PCNA binding through a conserved motif

Proliferating cell nuclear antigen (PCNA) has recently been identified as a target for the binding of several proteins. The cell cycle regulatory protein, p21, and the replication endonuclease, Fen1, have already been described as competing for PCNA binding. Two recent reports have identified DNA (cytosine-5)methyltransferase (MCMT) and the DNA repair endonuclease XPG as binding to PCNA.^1,2 The remarkable thing about these interactions is that they all seem to occur through a conserved motif that is likely to contact the same site on PCNA. This has fascinating implications for a regulatory network linking these diverse protein functions. BioEssays 20 :195–199, 1998. © 1998 John Wiley & Sons, Inc.     

Homotypic FADD interactions through a conserved RXDLL motif are required for death receptor-induced apoptosis

Death receptors in the TNF receptor superfamily signal for apoptosis via the ordered recruitment of FADD and caspase-8 to a death-inducing signaling complex (DISC). However, the nature of the protein-protein interactions in the signaling complex is not well defined. Here we show that FADD self-associates through a conserved RXDLL motif in the death effector domain (DED). Despite exhibiting similar binding to both Fas and caspase-8 and preserved overall secondary structure, FADD RDXLL motif mutants cannot reconstitute FasL- or TRAIL-induced apoptosis and fail to recruit caspase-8 into the DISC of reconstituted FADD-deficient cells. Abolishing self-association can transform FADD into a dominant-negative mutant that interferes with Fas-induced apoptosis and formation of microscopically visible receptor oligomers. These findings suggest that lateral interactions among adapter molecules are required for death receptor apoptosis signaling and implicate self-association into oligomeric assemblies as a key function of death receptor adapter proteins in initiating apoptosis.     

The DHHC domain: A new highly conserved cysteine-rich motif

A unique clone from a human pancreatic cDNA library was isolated and sequenced. Examination of the deduced polypeptide sequence of the clone showed a new form of cysteine-rich domain that included a region with the form of a Cys4 zinc-finger-like metal binding site followed by a complex Cys-His region. Searches of the Swiss-Protein data bank found a similar 48-residue domain in fifteen open reading frames deduced from A. thaliana, C. elegans, S. cerevisiae and S. pombe genomic sequences. The high degree of conservation of this domain (13 absolutely conserved and 17 highly conserved positions) suggests that it has an important function in the cell, possibly related to protein-protein or protein-DNA interactions. The gene recognized by the clone is is localized to human chromosome 16, and is conserved in vertebrates. The 2 Kb message is expressed in various human fetal and adult tissues. An antibody made to a peptide sequence of the deduced protein showed reactivity in immunoblots of monkey lung and retinal subcellular fractions and immunohistochemically in late fetal mouse tissues and a limited number of adult mouse tissues, including pancreatic islets, Leydig cells of the testis, and the plexiform layers of the retina.     

Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif

Hemin (iron protoporphyrin IX) is a necessary component of many proteins, functioning either as a cofactor or an intracellular messenger. Hemoproteins have diverse functions, such as transportation of gases, gas detection, chemical catalysis and electron transfer. Stanniocalcin 1 (STC1) is a protein involved in respiratory responses of the cell but whose mechanism of action is still undetermined. We examined the ability of STC1 to bind hemin in both its reduced and oxidized states and located Cys¹¹⁴ as the axial ligand of the central iron atom of hemin. The amino acid sequence differs from the established (Cys–Pro) heme regulatory motif (HRM) and therefore presents a novel heme binding motif (Cys–Ser). A STC1 peptide containing the heme binding sequence was able to inhibit both spontaneous and H₂O₂ induced decay of hemin. Binding of hemin does not affect the mitochondrial localization of STC1.     

A conserved cleavage-site motif in chloroplast transit peptides

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.     

A conserved helix-unfolding motif in the naturally unfolded proteins

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.     

A conserved motif controls nuclear localization of Drosophila Muscleblind

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.     

HtrA2 cleaves Apollon and induces cell death by IAP-binding motif in Apollon-deficient cells

Apollon/BRUCE is a giant IAP protein that has BIR and UBC domains in its amino- and carboxy-terminals, respectively. Apollon binds and ubiquitylates SMAC/DIABLO and caspase9, and regulates apoptosis by facilitating proteasomal degradation of these proteins. Apollon overexpression inhibits apoptosis, while its downregulation sensitizes cells to apoptosis, suggesting that Apollon level is important for apoptosis regulation. Here we show that HtrA2/Omi catalytically cleaves Apollon with its serine protease activity. Conversely, Apollon ubiquitylates and facilitates proteasomal degradation of HtrA2 that binds to Apollon through IAP-binding motif. Thus, Apollon and HtrA2 mutually downregulate each other. Expression of catalytically active, but not inactive, HtrA2 induced apoptosis in Apollon-expressing cells. In Apollon-deficient cells, however, expression of catalytically inactive HtrA2 mutant with IAP-binding motif also induced apoptosis. These results indicate that HtrA2 induces apoptosis in two different mechanisms, one with serine protease domain and the other with IAP-binding motif, in Apollon-deficient cells.     

A conserved cysteine motif essential for ceramide kinase function

Ceramide kinase (CerK) is a sphingolipid metabolizing enzyme very sensitive to oxidation; however, the determinants are unknown. We show here that the thiol-modifying agent N-ethyl-maleimide abrogates CerK activity in vitro and in a cell based assay, implying that important cysteine residues are accessible in purified as well as endogenous CerK. We replaced every 22 residues in human CerK, by an alanine, and measured activity in the resulting mutant proteins. This led to identification of a cluster of cysteines, C(347)XXXC(351)XXC(354), essential for CerK function. These findings are discussed based on homology modeling of the catalytic domain of CerK.     

Biotin synthase mechanism: mutagenesis of the YNHNLD conserved motif

Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin (DTB). The active form of the enzyme contains two iron-sulfur clusters, a [4Fe-4S](2+) cluster liganded by Cys-53, Cys-57, and Cys-60 and the S-adenosylmethionine (AdoMet or SAM) cosubstrate and a [2Fe-2S](2+) cluster liganded by Cys-97, Cys-128, Cys-188, and Arg-260. Single-point mutation of each of these six conserved cysteines produced inactive variants. In this work, mutants of other highly conserved residues from the Y(150)NHNLD motif are described. They have properties similar to those of the wild-type enzyme with respect to their cluster content and characteristics. For all of them, the as-isolated form, which contains an air-stable [2Fe-2S](2+) center, can additionally accommodate an air-sensitive [4Fe-4S](2+) center which is generated by incubation under anaerobic conditions with Fe(2+) and S(2-). Their spectroscopic properties are similar to those of the wild type. However, they are inactive, except the mutant H152A that exhibits a weak activity. We show that the mutants, inactive in producing biotin, are also unable to cleave AdoMet and to produce the deoxyadenosyl radical (AdoCH(2)(*)). In the case of H152A, a value of 5.5 +/- 0.4 is found for the 5'-deoxyadenosine (AdoCH(3)):biotin ratio, much higher than the value of 2.8 +/- 0.3 usually observed with the wild type. This reveals a greater contribution of the abortive process in which the AdoCH(2)(*) radical is quenched by hydrogen atoms from the protein or from some components of the system. Thus, in this case, the coupling between the production of AdoCH(2)(*) and its reaction with the hydrogen at C-6 and C-9 of DTB is less efficient than that in the wild type, probably because of geometry's perturbation within the active site.     

Spontaneous C-cleavage of a mini-intein without its conserved N-terminal motif A

Previously, the C-terminal fragment of a split intein was known to undergo controllable C-cleavage at its C-terminus only when the N-terminal fragment of the intein was added. Here we constructed a similar split intein from the Ssp DnaX intein, but we unexpectedly observed that its C-terminal 136-aa fragment could undergo spontaneous C-cleavage without the N-terminal fragment that was up to 15 aa long and contained the conserved intein motif A. This C-cleavage activity was significantly decreased by a mutation of the conserved Thr residue in the conserved intein motif B. These findings suggest a robust intein structure in the absence of motif A and a larger role of motif B in the third step of the protein splicing mechanism.     

A conserved acidic motif is crucial for enzymatic activity of protein O-mannosyltransferases

Protein O-mannosylation is an essential modification in fungi and mammals. It is initiated at the endoplasmic reticulum by a conserved family of dolichyl phosphate mannose-dependent protein O-mannosyltransferases (PMTs). PMTs are integral membrane proteins with two hydrophilic loops (loops 1 and 5) facing the endoplasmic reticulum lumen. Formation of dimeric PMT complexes is crucial for mannosyltransferase activity, but the direct cause is not known to date. In bakers' yeast, O-mannosylation is catalyzed largely by heterodimeric Pmt1p-Pmt2p and homodimeric Pmt4p complexes. To further characterize Pmt1p-Pmt2p complexes, we developed a photoaffinity probe based on the artificial mannosyl acceptor substrate Tyr-Ala-Thr-Ala-Val. The photoreactive probe was preferentially cross-linked to Pmt1p, and deletion of the loop 1 (but not loop 5) region abolished this interaction. Analysis of Pmt1p loop 1 mutants revealed that especially Glu-78 is crucial for binding of the photoreactive probe. Glu-78 belongs to an Asp-Glu motif that is highly conserved among PMTs. We further demonstrate that single amino acid substitutions in this motif completely abolish activity of Pmt4p complexes. In contrast, both acidic residues need to be exchanged to eliminate activity of Pmt1p-Pmt2p complexes. On the basis of our data, we propose that the loop 1 regions of dimeric complexes form part of the catalytic site.     

A conserved motif of vertebrate Y RNAs essential for chromosomal DNA replication

Noncoding Y RNAs are required for the reconstitution of chromosomal DNA replication in late G1 phase template nuclei in a human cell-free system. Y RNA genes are present in all vertebrates and in some isolated nonvertebrates, but the conservation of Y RNA function and key determinants for its function are unknown. Here, we identify a determinant of Y RNA function in DNA replication, which is conserved throughout vertebrate evolution. Vertebrate Y RNAs are able to reconstitute chromosomal DNA replication in the human cell-free DNA replication system, but nonvertebrate Y RNAs are not. A conserved nucleotide sequence motif in the double-stranded stem of vertebrate Y RNAs correlates with Y RNA function. A functional screen of human Y1 RNA mutants identified this conserved motif as an essential determinant for reconstituting DNA replication in vitro. Double-stranded RNA oligonucleotides comprising this RNA motif are sufficient to reconstitute DNA replication, but corresponding DNA or random sequence RNA oligonucleotides are not. In intact cells, wild-type hY1 or the conserved RNA duplex can rescue an inhibition of DNA replication after RNA interference against hY3 RNA. Therefore, we have identified a new RNA motif that is conserved in vertebrate Y RNA evolution, and essential and sufficient for Y RNA function in human chromosomal DNA replication.