共查询到20条相似文献,搜索用时 93 毫秒
1.
Almerinda Di Venere Thomas Horn Sabine Stehling Giampiero Mei Laura Masgrau Àngels González-Lafont Hartmut Kühn Igor Ivanov 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2013,1831(6):1079-1088
12/15-Lipoxygenases (12/15-LOX) have been implicated in inflammatory and hyperproliferative diseases but the numerous aspects of structural biology of these enzymes are far from clear. Early mutagenesis data and structural modeling of enzyme–substrate complexes suggested that Arg403, which is localized at the entrance of the putative substrate binding pocket, might interact with the fatty acid carboxylic group. On the other hand, side-chain of Arg403 is a part of an ionic network with the residues of α2-helix, which undergoes pronounced conformation changes upon inhibitor binding. To explore the role of Arg403 for catalysis in more detail we exchanged positively charged Arg403 to neutral Leu and quantified structural and functional consequences of the alteration at the site of mutation using fluorometric techniques. We found that a loss of electrostatic interaction between Arg403 and negatively charged amino acid residues of α2-helix has only minor impact on protein folding, but partially destabilized the tertiary structure of the enzyme. We hypothesize that interaction of Arg403 with the substrate's carboxylate might be involved in a complex mechanism triggering conformational changes of the α2-helix, which are required for formation of the catalytically competent dimer r12/15-LOX complex at pre-catalytic stages. 相似文献
2.
Kim KW Wang Z Busby J Tsuruda T Chen M Hale C Castro VM Svensson S Nybo R Xiong F Wang M 《Biochimica et biophysica acta》2006,1764(4):824-830
The catalytic motif (YSASK) at the active site of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is conserved across different species. The crystal structures of the human, guinea pig and mouse enzymes have been resolved to help identify the non-conserved residues at the active site. A tyrosine residue (Y177) upstream of the catalytic motif in human 11beta-HSD1 represents the largest difference at the active sites between the human and the rodent enzyme where the corresponding residue is glutamine. Although Y177 was postulated as a potential hydrogen bond donor in substrate binding in crystal structure-based modeling, no experimental evidence is available to support this notion. Here, we report that Y177 is not a hydrogen bond donor in substrate binding because removal of the hydroxyl group from its side chain by mutagenesis (Y177F) did not significantly change the Km value for cortisone. However, removal of the hydrophobic side chain by changing tyrosine to alanine (Y177A) or substitution with a hydrophilic side chain by changing tyrosine to glutamine (Y177Q) increased Km values for cortisone. These data suggest that Y177 is involved in substrate binding through its hydrophobic side chain but not by hydrogen bonding. In addition, the three mutations had little effect on the binding of the rodent substrate 11-dehydrocorticosterone, suggesting that Y177 does not confer substrate specificity. However, the same mutations reduced the affinity of the licorice derived 11beta-HSD1 inhibitor glycyrrhetinic acid by about 6- to 10-fold. Interestingly, the affinity of carbenoxolone, the hemisuccinate ester of glycyrrhetinic acid with a similar potency against the wildtype enzyme, was not drastically affected by the same mutations at Y177. These data suggest that Y177 has a unique role in inhibitor binding. Molecular modeling with glycyrrhetinic acid led to findings consistent with the experimental data and provided potential interaction mechanisms. Our data suggest that Y177 plays an important role in both substrate and inhibitor binding but it is unlikely a hydrogen bond donor for the substrate. 相似文献
3.
Andújar-Sánchez M Smith AW Clemente-Jimenez JM Rodriguez-Vico F Las Heras-Vazquez FJ Jara-Pérez V Cámara-Artigas A 《Biochemistry》2005,44(4):1174-1183
Glutathione S-transferases are a family of multifunctional enzymes involved in the metabolism of drugs and xenobiotics. Two tyrosine residues, Tyr 7 and Tyr 111, in the active site of the enzyme play an important role in the binding and catalysis of substrate ligands. The crystal structures of Schistosoma japonicum glutathione S-transferase tyrosine 7 to phenylalanine mutant [SjGST(Y7F)] in complex with the substrate glutathione (GSH) and the competitive inhibitor S-octylglutathione (S-octyl-GSH) have been obtained. These new structural data combined with fluorescence spectroscopy and thermodynamic data, obtained by means of isothermal titration calorimetry, allow for detailed characterization of the ligand-binding process. The binding of S-octyl-GSH to SjGST(Y7F) is enthalpically and entropically driven at temperatures below 30 degrees C. The stoichiometry of the binding is one molecule of S-octyl-GSH per mutant dimer, whereas shorter alkyl derivatives bind with a stoichiometry of two molecules per mutant dimer. The SjGST(Y7F).GSH structure showed no major structural differences compared to the wild-type enzyme. In contrast, the structure of SjGST(Y7F).S-octyl-GSH showed asymmetric binding of S-octyl-GSH. This lack of symmetry is reflected in the lower symmetry space group of the SjGST(Y7F).S-octyl-GSH crystals (P6(3)) compared to that of the SjGST(Y7F).GSH crystals (P6(3)22). Moreover, the binding of S-octyl-GSH to the A subunit is accompanied by conformational changes that may be responsible for the lack of binding to the B subunit. 相似文献
4.
de Lemos Esteves F Ruelle V Lamotte-Brasseur J Quinting B Frère JM 《Protein science : a publication of the Protein Society》2004,13(5):1209-1218
Xyl1 from Streptomyces sp. S38 belongs to the low molecular mass family 11 of endo-beta-1,4-xylanases. Its three-dimensional structure has been solved at 2.0 A and its optimum temperature and pH for enzymatic activity are 60 degrees C and 6.0, respectively. Aspergillus kawachii xylanase XynC belongs to the same family but is an acidophilic enzyme with an optimum pH of 2.0. Structural comparison of Xyl1 and XynC showed differences in residues surrounding the two glutamic acid side chains involved in the catalysis that could be responsible for the acidophilic adaptation of XynC. Mutations W20Y, N48D, A134E, and Y193W were introduced by site-directed mutagenesis and combined in multiple mutants. Trp 20 and Tyr 193 are involved in substrate binding. The Y193W mutation inactivated Xyl1 whereas W20Y decreased the optimum pH of Xyl1 to 5.0 and slightly increased its specific activity. The N48D mutation also decreased the optimum pH of Xyl1 by one unit. The A134E substitution did not induce any change, but when combined with N48D, a synergistic effect was observed with a 1.4 unit decrease in the optimum pH. Modeling showed that the orientations of residue 193 and of the fully conserved Arg 131 are different in acidophilic and "alkaline" xylanases whereas the introduced Tyr 20 probably modifies the pKa of the acid-base catalyst via residue Asn 48. Docking of a substrate analog in the catalytic site highlighted striking differences between Xyl1 and XynC in substrate binding. Hydrophobicity calculations showed a correlation between acidophilic adaptation and a decreased hydrophobicity around the two glutamic acid side chains involved in catalysis. 相似文献
5.
Blamey CJ Ceccarelli C Naik UP Bahnson BJ 《Protein science : a publication of the Protein Society》2005,14(5):1214-1221
Calcium- and integrin-binding protein 1 (CIB1) is involved in the process of platelet aggregation by binding the cytoplasmic tail of the alpha(IIb) subunit of the platelet-specific integrin alpha(Iib)beta(3). Although poorly understood, it is widely believed that CIB1 acts as a global signaling regulator because it is expressed in many tissues that do not express integrin alpha(Iib)beta(3). We report the structure of human CIB1 to a resolution of 2.3 A, crystallized as a dimer. The dimer interface includes an extensive hydrophobic patch in a crystal form with 80% solvent content. Although the dimer form of CIB1 may not be physiologically relevant, this intersub-unit surface is likely to be linked to alpha(IIb) binding and to the binding of other signaling partner proteins. The C-terminal domain of CIB1 is structurally similar to other EF-hand proteins such as calmodulin and calcineurin B. Despite structural homology to the C-terminal domain, the N-terminal domain of CIB1 lacks calcium-binding sites. The structure of CIB1 revealed a complex with a molecule of glutathione in the reduced state bond to the N-terminal domain of one of the two subunits poised to interact with the free thiol of C35. Glutathione bound in this fashion suggests CIB1 may be redox regulated. Next to the bound GSH, the orientation of residues C35, H31, and S48 is suggestive of a cysteine-type protein phosphatase active site. The potential enzymatic activity of CIB1 is discussed and suggests a mechanism by which it regulates a wide variety of proteins in cells in addition to platelets. 相似文献
6.
Galit Lazer Liron Pe'er Marganit Farago Kazuya Machida Bruce J. Mayer Shulamit Katzav 《The Journal of biological chemistry》2010,285(30):23075-23085
The guanine nucleotide exchange factor (GEF) Vav1 is an essential signal transducer protein in the hematopoietic system, where it is expressed physiologically. It is also involved in several human malignancies. Tyrosine phosphorylation at the Vav1 amino terminus plays a central role in regulating its activity; however, the role of carboxyl terminal tyrosine residues is unknown. We found that mutation of either Tyr-826 (Y826F) or Tyr-841 (Y841F) to phenylalanine led to loss of Vav1 GEF activity. When these Vav1 mutants were ectopically expressed in pancreatic cancer cells lacking Vav1, they failed to induce growth in agar, indicating loss of transforming potential. Furthermore, although Y841F had no effect on Vav1-stimulated nuclear factor of activated T cells (NFAT) activity, Y826F doubled NFAT activity when compared with Vav1, suggesting that Tyr-826 mediates an autoinhibitory effect on NFAT activity. SH2 profiling revealed that Shc, Csk, Abl, and Sap associate with Tyr-826, whereas SH2-B, Src, Brk, GTPase-activating protein, and phospholipase C-γ associate with Tyr-841. Although the mutations in the Tyr-826 and Tyr-841 did not affect the binding of the carboxyl SH3 of Vav1 to other proteins, binding to several of the proteins identified by the SH2 profiling was lost. Of interest is Csk, which associates with wild-type Vav1 and Y841F, yet it fails to associate with Y826F, suggesting that loss of binding between Y826F and Csk might relieve an autoinhibitory effect, leading to increased NFAT. Our data indicate that GEF activity is critical for the function of Vav1 as a transforming protein but not for NFAT stimulation. The association of Vav1 with other proteins, detected by SH2 profiling, might affect other Vav1-dependent activities, such as NFAT stimulation. 相似文献
7.
Assembly of interferon-β enhanceosome from its individual protein components and of enhancer DNA has been studied in solution using a combination of fluorescence anisotropy, microcalorimetry, and CD titration. It was shown that the enhancer binds only one full-length phosphomimetic IRF-3 dimer at the PRDIII-PRDI sites, and this binding does not exhibit cooperativity with binding of the ATF-2/c-Jun bZIP (leucine zipper dimer with basic DNA recognition segments) heterodimer at the PRDIV site. The orientation of the bZIP pair is, therefore, not determined by the presence of the IRF-3 dimer, but is predetermined by the asymmetry of the PRDIV site. In contrast, bound IRF-3 dimer interacts strongly with the NF-κB (p50/p65) heterodimer bound at the neighboring PRDII site. The orientation of bound NF-κB is also predetermined by the asymmetry of the PRDII site and is the opposite of that found in the crystal structure. The HMG-I/Y protein, proposed as orchestrating enhanceosome assembly, interacts specifically with the PRDII site of the interferon-β enhancer by inserting its DNA-binding segments (AT hooks) into the minor groove, resulting in a significant increase in NF-κB binding affinity for the major groove of this site. 相似文献
8.
Insulin-degrading enzyme (IDE) exists primarily as a dimer being unique among the zinc metalloproteases in that it exhibits allosteric kinetics with small synthetic peptide substrates. In addition the IDE reaction rate is increased by small peptides that bind to a distal site within the substrate binding site. We have generated mixed dimers of IDE in which one or both subunits contain mutations that affect activity. The mutation Y609F in the distal part of the substrate binding site of the active subunit blocks allosteric activation regardless of the activity of the other subunit. This effect shows that substrate or small peptide activation occurs through a cis effect. A mixed dimer composed of one wild-type subunit and the other subunit containing a mutation that neither permits substrate binding nor catalysis (H112Q) exhibits the same turnover number per active subunit as wild-type IDE. In contrast, a mixed dimer in which one subunit contains the wild-type sequence and the other contains a mutation that permits substrate binding, but not catalysis (E111F), exhibits a decrease in turnover number. This indicates a negative trans effect of substrate binding at the active site. On the other hand, activation in trans is observed with extended substrates that occupy both the active and distal sites. Comparison of the binding of an amyloid β peptide analog to wild-type IDE and to the Y609F mutant showed no difference in affinity, indicating that Y609 does not play a significant role in substrate binding at the distal site. 相似文献
9.
Sphingomonas sp. A1 alginate lyase A1-II′, a member of polysaccharide lyase family 7, shows a broad substrate specificity acting on poly α-L-guluronate (poly(G)), poly β-D-mannuronate (poly(M)) and the heteropolymer (poly(MG)) in alginate molecules. A1-II′ with a glove-like β-sandwich as a basic scaffold forms a cleft covered with two lid loops (L1 and L2). Here, we demonstrate the loop flexibility for substrate binding and structural determinants for broad substrate recognition and catalytic reaction. The two loops associate mutually over the cleft through the formation of a hydrogen bond between their edges (Asn141 and Asn199). A double mutant, A1-II′ N141C/N199C, has a disulfide bond between Cys141 and Cys199, and shows little enzyme activity. Adding dithiothreitol to the enzyme reaction mixture leads to a tenfold increase in its molecular activity, suggesting the significance of flexibility in lid loops for accommodating the substrate into the active cleft. In alginate trisaccharide (GGG or MMG)-bound A1-II′ Y284F, the enzyme interacts appropriately with substrate hydroxyl groups at subsites + 1 and + 2 and accommodates G or M, while substrate carboxyl groups are strictly recognized by specific residues. This mechanism for substrate recognition enables A1-II′ to show the broad substrate specificity. The structure of A1-II′ H191N/Y284F complexed with a tetrasaccharide bound at subsites − 1 to + 3 suggests that Gln189 functions as a neutralizer for the substrate carboxyl group, His191 as a general base, and Tyr284 as a general acid. This is, to our knowledge, the first report on the structure and function relationship in family 7. 相似文献
10.
Nakaishi A Hirose M Yoshimura M Oneyama C Saito K Kuki N Matsuda M Honma N Ohnishi H Matozaki T Okada M Nakagawa A 《Journal of molecular biology》2008,375(3):650-660
SRC homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1 or SIRPα/BIT) is an immunoglobulin (Ig) superfamily transmembrane receptor and a member of the signal regulatory protein (SIRP) family involved in cell-cell interaction. SHPS-1 binds to its ligand CD47 to relay an inhibitory signal for cellular responses, whereas SIRPβ, an activating member of the same family, does not bind to CD47 despite sharing a highly homologous ligand-binding domain with SHPS-1. To address the molecular basis for specific CD47 recognition by SHPS-1, we present the crystal structure of the ligand-binding domain of murine SHPS-1 (mSHPS-1). Folding topology revealed that mSHPS-1 adopts an I2-set Ig fold, but its overall structure resembles IgV domains of antigen receptors, although it has an extended loop structure (C′E loop), which forms a dimer interface in the crystal. Site-directed mutagenesis studies of mSHPS-1 identified critical residues for CD47 binding including sites in the C′E loop and regions corresponding to complementarity-determining regions of antigen receptors. The structural and functional features of mSHPS-1 are consistent with the human SHPS-1 structure except that human SHPS-1 has an additional β-strand D. These results suggest that the variable complementarity-determining region-like loop structures in the binding surface of SHPS-1 are generally required for ligand recognition in a manner similar to that of antigen receptors, which may explain the diverse ligand-binding specificities of SIRP family receptors. 相似文献
11.
Aitio O Hellman M Kesti T Kleino I Samuilova O Pääkkönen K Tossavainen H Saksela K Permi P 《Journal of molecular biology》2008,382(1):167-178
We have determined the solution structure of epidermal growth factor receptor pathway substrate 8 (Eps8) L1 Src homology 3 (SH3) domain in complex with the PPVPNPDYEPIR peptide from the CD3ε cytoplasmic tail. Our structure reveals the distinct structural features that account for the unusual specificity of the Eps8 family SH3 domains for ligands containing a PxxDY motif instead of canonical PxxP ligands. The CD3ε peptide binds Eps8L1 SH3 in a class II orientation, but neither adopts a polyproline II helical conformation nor engages the first proline-binding pocket of the SH3 ligand binding interface. Ile531 of Eps8L1 SH3, instead of Tyr or Phe residues typically found in this position in SH3 domains, renders this hydrophobic pocket smaller and nonoptimal for binding to conventional PxxP peptides. A positively charged arginine at position 512 in the n-Src loop of Eps8L1 SH3 plays a key role in PxxDY motif recognition by forming a salt bridge to D7 of the CD3ε peptide. In addition, our structural model suggests a hydrogen bond between the hydroxyl group of the aromatic ring of Y8 and the carboxyl group of E496, thus explaining the critical role of the PxxDY motif tyrosine residue in binding to Eps8 family SH3. These finding have direct implications also for understanding the atypical binding specificity of the amino-terminal SH3 of the Nck family proteins. 相似文献
12.
Koskiniemi H Metsä-Ketelä M Dobritzsch D Kallio P Korhonen H Mäntsälä P Schneider G Niemi J 《Journal of molecular biology》2007,372(3):633-648
Angucyclines are aromatic polyketides produced in Streptomycetes via complex enzymatic biosynthetic pathways. PgaE and CabE from S. sp PGA64 and S. sp. H021 are two related homo-dimeric FAD and NADPH dependent aromatic hydroxylases involved in the early steps of the angucycline core modification. Here we report the three-dimensional structures of these two enzymes determined by X-ray crystallography using multiple anomalous diffraction and molecular replacement, respectively, to resolutions of 1.8 A and 2.7 A. The enzyme subunits are built up of three domains, a FAD binding domain, a domain involved in substrate binding and a C-terminal thioredoxin-like domain of unknown function. The structure analysis identifies PgaE and CabE as members of the para-hydroxybenzoate hydroxylase (pHBH) fold family of aromatic hydroxylases. In contrast to phenol hydroxylase and 3-hydroxybenzoate hydroxylase that utilize the C-terminal domain for dimer formation, this domain is not part of the subunit-subunit interface in PgaE and CabE. Instead, dimer assembly occurs through interactions of their FAD binding domains. FAD is bound non-covalently in the "in"-conformation. The active sites in the two enzymes differ significantly from those of other aromatic hydroxylases. The volumes of the active site are significantly larger, as expected in view of the voluminous tetracyclic angucycline substrates. The structures further suggest that substrate binding and catalysis may involve dynamic rearrangements of the middle domain relative to the other two domains. Site-directed mutagenesis studies of putative catalytic groups in the active site of PgaE argue against enzyme-catalyzed substrate deprotonation as a step in catalysis. This is in contrast to pHBH, where deprotonation/protonation of the substrate has been suggested as an essential part of the enzymatic mechanism. 相似文献
13.
CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin alpha5beta1 总被引:3,自引:0,他引:3
Hoshijima M Hattori T Inoue M Araki D Hanagata H Miyauchi A Takigawa M 《FEBS letters》2006,580(5):1376-1382
Searching for CCN family protein 2/connective tissue growth factor (CCN2/CTGF) interactive proteins by yeast-two-hybrid screening, we identified fibronectin 1 gene product as a major binding partner of CCN2/CTGF in the chondrosarcoma-derived chondrocytic cell line HCS-2/8. Only the CT domain of CCN2/CTGF bound directly to fibronectin (FN). CCN2/CTGF and its CT domain enhanced the adhesion of HCS-2/8 cells to FN in a dose-dependent manner. The CCN2/CTGF-enhancing effect on cell adhesion to FN was abolished by a blocking antibody against alpha5beta1 integrin (alpha5beta1), but not by one against anti-alphavbeta3 integrin. These findings suggest for the first time that CCN2/CTGF enhances chondrocyte adhesion to FN through direct interaction of its C-terminal CT domain with FN, and that alpha5beta1 is involved in this adhesion. 相似文献
14.
The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 A resolution structure of Kaposi's sarcoma-associated herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 A2 of surface are buried in the newly formed S4 pocket when tyrosine binds at this site. The peptide inhibitor also induces a rearrangement of residues that stabilizes the oxyanion hole and the dimer interface. Concomitant with the structural changes, an increase in catalytic efficiency of the enzyme results upon extended substrate binding. A nearly 20-fold increase in kcat/KM results upon extending the peptide substrate from a tetrapeptide to a hexapeptide exclusively due to a KM effect. This suggests that the mechanism by which herpesvirus proteases achieve their high specificity is by using extended substrates to modulate both the structure and activity of the enzyme. 相似文献
15.
Sendovski M Kanteev M Ben-Yosef VS Adir N Fishman A 《Journal of molecular biology》2011,405(1):227-237
Tyrosinase is a member of the type 3 copper enzyme family that is involved in the production of melanin in a wide range of organisms. The crystal structures of a tyrosinase from Bacillus megaterium were determined at a resolution of 2.0-2.3 Å. The enzyme crystallized as a dimer in the asymmetric unit and was shown to be active in crystal. The overall monomeric structure is similar to that of the monomer of the previously determined tyrosinase from Streptomyces castaneoglobisporus, but it does not contain an accessory Cu-binding “caddie” protein. Two Cu(II) ions, serving as the major cofactors within the active site, are coordinated by six conserved histidine residues. However, determination of structures under different conditions shows varying occupancies and positions of the copper ions. This apparent mobility in copper binding modes indicates that there is a pathway by which copper is accumulated or lost by the enzyme. Additionally, we suggest that residues R209 and V218, situated in a second shell of residues surrounding the active site, play a role in substrate binding orientation based on their flexibility and position. The determination of a structure with the inhibitor kojic acid, the first tyrosinase structure with a bound ligand, revealed additional residues involved in the positioning of substrates in the active site. Comparison of wild-type structures with the structure of the site-specific variant R209H, which possesses a higher monophenolase/diphenolase activity ratio, lends further support to a previously suggested mechanism by which monophenolic substrates dock mainly to CuA. 相似文献
16.
Coliphage T4 endonuclease II (EndoII), encoded by gene denA, is a small (16 kDa, 136 aa) enzyme belonging to the GIY-YIG family of endonucleases, which lacks a C-terminal domain corresponding to that providing most of the binding energy in the structurally characterized GIY-YIG endonucleases, I-TevI and UvrC. In vivo, it is involved in degradation of host DNA, permitting scavenging of host-derived nucleotides for phage DNA synthesis. EndoII primarily catalyzes single-stranded nicking of DNA; 5- to 10-fold less frequently double-stranded breaks are produced. The Glu118Ala mutant of EndoII was crystallized in space group P21 with four monomers in the asymmetric unit. The fold of the EndoII monomer is similar to that of the catalytic domains of UvrC and I-TevI. In contrast to these enzymes, EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain not present in UvrC or I-TevI providing most of the dimerization and tetramerization interfaces. A bound phosphate ion in one of the four active sites of EndoII likely mimics the scissile phosphate in a true substrate complex. In silico docking experiments showed that a protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. The positioning of these sites within the EndoII primary dimer suggests that the substrate would bind to a primary EndoII dimer diagonally over the active sites, requiring significant distortion of the enzyme or the substrate DNA, or both, for simultaneous nicking of both DNA strands. The scarcity of potential nucleic acid binding residues between the active sites indicates that EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks. Mutations analyzed in earlier functional studies are discussed in their structural context. 相似文献
17.
The hydrogen bonding of polar side-chains has emerged as an important theme for membrane protein interactions. The crystal structure of the dimeric state of the transmembrane beta-barrel protein outer membrane phospholipase A (OMPLA) revealed an intermolecular hydrogen bond mediated by a highly conserved glutamine side-chain (Q94). It has been shown that the introduction of a polar residue can drive the association of model helices, and by extension it was presumed that the glutamine hydrogen bond played a key role in stabilizing the OMPLA dimer. However, a thermodynamic investigation using sedimentation equilibrium ultracentrifugation in detergent micelles reveals that the hydrogen bond plays only a very modest role in stabilizing the dimer. The Q94 side-chain is hydrogen bonded intramolecularly to residues Y92 and S96, but amino acid substitutions at these positions suggest these intramolecular interactions are not responsible for attenuating the strength of the intermolecular Q94 hydrogen bond. Other substitutions suggested that hydration of the local environment around Q94 may be responsible for the modest strength of the hydrogen bond. Heat inactivation experiments with the variants suggest that the Y92-Q94-S96 network may instead be important for thermal stability of the monomer. These results highlight the context dependence and broad range of interactions that can be mediated by polar residues in membrane proteins. 相似文献
18.
Andrew J. Annalora David B. Goodin Qinghai Zhang C. David Stout 《Journal of molecular biology》2010,396(2):441-451
Cytochrome P450 (CYP) 24A1 catalyzes the side-chain oxidation of the hormonal form of vitamin D. Expression of CYP24A1 is up-regulated to attenuate vitamin D signaling associated with calcium homeostasis and cellular growth processes. The development of therapeutics for disorders linked to vitamin D insufficiency would be greatly facilitated by structural knowledge of CYP24A1. Here, we report the crystal structure of rat CYP24A1 at 2.5 Å resolution. The structure exhibits an open cleft leading to the active-site heme prosthetic group on the distal surface that is likely to define the path of substrate access into the active site. The entrance to the cleft is flanked by conserved hydrophobic residues on helices A′ and G′, suggesting a mode of insertion into the inner mitochondrial membrane. A docking model for 1α,25-dihydroxyvitamin D3 binding in the open form of CYP24A1 that clarifies the structural determinants of secosteroid recognition and validates the predictive power of existing homology models of CYP24A1 is proposed. Analysis of CYP24A1's proximal surface identifies the determinants of adrenodoxin recognition as a constellation of conserved residues from helices K, K″, and L that converge with an adjacent lysine-rich loop for binding the redox protein. Overall, the CYP24A1 structure provides the first template for understanding membrane insertion, substrate binding, and redox partner interaction in mitochondrial P450s. 相似文献
19.
Phosphofructokinase-1 and -2 (Pfk-1 and Pfk-2, respectively) from Escherichia coli belong to different homologous superfamilies. However, in spite of the lack of a common ancestor, they share the ability to catalyze the same reaction and are inhibited by the substrate MgATP. Pfk-2, an ATP-dependent 6-phosphofructokinase member of the ribokinase-like superfamily, is a homodimer of 66 kDa subunits whose oligomerization state is necessary for catalysis and stability. The presence of MgATP favors the tetrameric form of the enzyme. In this work, we describe the structure of Pfk-2 in its inhibited tetrameric form, with each subunit bound to two ATP molecules and two Mg ions. The present structure indicates that substrate inhibition occurs due to the sequential binding of two MgATP molecules per subunit, the first at the usual site occupied by the nucleotide in homologous enzymes and the second at the allosteric site, making a number of direct and Mg-mediated interactions with the first. Two configurations are observed for the second MgATP, one of which involves interactions with Tyr23 from the adjacent subunit in the dimer and the other making an unusual non-Watson-Crick base pairing with the adenine in the substrate ATP. The oligomeric state observed in the crystal is tetrameric, and some of the structural elements involved in the binding of the substrate and allosteric ATPs are also participating in the dimer-dimer interface. This structure also provides the grounds to compare analogous features of the nonhomologous phosphofructokinases from E. coli. 相似文献
20.
In an attempt to clarify the protective effect of puerarin on toxin-insulted dopaminergic neuronal death, this present study was carried out by using a typical Parkinson's disease (PD) model - 1-methyl-4-phenylpyridinium iodide (MPP(+))-induced dopaminergic SH-SY5Y cellular model. Data are presented, which showed that puerarin up-regulated Akt phosphorylation in both of MPP(+)-treated and non-MPP(+)-treated cells. The presence of PI3K inhibitor LY294002 completely blocked puerarin-induced activation of Akt phosphorylation. Moreover, puerarin decreased MPP(+)-induced cell death, which was blocked by phosphoinositide 3-kinase (PI3K) inhibitor LY294002. We further demonstrated that puerarin protected against MPP(+)-induced p53 nuclear accumulation, Puma (p53-upregulated mediator of apoptosis) and Bax expression and caspase-3-dependent programmed cell death (PCD). This protection was blocked by applying a PI3K/Akt inhibitor. Additionally, it was Pifithrin-α, but not Pifithrin-μ, which blocked MPP(+)-induced Puma and Bax expression, caspase-3 activation and cell death. Collectively, these data suggest that the activation of PI3K/Akt pathway is involved in the protective effect of puerarin against MPP(+)-induced neuroblastoma SH-SY5Y cell death through inhibiting nuclear p53 accumulation and subsequently caspase-3-dependent PCD. Puerarin might be a potential therapeutic agent for PD. 相似文献