues essential for Lit activity, and probe the involvement of the Gol sequence in the activation of enzymatic activity. Lit activity was inhibited by the zinc chelator 1,10-phenanthroline, consistent with the suggestion that it is a metallopeptidase. Preliminary covalent modification experiments found that Lit was susceptible to inactivation by diethyl pyrocarbonate, with about three histidines reversibly modified, one of which was found to be essential for proteolytic activity. Subsequently, 13 mutants of the Lit enzyme were constructed that included all 10 histidines as well as other residues within the metallopeptidase motif. This demonstrated that the residues within the HEXXH motif are required for Lit activity and further defined the essential catalytic core as H(160)EXXHX(67)H, with additional residues such as His169 being important but not essential for activity. Kinetic analysis of Lit activation by a synthetic Gol peptide highlighted that elevated concentrations of the peptide (>10-fold above activation K(M)) are inhibitory to Lit, with this effect also seen in partially active Lit mutants. The susceptibility of Lit to inhibition by its own activating peptide suggests that the Gol sequence may be able to bind nonproductively to the enzyme at high concentration. We discuss these data in the context of the currently understood models for Gol-mediated activation of the Lit peptidase and its mechanism of action.     

Substrate specificity of rat brain neurolysin disclosed by molecular display system and putative substrates in rat tissues

To search for the substrates, other than neurotensin, of rat brain neurolysin, a novel method of determining peptidase activity was developed using a yeast molecular display system. This is a useful and convenient method of handling homogenously pure proteins to evaluate the properties of neurolysin. The neurolysin gene was ligated to the C-terminal half of the α-agglutinin gene with a FLAG tag sequence and a yeast cell-surface molecular displaying plasmid was constructed. Display of neurolysin with correct folding and appropriate activity was verified by immunofluorescence staining and activity measurement of a bradykinin-related peptide. The cleavage sites of peptides were determined by high-performance liquid chromatography (HPLC) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results showed the amino acid preferences of hydrophobic, aromatic, and basic residues, which were the same as those of soluble neurolysin. Moreover, this method clearly showed the presence of two recognition motifs in neurolysin. By using these motifs, novel substrate candidates of neurolysin in rat tissues were screened, and several bioactive peptides that regulate feeding were found. We also discussed the ubiquitous distribution of neurolysin in rat tissues and the functions of substrate candidate peptides.     

Two independent amsacrine-resistant human myeloid leukemia cell lines share an identical point mutation in the 170 kDa form of human topoisomerase II.

Cloning and sequencing of cDNA segments of human TOP2 gene encoding the 170 kDa form of human DNA topoisomerase II show that Arg486 of the enzyme has been mutated to a lysine in the enzyme from two human leukemia cell lines HL-60/AMSA and KBM-3/AMSA, which were independently selected for resistance to the antitumor drug amsacrine (4'-[9-acridinylamino]-methanesulfon-m-anisidide, mAMSA). Sequence identity comparisons between eukaryotic DNA topoisomerase II and bacterial gyrase (bacterial DNA topoisomerase II) indicate that the position of the common mutation observed in mAMSA-resistant human TOP2 corresponds to that of the point mutation nal-31 in the Escherichia coli gyrase B gene, which confers resistance to nalidixic acid. Because mAMSA and nalidixic acid are known to act on their respective targets by a common mechanism of trapping the covalent enzyme-DNA intermediates, these results provide strong evidence that the 170 kDa form of human DNA topoisomerase II is a major cellular target of mAMSA, and that Arg486 of this enzyme is involved in mAMSA-mediated trapping of the covalent enzyme-DNA complex.     

Crystal structure of the cold-active aminopeptidase from Colwellia psychrerythraea, a close structural homologue of the human bifunctional leukotriene A4 hydrolase

The crystal structure of a cold-active aminopeptidase (ColAP) from Colwellia psychrerythraea strain 34H has been determined, extending the number of crystal structures of the M1 metallopeptidase family to four among the 436 members currently identified. In agreement with their sequence similarity, the overall structure of ColAP displayed a high correspondence with leukotriene A4 hydrolase (LTA4H), a human bifunctional enzyme that converts leukotriene A4 (LTA4) in the potent chemoattractant leukotriene B4. Indeed, both enzymes are composed of three domains, an N-terminal saddle-like domain, a catalytic thermolysin-like domain, and a less conserved C-terminal alpha-helical flat spiral domain. Together, these domains form a deep cavity harboring the zinc binding site formed by residues included in the conserved HEXXHX(18)H motif. A detailed structural comparison of these enzymes revealed several plausible determinants of ColAP cold adaptation. The main differences involve specific amino acid substitutions, loop content and solvent exposure, complexity and distribution of ion pairs, and differential domain flexibilities. Such elements may act synergistically to allow conformational flexibility needed for an efficient catalysis in cold environments. Furthermore, the region of ColAP corresponding to the aminopeptidase active site of LTA4H is much more conserved than the suggested LTA4 substrate binding region. This observation supports the hypothesis that this region of the LTA4H active site has evolved in order to fit the lipidic substrate.     

Metallopeptidase,neurolysin, as a novel molecular tool for analysis of properties of cancer-producing matrix metalloproteinases-2 and -9

To compare the substrate preferences of rat brain neurolysin and cancer-producing matrix metalloproteinases (MMPs), which have the same architecture in their catalytic domains, the cleavage activity of neurolysin toward MMP-specific fluorescence-quenching peptides was quantitatively measured. The results show that neurolysin effectively cleaved MOCAc [(7-methoxy coumarin-4-yl) acetyl]-RPKPYANvaWMK(Dnp[2,4-dinitrophenyl])-NH₂, a specific substrate of MMP-2 and MMP-9, but hardly cleaved MOCAc-RPKPVENvaWRK(Dnp)-NH₂, a specific substrate of MMP-3, suggesting that neurolysin has a similar substrate preference to MMP-2 and MMP-9. A structural comparison between neurolysin and MMP-9 showed the similar key amino acid residues for substrate recognition. The possible application of neurolysin displayed on the yeast cell surface, as a safe protein alternative to MMP-2 and MMP-9 which induce cancer cell growth, invasion, and metastasis, to analysis of properties of the MMPs, including the screening of inhibitors and analysis of inhibition mechanism etc., are also discussed.     

Purification and substrate specificity of an endopeptidase from the human oral spirochete Treponema denticola ATCC 35405, active on furylacryloyl-Leu-Gly-Pro-Ala and bradykinin.

An endopeptidase was purified to homogeneity from the cell extracts of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised dialysis, anion exchange fast protein liquid chromatography (FPLC), hydroxylapatite FPLC, immobilized metal affinity FPLC, FPLC chromatofocusing, and two consecutive gel permeation FPLC steps. The enzyme is a 62-kDa protein with an isoelectric point of 6.5-7.0. Experiments with enzyme inhibitors suggest that this enzyme is a metallopeptidase and that its activity is not dependent on sulfhydryl or serine residues. The enzyme is active on furylacryloyl-Leu-Gly-Pro-Ala (FALGPA; pH optimum near 6.25), bradykinin (Bk), and several Bk-related peptides. In FALGPA, the cleavage site is the Leu-Gly bond. An imino acid is absolutely necessary in position P'2. The shortest hydrolyzed peptide was FALGPA, the hydrolysis of which is strongly and competitively inhibited by Bk (Ki = 5.0 microM). The pyrophosphate ion and phosphoramidon also inhibited the hydrolysis of FALGPA. The enzyme does not hydrolyze all typical synthetic collagenase substrates, Azocoll, Azocasein, or Type I and Type IV collagens, or any other proteins tested. In Bk-related peptides, the hydrolyzed bond was Phe5-Ser6. Since a Bk antagonist and a Bk-potentiating pentapeptide also were good substrates, it is possible that the enzyme hydrolyzes Bks and related peptides only because of the coincidental, specific amino acid sequence of those substrates. A proposal is made that since a substantial portion of the amino acid sequence of FALGPA is present in collagen (and additionally acknowledging that the furylacryloyl residue structurally resembles that of proline), the natural substrates of this enzyme may be small, soluble collagen fragments produced by other enzymes from periodontal connective tissue, and that such peptides are important for the nutrition and pathogenicity of T. denticola.     

Active and regulatory sites of cytosolic 5'-nucleotidase

Cytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap?A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap?A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation.     

Potencies of phosphine peptide inhibitors of mammalian thimet oligopeptidase and neurolysin on two bacterial pz peptidases

Pz peptidases A and B, from a thermophile Geobacillus collagenovorans MO-1, recognize collagen-specific tripeptide units (Gly-Pro-Xaa). They share similarities in function but extremely low identities in primary sequence with mammalian thimet oligopeptidase (TOP) and neurolysin. Three phosphine peptide inhibitors that selectively inhibit TOP and neurolysin on two bacterial Pz peptidases were investigated. They showed potent inhibition of both Pz peptidases in a range from 10 to 100 nM.     

Characterization of the binding interface between ubiquitin and class I human ubiquitin-conjugating enzyme 2b by multidimensional heteronuclear NMR spectroscopy in solution.

Ubiquitin-conjugating enzymes (Ubc) are involved in ubiquitination of proteins in the protein degradation pathway of eukaryotic cells. Ubc transfers the ubiquitin (Ub) molecules to target proteins by forming a thioester bond between their active site cysteine residue and the C-terminal glycine residue of ubiquitin. Here, we report on the NMR assignment and secondary structure of class I human ubiquitin-conjugating enzyme 2b (HsUbc2b). Chemical shift perturbation studies allowed us to map the contact area and binding interface between ubiquitin and HsUbc2b by1H-15N HSQC NMR spectroscopy. The serine mutant of the active site Cys88 of HsUbc2b was employed to obtain a relatively stable covalent ubiquitin complex of HsUbc2b(C88S). Changes in chemical shifts of amide protons and nitrogen atoms induced by the formation of the covalent complex were measured by preparing two segmentally labeled complexes with either ubiquitin or HsUbc2b(C88S)15N-labeled. In ubiquitin, the interaction is primarily sensed by the C-terminal segment Val70 - Gly76, and residues Lys48 and Gln49. The surface area on ubiquitin, as defined by these residues, overlaps partially with the presumed binding site with ubiquitin-activating enzyme (E1). In HsUbc2b, most of the affected residues cluster in the vicinity of the active site, namely, around the active site Cys88 itself, the second alpha-helix, and the flexible loop which connects helices alpha2 and alpha3 and which is adjacent to the active site. An additional site on HsUbc2b for a weak interaction with ubiquitin could be detected in a titration study where the two proteins were not covalently linked. This site is located on the backside of HsUbc2b opposite to the active site and is part of the beta-sheet. The covalent and non-covalent interaction sites are clearly separated on the HsUbc2b surface, while no such clear-cut segregation of the interaction area was observed on ubiquitin.     

Identification of the residues in the helix F/G loop important to catalytic function of membrane-bound prostacyclin synthase

A topological model of prostaglandin I(2) synthase (PGIS) was created by homology modeling. This model, along with site-specific antibodies and other topology studies, has suggested that the residue(s) within helix F/G loop of PGIS may be involved in forming the substrate access channel and located in a position that influences the membrane-bound PGIS catalytic function (1). To test this hypothesis, we have explored an approach to identify the residues of the helix F/G loop important to enzyme activity of the membrane-bound PGIS by a combination of 2-D NMR experiment and mutagenesis methods. Using the distance measured from the model as a guide, the helix F/G loop was mimicked in a synthetic peptide by introducing a spacer to maintain a distance of about 7 A between the N- and the C-termini (PGIS residues 208 and 230). The peptide was used to interact with the enzyme substrate analogue, U46619. High-resolution 2-D NMR experiments were performed to determine the contacts between the peptide and U46619. The interaction between the constrained F/G loop peptide and U46619 was confirmed by the observation of the conformational changes of the peptide and U46619 using the comparison of the cross-peaks between the NOESY spectra of U46619 with the peptide, without the peptide, and the peptide alone. Through the combination of the 2-D NMR experiments, completed (1)H NMR assignments of the F/G loop segment in the presence and absence of U46619 were obtained, and these data were used to predict the contact residues (Leu214 and Pro215) of the F/G loop with PGIS substrate. The predicted influence of residues on enzyme catalytic activity in membrane-bound environments was confirmed by the mutagenesis of the F/G loop residues of human PGIS. These observations support that the F/G loop is involved in forming the substrate access channel for membrane-bound PGIS and suggests that the NMR experiment-based mutagenesis approach may be applied to study structure and function relationships for other proteins.     

Ion transport and ligand binding by the Na-K-Cl cotransporter, structure-function studies

The cation-Cl cotransporters (CCCs) mediate the coupled movement of Na and/or K to that of Cl across the plasmalemma of animal cells. Eight CCCs have been identified to date: two Na-K-Cl cotransporters (NKCC), four K-Cl cotransporters (KCCs), one Na-Cl cotransporter (NCC) and one CCC interacting protein (CIP). All of the NKCCs and KCCs are inhibited by loop diuretics; mercury and other modifying agents are also known to block NKCC-mediated transport. In this work, we have utilized a mutational approach to study the interaction between different substrates and the NKCCs. We relied on the strategy of exchanging domains between functionally distinct carriers (the shark NKCCl and the human NKCCl) to identify residues or group of residues that are involved in the interaction with ions, loop diuretics and Hg. Our results show that the N- and C-termini have no role in determining the species differences in ion transport and bumetanide binding. On the other hand, the interaction between Hg and the NKCCs is found to partially involve the C-terminus through residues that contain available sulfhydryl groups. Within the transmembrane segments, variant residues in the 2nd, 4th and 7th predicted alpha-helices are shown to encode the differences in ion transport between the shark and the human cotransporters. For loop diuretic binding, several regions throughout the central domain appear to be involved. Interestingly, these regions are not the same as those involved in cation or anion transport, and in Hg binding.