共查询到20条相似文献,搜索用时 15 毫秒
1.
Dunlop MH Dray E Zhao W Tsai MS Wiese C Schild D Sung P 《The Journal of biological chemistry》2011,286(43):37328-37334
Homologous recombination (HR) reactions mediated by the RAD51 recombinase are essential for DNA and replication fork repair, genome stability, and tumor suppression. RAD51-associated protein 1 (RAD51AP1) is an important HR factor that associates with and stimulates the recombinase activity of RAD51. We have recently shown that RAD51AP1 also partners with the meiotic recombinase DMC1, displaying isoform-specific interactions with DMC1. Here, we have characterized the DMC1 interaction site in RAD51AP1 by a series of truncations and point mutations to uncover a highly conserved WVPP motif critical for DMC1 interaction but dispensable for RAD51 association. This RAD51AP1 motif is reminiscent of the FVPP motif in the tumor suppressor protein BRCA2 that mediates DMC1 interaction. These results further implicate RAD51AP1 in meiotic HR via RAD51 and DMC1. 相似文献
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A conserved motif in the hexosyltransferases 总被引:1,自引:1,他引:0
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Ohyama K Yamano Y Sano T Nakagomi Y Wada M Inagami T 《Biochemical and biophysical research communications》2002,292(2):362-367
To delineate the functional importance of the highly conserved triplet amino acid sequence, Asp-Arg-Tyr (DRY) among G protein-coupled receptors in the second intracellular loop, these residues of rat angiotensin II (Ang II) receptor type 1A (AT(1A)) were changed by alanine or glycine by site-directed mutagenesis. These mutant receptors were stably expressed in CHO-K1 cells, and the binding of Ang II, GTP effect, InsP(3) production, and the acidification of the medium in response to Ang II were determined. The effects of GTPgammaS on Ang II binding in the mutant receptors D125A and D125G were markedly reduced. InsP(3) production of the mutant D125A, D125G, R126A, and R126G was markedly reduced. Extracellular acidification of D125A was not distinguishable from untransfected CHO-K1 cells. Mutant Y127A was able to produce InsP(3) and acidify medium comparable with wild type AT(1A). These results indicate as follows; Asp(125) is essential for intracellular signal transduction involving G protein coupling, Arg(126) is essential for coupling of G(q) protein but not other G proteins, and Tyr(127) is not important for G protein coupling. 相似文献
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PilT is a hexameric ATPase required for type IV pilus retraction in gram-negative bacteria. Retraction of type IV pili mediates intimate attachment to and signaling in host cells, surface motility, biofilm formation, natural transformation, and phage sensitivity. We investigated the in vivo and in vitro roles of each amino acid of the distinct, highly conserved C-terminal AIRNLIRE motif in PilT. Substitution of amino acids A288, I289, L292, and I293 as well as a double substitution of R290 and R294 abolished Pseudomonas aeruginosa PilT function in vivo, as measured by a loss of surface motility and phage sensitivity. When introduced into purified Aquifex aeolicus PilT, substitutions in the AIRNLIRE motif did not disrupt ATPase activity or oligomerization. In contrast, a K136Q substitution in the broadly conserved nucleotide binding motif prevented PilT function in vivo as well as in vitro. We propose that the AIRNLIRE motif forms an amphipathic alpha helix which transmits signals between a surface-exposed protein interaction site and the ATPase core of PilT, and we recognize a potential functional homology in other type II secretion ATPases. 相似文献
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Begley GS Furie BC Czerwiec E Taylor KL Furie GL Bronstein L Stenflo J Furie B 《The Journal of biological chemistry》2000,275(46):36245-36249
The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the posttranslational conversion of glutamic acid to gamma-carboxyglutamic acid, an amino acid critical to the function of the vitamin K-dependent blood coagulation proteins. Given the functional similarity of mammalian vitamin K-dependent carboxylases and the vitamin K-dependent carboxylase from Conus textile, a marine invertebrate, we hypothesized that structurally conserved regions would identify sequences critical to this common functionality. Furthermore, we examined the diversity of animal species that maintain vitamin K-dependent carboxylation to generate gamma-carboxyglutamic acid. We have cloned carboxylase homologs in full-length or partial form from the beluga whale (Delphinapterus leucas), toadfish (Opsanus tau), chicken (Gallus gallus), hagfish (Myxine glutinosa), horseshoe crab (Limulus polyphemus), and cone snail (Conus textile) to compare these structures to the known bovine, human, rat, and mouse cDNA sequences. Comparison of the predicted amino acid sequences identified a nearly perfectly conserved 38-amino acid residue region in all of these putative carboxylases. In addition, this amino acid motif is also present in the Drosophila genome and identified a Drosophila homolog of the gamma-carboxylase. Assay of hagfish liver demonstrated vitamin K-dependent carboxylase activity in this hemichordate. These results demonstrate the broad distribution of the vitamin K-dependent carboxylase gene, including a highly conserved motif that is likely critical for enzyme function. The vitamin K-dependent biosynthesis of gamma-carboxyglutamic acid appears to be a highly conserved function in the animal kingdom. 相似文献
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Human epidermal keratinocytes are one of the few cell types that express the beta1B splice variant of the beta1 integrin subunit. Although in transfection experiments beta1B acts as a dominant negative inhibitor of cell adhesion, we found that beta1B was expressed at very low levels in keratinocytes, both in vivo and in culture, and had a predominantly cytoplasmic distribution, concentrated within the endoplasmic reticulum. To examine why beta1B accumulated in the cytoplasm, we prepared chimeras between CD8alpha and the beta1A and beta1B integrin cytoplasmic domains. In transfected HeLa cells, both constructs reached the cell surface but the rate of maturation of the beta1B chimera was considerably retarded relative to beta1A. The beta1B cytoplasmic domain contains two lysine residues that resemble the double lysine motif characteristic of many proteins that are resident within the endoplasmic reticulum. Mutation of each lysine individually to serine had no effect on CD8beta1B maturation, but when both residues were mutated the rate of CD8beta1B maturation increased to that of CD8beta1A. Further analysis of beta1B function in keratinocytes must, therefore, take into account the low abundance of the isoform relative to beta1A and the potential for beta1B to accumulate in the endoplasmic reticulum. 相似文献
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Ding ZC Chen D Ni FY Zheng Q Cai B Yao WH Wang Y Zhou GM Huang ZX 《Biochemical and biophysical research communications》2008,372(4):779-784
It has been reported that the 6CPCP9 motif near the N-terminus is pivotal to the inhibitory activity of human neuronal growth inhibitory factor (hGIF). In order to better understand the biological significance of this region on the structure, property and function of hGIF, we introduced a highly flexible residue, Gly, either in front of the 6CPCP9 motif (the IG6 mutant, TGCPCP) or in the middle of it (the IG8 mutant, TCPGCP) and investigated their structural and metal binding properties in detail. The results showed that the overall structure and the stability of the metal-thiolate clusters of the two mutants were comparable to that of hGIF. However, the bioassay results showed that the bioactivity of the IG6 mutant decreased significantly, while the bioactivity of the IG8 mutant was almost abolished. Molecular dynamics simulation results showed that the backbone of the IG6 mutant exhibited high similarity to that of hGIF, and the two prolines could still induce structural constraints on the 6CPCP9 tetrapeptide and form a similar conformation with that of hGIF, however, the conformation of the first five amino acid residues in the N-terminus was quite different. In hGIF, the five residues are twisted and form a restricted conformation, while in the IG6 mutant this peptide extends more naturally and smoothly, which is similar to that of MT2. As to the IG8 mutant, the Gly insertion broke the 6CPCP9 motif, thus probably abolishing the interactions with other molecules and eliminating its inhibitory activity. Based on these results, we suggested that although the structure adopted by the 6CPCP9 motif is the determinant factor of the inhibitory bioactivity of hGIF, other residues within the N-terminal fragment (residue 1-13) may also influence the peptide conformation and contribute to the protein’s bioactivity. 相似文献
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Site-specific mutagenesis of conserved residues within Walker A and B sequences of Escherichia coli UvrA protein. 总被引:1,自引:0,他引:1
UvrA is the ATPase subunit of the DNA repair enzyme (A)BC excinuclease. The amino acid sequence of this protein has revealed, in addition to two zinc fingers, three pairs of nucleotide binding motifs each consisting of a Walker A and B sequence. We have conducted site-specific mutagenesis, ATPase kinetic analyses, and nucleotide binding equilibrium measurements to correlate these sequence motifs with activity. Replacement of the invariant Lys by Ala in the putative A sequences indicated that K37 and K646 but not K353 are involved in ATP hydrolysis. In contrast, substitution of the invariant Asp by Asn in the B sequences at positions D238, D513, or D857 had little effect on the in vivo activity of the protein. Nucleotide binding studies revealed a stoichiometry of 0.5 ADP/UvrA monomer while kinetic measurements on wild-type and mutant proteins showed that the active form of UvrA is a dimer with 2 catalytic sites which interact in a positive cooperative manner in the presence of ADP; mutagenesis of K37 but not of K646 attenuated this cooperativity. Loss of ATPase activity was about 75% in the K37A, 86% in the K646A mutant, and 95% in the K37A-K646A double mutant. These amino acid substitutions had only a marginal effect on the specific binding of UvrA to damaged DNA but drastically reduced its ability to deliver UvrB to the damage site. We find that the deficient UvrB loading activity of these mutant UvrA proteins results from their inability to associate with UvrB in the form of (UvrA)2(UvrB)1 complexes. We conclude that UvrA forms a dimer with two ATPase domains involving K37 and K646 and that the work performed by ATP hydrolysis is the delivery of UvrB to the damage site on DNA. 相似文献
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Stenberg G Dragani B Cocco R Mannervik B Aceto A 《The Journal of biological chemistry》2000,275(14):10421-10428
The specific (i, i+5) hydrophobic staple interaction involving a helix residue and a second residue located in the turn preceding the helix is a recurrent motif at the N terminus of alpha-helices. This motif is strictly conserved in the core of all soluble glutathione transferases (GSTs) as well as in other protein structures. Human GSTP1-1 variants mutated in amino acid Ile(149) and Tyr(154) of the hydrophobic staple motif of the alpha6-helix were analyzed. In particular, a double mutant cycle analysis has been performed to evaluate the role of the hydrophobic staple motif in the refolding process. The results show that this local interaction, by restricting the number of conformations of the alpha6-helix relative to the alpha1-helix, favors the formation of essential interdomain interactions and thereby accelerates the folding process. Thus, for the first time it is shown that the hydrophobic staple interaction has a role in the folding process of an intact protein. In P(i) class GSTs, Tyr(154) appears to be of particular structural importance, since it interacts with conserved residues Leu(21), Asp(24), and Gln(25) of the adjacent alpha1-helix which contributes to the active site. Human GSTP1-1 variants L21A and Y154F have also been analyzed in order to distinguish the role of interdomain interactions from that of the hydrophobic staple. The experimental results reported here suggest that the strict conservation of the hydrophobic staple motif reflects an evolutionary pressure for proteins to fold rapidly. 相似文献
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Zetina CR 《Proteins》2001,44(4):479-483
Among the naturally unfolded proteins there are many polypeptides that retain an extended conformation in the absence of any apparent signal. Using sequence alignment and secondary structure prediction tools, a conserved (LS/SL)(D/E)(D/E)(D/E)X(E/D) motif is uncovered in the vicinity of the N-terminus of their unfolded helices. A comparison of these data with published observations allows one to propose that the (LS/SL)(D/E)(D/E)(D/E)X(E/D) motif is a helix-unfolding signal. Furthermore, the strong similarity between this motif and the STXXDE casein kinase II phosphorylation site suggests a regulatory mechanism for the naturally unfolded proteins within the cell. 相似文献
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Kinebuchi T Kagawa W Kurumizaka H Yokoyama S 《The Journal of biological chemistry》2005,280(31):28382-28387
The DMC1 protein, a eukaryotic homologue of RecA that shares significant amino acid identity with RAD51, exhibits two oligomeric DNA binding forms, an octameric ring and a helical filament. In the crystal structure of the octameric ring form, the DMC1 N-terminal domain (1-81 amino acid residues) was highly flexible, with multiple conformations. On the other hand, the N-terminal domain of Rad51 makes specific interactions with the neighboring ATPase domain in the helical filament structure. To gain insights into the functional role of the N-terminal domain of DMC1, we prepared a deletion mutant, DMC1-(82-340), that lacks the N-terminal 81 amino acid residues from the human DMC1 protein. Analytical ultracentrifugation experiments revealed that, whereas full-length DMC1 forms a octamer, DMC1-(82-340) is a heptamer. Furthermore, DNA binding experiments showed that DMC1-(82-340) was completely defective in both single-stranded and double-stranded DNA binding activities. Therefore, the N-terminal domain of DMC1 is required for the formation of the octamer, which may support the proper DNA binding activity of the DMC1 protein. 相似文献
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A conserved cleavage-site motif in chloroplast transit peptides 总被引:44,自引:0,他引:44
A collection of 32 stroma-targeting chloroplast transit peptides with known cleavage sites have been analysed in terms of amino acid preferences in the vicinity of the processing site. A loosely conserved consensus motif (Val/Ile)-X-(Ala/Cys) decreases Ala is found in the majority of the transit peptides. About 30% of the sequences have a perfect match to the consensus. When such a match is found, there is a 90% probability that it specifies the correct cleavage site. 相似文献
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Human Muscleblind-like proteins are alternative splicing regulators that are functionally altered in the RNA-mediated disease
myotonic dystrophy. There are different Muscleblind protein isoforms in Drosophila and we previously determined that these have different subcellular localizations in the COS-M6 cell line. Here, we describe
the conservation of the sequence motif KRAEK in isoforms C and E and propose a specific function for this motif. Different
Muscleblind isoforms localize to the peri-plasma membrane (MblA), cytoplasm (MblB), or show no preference for the nuclear
or cytoplasmic compartment (MblC and MblD) in Drosophila S2 cells transiently transfected with Musclebind expression plasmids. Mutation of the KRAEK motif reduces MblC nuclear localization,
whereas fusion of a single KRAEK motif to the heterologous protein β-galactosidase is sufficient to target the reporter protein
to the nucleus of S2 cells. This motif is not exclusive to Muscleblind proteins and is detected in several other protein types.
Taken together, these results suggest that the KRAEK motif regulates nuclear translocation of Muscleblind and may constitute
a new class of nuclear localization signal. 相似文献
20.
Noguchi M Takata T Kimura Y Manno A Murakami K Koike M Ohizumi H Hori S Kakizuka A 《The Journal of biological chemistry》2005,280(50):41332-41341
Valosin-containing protein (p97/VCP) has been proposed as playing crucial roles in a variety of physiological and pathological processes such as cancer and neurodegeneration. We previously showed that VCP(K524A), an ATPase activity-negative VCP mutant, induced vacuolization, accumulation of ubiquitinated proteins, and cell death, phenotypes commonly observed in neurodegenerative disorders. However, any regulatory mechanism of its ATPase activity has not yet been clarified. Here, we show that oxidative stress readily inactivates VCP ATPase activity. With liquid chromatography/tandem mass spectrometry, we found that at least three cysteine residues were modified by oxidative stress. Of them, the 522nd cysteine (Cys-522) was identified as the site responsible for the oxidative inactivation of VCP. VCP(C522T), a single-amino acid substitution mutant from cysteine to threonine, conferred almost complete resistance to the oxidative inactivation. In response to oxidative stress, VCP strengthened the interaction with Npl4 and Ufd1, both of which are essential in endoplasmic reticulum-associated protein degradation. Cys-522 is located in the second ATP binding motif and is highly conserved in multicellular but not unicellular organisms. Cdc48p (yeast VCP) has threonine in the corresponding amino acid, and it showed resistance to the oxidative inactivation in vitro. Furthermore, a yeast mutant (delta cdc48 + cdc48[T532C]) was shown to be susceptible to oxidants-induced growth inhibition and cell death. These results clearly demonstrate that VCP ATPase activity is regulated by the oxidative modification of the Cys-522 residue. This regulatory mechanism may play a key role in the conversion of oxidative stress to endoplasmic reticulum stress response in multicellular organisms and also in the pathological process of various neurodegenerative disorders. 相似文献