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1.
The desmin-specific calpain I from chicken gizzard smooth muscle is a dimer of 83 and 35 kDalton subunits. A monoclonal antibody to the large subunit did not cross-react with chicken gizzard and hamster skeletal muscle calpain II, but it did recognize hamster skeletal muscle desmin-specific calpain I and the denatured calpain II from chicken gizzard smooth muscle. These results indicate that different desmin-specific calpains have similar large subunits which differ significantly from the large subunit of calpain II in the same tissue.  相似文献   

2.
Mutations in the KCNQ2 gene cause myokymia and neonatal epilepsy, indicating that this K(+) channel regulates the excitability of lower motoneurons and CNS neurons. Little is known about the parameters that direct the assembly of this multimeric molecule and other KCNQ subunits. Here, we show that the carboxy-terminal subunit interaction domain of KCNQ2 autonomously folds and assembles into tetramers. This domain contains a bipartite coiled-coil motif. Whereas structural integrity of the second coiled-coil motif is crucial for tetramer formation, that of the first motif is less important. These data suggest a crucial role of coiled-coil motifs in tetrameric KCNQ channel assembly.  相似文献   

3.
PDE4 inhibitors have been identified as therapeutic targets for a variety of conditions, particularly inflammatory diseases. We have serendipitously identified a novel class of phosphodiesterase 4 (PDE4) inhibitor during a study to discover antagonists of the parathyroid hormone receptor. X-ray crystallographic studies of PDE4D2 complexed to four potent inhibitors reveal the atomic details of how they inhibit the enzyme and a notable contrast to another recently reported thiophene-based inhibitor.  相似文献   

4.
5.
The calpains are a family of cysteine proteases with closely related amino acid sequences, but a wide range of Ca(2+) requirements (K(d)). For m-calpain, K(d) is approximately 325microM, for mu-calpain it is approximately 50microM, and for calpain 3 it is not strictly known but may be approximately 0.1microM. On the basis of previous structure determination of m-calpain we postulated that two regions of the calpain large subunits, the N-terminal peptide (residues 1-20) and a domain III-IV linker peptide (residues 514-530 in m-calpain) were important in defining K(d). The mutations Lys10Thr in the N-terminal peptide, and Glu517Pro in the domain linker peptide, reduced K(d) of m-calpain by 30% and 42%, respectively, revealing that these two regions are functionally important. The increased Ca(2+)-sensitivity of these mutants demonstrate that the Lys10-Asp148 salt link and the short beta-sheet interaction involving Glu517 are factors contributing to the high K(d) of m-calpain. Though these two regions are physically remote from the active site and Ca(2+)-binding site, they play significant roles in regulating the response of calpain to Ca(2+). Differences in these interactions in mu-calpain and in calpain 3 are also consistent with their progressively lower K(d) values.  相似文献   

6.
Calpains are involved in a variety of calcium-regulated cellular processes, such as signal transduction, cell proliferation, differentiation, and apoptosis. Excessive calpain activation contributes to serious cellular damage and has been reported in many pathological conditions. 4-Quinolinone 2-carboxamide derivatives were prepared and evaluated for mu-calpain inhibitory activities. Of the compounds synthesized, 3a and 3k, which possess a primary amide and 4-methoxyphenethyl amide at P1' region, were found to most potently inhibit mu-calpain with IC50 values of 0.71+/-0.07 and 0.73+/-0.23 microM, respectively. On the other hand, the incorporation of pyridine-containing amides decreased inhibitory activity.  相似文献   

7.
HIV-1 integrase (IN) is a validated therapeutic target for antiviral drug design. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands the discovery of novel inhibitors that are structurally as well mechanistically different. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC(50)=5 μM) with more than 40-fold selectivity for the strand transfer over the 3'-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC(50) value of 8 μM. Using molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. Furthermore, the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site on IN. This work provides a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors.  相似文献   

8.
We structurally characterized the adducts of the catalytic domain of matrix metalloproteinase-3 (MMP3) with three different nonpeptidic inhibitors by solving the solution structure of one adduct [MMP3–N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid] and then by calculating structural models of the other two adducts using a reduced set of experimental NMR data, following a recently proposed procedure (Bertini et al. in J. Med. Chem. 48:7544–7559, 2005). The inhibitors were selected with the criteria of maintaining in all of them the same zinc-coordinating moiety and of selectively changing the substituents and/or the functional groups. The backbone dynamics on various time scales have been characterized as well. The comparison among these structures and with others previously reported allowed us to elucidate fine details of inhibitor–receptor interactions and to develop some criteria, which could guide in optimizing the design of selective inhibitors. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

9.
The inactivation of the catalytic subunit from rabbit muscle cAMP-dependent protein kinase by the chloromethyl ketones from lysine and phenylalanine (TLCK and TPCK; A. Kupfer et al. (1979) Proc. Natl. Acad. Sci. USA 76, 3073) has been confirmed for the same enzyme from rat muscle. However, other structurally not related protease inhibitors, antipain and leupeptin, did not inhibit the catalytic subunit from rat muscle. Thus it seems to be critical to attribute the interference of protease inhibitors with complex biological phenomena like tumorigenesis etc. generally to the inhibition of protein kinases.  相似文献   

10.
Class IA phosphoinositide 3-kinases (PI3Ks) represent a group of heterodimeric lipid kinases with important functions in cellular signal transduction. The regulatory p85 subunit constitutively binds to the catalytic p110 subunit and mediates the recruitment of the heterodimer to various membrane-localized proteins upon activation by a vast array of stimuli. The functional characterization of protein domains that mediate p85 function has been hampered by a lack of structural data. Therefore, we investigated a 35-aa region in the inter-SH2 domain of p85, reported to be necessary for binding of p110, by site-directed mutagenesis and evaluated the importance of individual amino acids for PI3K heterodimer formation. This approach led to the identification of an 11-aa region required for p110 binding in vitro and mesoderm induction during early Xenopus development in vivo. Further analyses revealed two pairs of hydrophobic amino acids within this region, which are particularly important for high-affinity intersubunit interaction. Thus, our data provide further insight into the molecular mechanisms of PI3K intersubunit interaction and led to the identification of new p85 mutant proteins with varying degrees of dominant-negative effects that will be helpful for future PI3K-related research.  相似文献   

11.
The effects on mitochondrial respiration and complex I NADH oxidase activity of cubebin and derivatives were evaluated. The compounds inhibited the state 3 glutamate/malate-supported respiration of hamster liver mitochondria with IC50 values ranging from 12.16 to 83.96 μM. NADH oxidase reaction was evaluated in submitochondrial particles. The compounds also inhibited this activity, showing the same order of potency observed for effects on state 3 respiration, as well as a tendency towards a non-competitive type of inhibition (KI values ranging from 0.62 to 16.1 μM). A potential binding mode of these compounds with complex I subunit B8, assessed by docking calculations, is proposed.  相似文献   

12.
The effects on mitochondrial respiration and complex I NADH oxidase activity of cubebin and derivatives were evaluated. The compounds inhibited the state 3 glutamate/malate-supported respiration of hamster liver mitochondria with IC(50) values ranging from 12.16 to 83.96 microM. NADH oxidase reaction was evaluated in submitochondrial particles. The compounds also inhibited this activity, showing the same order of potency observed for effects on state 3 respiration, as well as a tendency towards a non-competitive type of inhibition (K(I) values ranging from 0.62 to 16.1 microM). A potential binding mode of these compounds with complex I subunit B8, assessed by docking calculations, is proposed.  相似文献   

13.
It was previously shown that the catalytic subunit of the plant toxin viscumin induces aggregation of small unilamellar liposomes and this process is inhibited by the mab_TA7 monoclonal antibody produced to the denatured catalytic subunit of viscumin (Agapov, I.I. et al., FEBS Lett., 1999, vol. 464, pp. 63-66). The interaction of the synthetic F101-T105 and A96-T105 fragments of the viscumin catalytic subunit with the mab_TA7 monoclonal antibody was studied by 1H NMR spectroscopy. The results of this study demonstrated that only the A96-T105 fragment is capable of binding to mab_TA7. A nuclear Overhauser effect observed in the antigen-antibody complex and registered on the resonances of the free peptide and exchanging between the free state and the antibody-bound state was analyzed; the mab_TA7 antigen determinant (H99-T105) was identified; and its conformation and orientation within the complex with the antibody were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.  相似文献   

14.
Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca(2+) ions, of the (alphabetagammadelta)(4) phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory alpha and catalytic gamma subunits. The gamma subunit is also known to interact with the delta subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca(2+) ions. In this study, we have used two-hybrid screening and chemical cross-linking to dissect the regulatory quaternary interactions involving these subunits. The yeast two-hybrid system indicated that regions near the C termini of the gamma (residues 343-386) and alpha (residues 1060-1237) subunits interact. The association of this region of alpha with gamma was corroborated by the isolation of a cross-linked fragment of alpha containing residues 1015-1237 from an alpha-gamma dimer that had been formed within the PhK holoenzyme by formaldehyde, a nearly zero-length cross-linker. Because the region of gamma that we found to interact with alpha has previously been shown to contain a high affinity binding site for calmodulin (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156-17163), we tested the influence of Ca(2+) on the conformation of the alpha subunit and found that the region of alpha that interacts with gamma was, in fact, perturbed by Ca(2+). The results herein support the existence of a Ca(2+)-sensitive communication network among the delta, gamma, and alpha subunits, with the regulatory domain of gamma being the primary mediator. The similarity of such a Ca(2+)-dependent network to the interactions among troponin C, troponin I, and actin is discussed in light of the known structural and functional similarities between troponin I and the gamma subunit of PhK.  相似文献   

15.
S Di Bernardo  T Yagi 《FEBS letters》2001,508(3):385-388
When Paracoccus denitrificans membranes were treated with a crosslinker, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), a cross-linked product of M(r) approximately 31 kDa was found which reacted with antibodies against the hydrophobic subunit Nqo7 and the connector subunit Nqo6. NaI treatment of the Paracoccus membranes before, but not after, the crosslinking step prevented the formation of the 31 kDa band. When Nqo7 and Nqo6 were coexpressed in Escherichia coli, both subunits were located in the membrane fraction. MBS treatment of the E. coli membranes generated the 31 kDa band as in the Paracoccus membranes. These results indicate that Nqo7 interacts with probable N2-binding Nqo6.  相似文献   

16.
Winger JA  Marletta MA 《Biochemistry》2005,44(10):4083-4090
The catalytic domains (alpha(cat) and beta(cat)) of alpha1beta1 soluble guanylate cyclase (sGC) were expressed in Escherichia coli and purified to homogeneity. alpha(cat), beta(cat), and the alpha(cat)beta(cat) heterodimeric complex were characterized by analytical gel filtration and circular dichroism spectroscopy, and activity was assessed in the absence and presence of two different N-terminal regulatory heme-binding domain constructs. Alpha(cat) and beta(cat) were inactive separately, but together the domains exhibited guanylate cyclase activity. Analysis by gel filtration chromatography demonstrated that each of the approximately 25-kDa domains form homodimers. Heterodimers were formed when alpha(cat) and beta(cat) were combined. Results from circular dichroism spectroscopy indicated that no major structural changes occur upon heterodimer formation. Like the full-length enzyme, the alpha(cat)beta(cat) complex was more active in the presence of Mn(2+) as compared to the physiological cofactor Mg(2+), although the magnitude of the difference was much larger for the catalytic domains than for the full-length enzyme. The K(M) for Mn(2+)-GTP was measured to be 85 +/- 18 microM, and in the presence of Mn(2+)-GTP, the K(D) for the alpha(cat)beta(cat) complex was 450 +/- 70 nM. The N-terminal heme-bound regulatory domain of the beta1 subunit of sGC inhibited the activity of the alpha(cat)beta(cat) complex in trans, suggesting a domain-scale mechanism of regulation by NO. A model in which binding of NO to sGC causes relief of an autoinhibitory interaction between the regulatory heme-binding domain and the catalytic domains of sGC is proposed.  相似文献   

17.
The interaction between the inhibitor protein and the catalytic subunit of the cAMP-dependent protein kinase has been investigated by steady state kinetics and by an assessment of the requirement of this interaction for ATP. By analysis for tightly bound inhibitors, inhibition by the inhibitor protein was shown to be competitive versus peptide substrate and uncompetitive versus Mg X ATP2-. This, together with the observations of Gronot et al. (Gronot, J., Mildvan, A.S., Bramson, H. N., Thomas, N., and Kaiser, E.T. (1981) Biochemistry 20, 602-610) and those given in the accompanying paper (Whitehouse, S., Feramisco, J.R., Casnellie, J.E., Krebs, E.G., and Walsh, D.A. (1983) J. Biol. Chem. 258, 3693-3701), would indicate that the probable reaction mechanism of the protein kinase is ordered with the nucleotide binding first and that the inhibitor protein blocks catalysis by interaction with the catalytic subunit-Mg X ATP complex. The Ki for this interaction at saturating Mg X ATP and zero peptide substrate is 0.49 nM. Multiple inhibition analysis in the presence of 5'-adenylimidodiphosphate (AMP X PNP) indicates that the inhibitor protein does not interact with a catalytic subunit-AMP X PNP complex. The requirement for ATP for the inhibitor protein-catalytic subunit interaction has also been demonstrated by direct binding measurements and by the observation that the efficiency of the inhibitor protein is increased by preincubation of the inhibitor protein, catalytic subunit, and ATP in the absence of peptide substrate. By either measurement, the catalytic subunit in the presence of the inhibitor protein, was shown to exhibit an apparent Kd of 20 approximately 60 nM for ATP; this value is two orders of magnitude higher than the affinity for ATP by the catalytic subunit alone. This high apparent affinity of the catalytic subunit for ATP (in the presence of the inhibitor) does not require that there be a specific binding site on the inhibitor protein for some moiety of the ATP but may simply be a reflection of the formation of a catalytic subunit-Mg X ATP X inhibitor protein complex with resultant displacement of the equilibrium of ATP binding to the protein kinase.  相似文献   

18.
The stromelysin-1 catalytic domain(83-247) (SCD) is stable for at least 16 h at pHs 6.0-8.4. At pHs 5.0 and 9.0 there is exponential irreversible denaturation with half lives of 38 and 68 min respectively. At pHs 4.5 and 10.0 irreversible denaturation is biphasic. At 25°C, C-terminal truncation of stromelysin-1 decreases the stability of the stromelysin-1 catalytic domain at pH values >8.4 and <6.0. We describe the conversion of the carboxylate group of (βR)-β-[[[(1S)-1-[[[(1S)-2-Methoxy-1-phenylethyl]amino]carbonyl]-2,2-dimethylpropyl]amino]carbonyl]-2-methyl-[1,1'-biphenyl]-4-hexanoic acid (UK-370106-COOH) a potent inhibitor of the metalloprotease stromelysin-1 to a glyoxal group (UK-370106-CO(13)CHO). At pH 5.5-6.5 the glyoxal inhibitor is a potent inhibitor of stromelysin-1 (K(i)=~1μM). The aldehyde carbon of the glyoxal inhibitor was enriched with carbon-13 and using carbon-13 NMR we show that the glyoxal aldehyde carbon is fully hydrated when it is in aqueous solutions (90.4ppm) and also when it is bound to SCD (~92.0ppm). We conclude that the hemiacetal hydroxyl groups of the glyoxal inhibitor are not ionised when the glyoxal inhibitor is bound to SCD. The free enzyme pK(a) values associated with inhibitor binding were 5.9 and 6.2. The formation and breakdown of the signal at ~92ppm due to the bound UK-370106-CO(13)CHO inhibitor depends on pK(a) values of 5.8 and 7.8 respectively. No strong hydrogen bonds are present in free SCD or in SCD-inhibitor complexes. We conclude that the inhibitor glyoxal group is not directly coordinated to the catalytic zinc atom of SCD.  相似文献   

19.
We have determined 2.2 Å resolution crystal structure of Thermotoga maritima CheB methylesterase domain to provide insight into the interaction mode between CheB and chemoreceptors. T. maritima CheB methylesterase domain has identical topology of a modified doubly-wound α/β fold that was observed from the previously reported Salmonella typhimurium counterpart, but the analysis of the electrostatic potential surface near the catalytic triad indicated considerable charge distribution difference. As the CheB demethylation consensus sites of the chemoreceptors, the CheB substrate, are not uniquely conserved between T. maritima and S. typhimurium, such surfaces with differing electrostatic properties may reflect CheB regions that mediate protein–protein interaction. Via the computational docking of the two T. maritima and S. typhimurium CheB structures to the respective T. maritima and Escherichia coli chemoreceptors, we propose a CheB:chemoreceptor interaction mode.  相似文献   

20.
Liu W  Linn S 《Nucleic acids research》2000,28(21):4180-4188
Human DNA polymerase epsilon (pol ) normally contains a 261-kDa catalytic subunit (p261), but from some sources it is isolated as a 140-kDa catalytic core of p261. This shortened form possesses normal or somewhat enhanced polymerase activity and its significance is unknown. We report here that caspase-3 and calpain can form p140 from p261 in vitro and in vivo and that during early stages of apoptosis induced in Jurkat cells by staurosporine or anti-Fas-activating antibody, p261 is cleaved into p140 by caspase-3. At later stages, activated calpain might also contribute to this conversion. The sites of cleavage by caspase-3 have been identified, and mutations at these ‘DEAD boxes’ resulted in cleavage-resistant enzyme. Cleavage at these sites separates the ‘N-terminal catalytic core’ from the ‘C-terminal’ regions described for p261. Cleavage does not occur during necrosis or following exposure to H2O2 or methanesulfonic acid methyl ester. p140 is unlikely to be able to functionally replace p261 in vivo, since it does not bind to PCNA or the other pol subunits.  相似文献   

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