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PubMed2001

Structure of the C-terminal sterile alpha-motif (SAM) domain of human p73 alpha.

p73 is a homologue of the tumour suppressor p53 and contains all three functional domains of p53. The alpha-splice variant of p73 (p73 alpha) contains near its C-terminus an additional structural domain known as the sterile alpha-motif (SAM) that is probably responsible for regulating p53-like functions of p73. Here, the 2.54 A resolution crystal structure of this protein domain is reported. The crystal structure and the published solution structure have the same five-helix bundle fold that is characteristic of all SAM-domain structures, with an overall r.m.s.d. of 1.5 A for main-chain atoms. The hydrophobic core residues are well conserved, yet some large local differences are observed. The crystal structure reveals a dimeric organization, with the interface residues forming a mini four-helix bundle. However, analysis of solvation free energies and the surface area buried upon dimer formation indicated that this arrangement is more likely to be an effect of crystal packing rather than reflecting a physiological state. This is consistent with the solution structure being a monomer. The p73 alpha SAM domain also contains several interesting structural features: a Cys-X-X-Cys motif, a 3(10)-helix and a loop that have elevated B factors, and short tight inter-helical loops including two beta-turns; these elements are probably important in the normal function of this domain.

Amino Acid Motifs↗
PubMed1995

CHUK, a conserved helix-loop-helix ubiquitous kinase, maps to human chromosome 10 and mouse chromosome 19.

Helix-loop-helix proteins contain stretches of DNA that encode two amphipathic alpha-helices joined by a loop structure and are involved in protein dimerization and transcriptional regulation essential to a variety of cellular processes. CHUK, a newly described conserved helix-loop-helix ubiquitous kinase, was mapped by somatic cell hybrid analyses to human Chr 10q24-q25. Chuk and a related sequence, Chuk-rs1, were mapped to mouse chromosomes 19 and 16, respectively, by a combination of somatic cell hybrid, recombinant inbred, and backcross analyses.

Animals↗
PubMed1992

Strong electrostatic loop-helix interactions in bundle motif protein structures.

Based on CHARMM potential (Brooks et al., 1983) an energetic analysis has been carried out for four typical 4-alpha-helix bundle proteins, i.e., methemerythrin, cytochrome b-562, cytochrome c', and bovine somatotropin. The bovine somatotropin possesses long loops, but all the other three proteins have short loops. It was found that in all these four 4-alpha-helix bundle motif structures the interaction between loops and helices was much stronger than the interaction among the four helices themselves. Particularly for the electrostatic interaction energy, the loop-helix interaction is overwhelmingly stronger than the interhelix interaction although the latter involves the favorable helix dipole interaction due to the antiparallel arrangement of neighboring alpha-helices. The present study indicates that such a conclusion holds true regardless of what loops, long or short, are in the 4-alpha-helix bundle protein, and also regardless of which empirical potential, ECEPP or CHARMM, is used for calculations although in CHARMM the electrostatic energy is much more heavily emphasized than in ECEPP. Therefore, no appropriate conclusion can be drawn in arguing whether the dipole interaction among the four alpha-helices play a stabilizing role or destabilizing role for a 4-alpha-helix bundle protein without taking into consideration the effect of interaction between helices and loops. The calculated results reported here provide, from a different point of view, insights that might be useful for revealing the essence of the driving forces during the folding of proteins.

Animals↗
PubMed2005

The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding.

ATP-binding cassette (ABC) transporters represent one of the largest families of proteins, and transport a variety of substrates ranging from ions to amphipathic anticancer drugs. The functional unit of an ABC transporter is comprised of two transmembrane domains and two cytoplasmic ABC ATPase domains. The energy of the binding and hydrolysis of ATP is used to transport the substrates across membranes. An ABC domain consists of conserved regions, the Walker A and B motifs, the signature (or C) region and the D, H and Q loops. We recently described the A-loop (Aromatic residue interacting with the Adenine ring of ATP), a highly conserved aromatic residue approximately 25 amino acids upstream of the Walker A motif that is essential for ATP-binding. Here, we review the mutational analysis of this subdomain in human P-glycoprotein as well as homology modeling, structural and data mining studies that provide evidence for a functional role of the A-loop in ATP-binding in most members of the superfamily of ABC transporters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

USF2/FIP associates with the b-Zip transcription factor, c-Maf, via its bHLH domain and inhibits c-Maf DNA binding activity.

In screening for proteins that interact with the basic zipper (bZip) transcription factor, c-Maf, we isolated USF2/FIP. USF2 is a member of the bHLH-Zip protein family, possessing a basic (b) DNA binding region, a helix-loop-helix (HLH) motif, and a leucine zipper (Zip) structure. Mutants of USF2 that lacked a Zip formed heterodimers with c-Maf, but did not homodimerize. Deletion of the USF2 basic region or mutation of its helices abrogated its binding to c-Maf, but had no effect on homodimerization. A functional c-Maf bZip motif was necessary for both homodimerization and heterodimerization with USF2. These data suggest a tetrameric configuration for Maf-USF2 complexes. In the presence of USF2, the DNA binding activity of c-Maf was markedly reduced. Therefore, USF2 and c-Maf may interact to regulate gene expression.

Basic-Leucine Zipper Transcription Factors↗
PubMed2003

An alternate pathway for recruiting template RNA to the brome mosaic virus RNA replication complex.

The multidomain RNA replication protein 1a of brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, plays key roles in assembly and function of the viral RNA replication complex. 1a, which encodes RNA capping and helicase-like domains, localizes to endoplasmic reticulum membranes, recruits BMV 2a polymerase and viral RNA templates, and forms membrane-bound, capsid-like spherules in which RNA replication occurs. cis-acting signals necessary and sufficient for RNA recruitment by 1a have been mapped in BMV genomic RNA2 and RNA3. Both signals comprise an extended stem-loop whose apex matches the conserved sequence and structure of the TPsiC stem-loop in tRNAs (box B). Mutations show that this box B motif is crucial to 1a responsiveness of wild-type RNA2 and RNA3. We report here that, unexpectedly, some chimeric mRNAs expressing the 2a polymerase open reading frame from RNA2 were recruited by 1a to the replication complex and served as templates for negative-strand RNA synthesis, despite lacking the normally essential, box B-containing 5' signal. Further studies showed that this template recruitment required high-efficiency translation of the RNA templates. Moreover, multiple small frameshifting insertion or deletion mutations throughout the N-terminal region of the open reading frame inhibited this template recruitment, while an in-frame insertion did not. Providing 2a in trans did not restore template recruitment of RNAs with frameshift mutations. Only those deletions in the N-terminal region of 2a that abolished 2a interaction with 1a abolished template recruitment of the RNA. These and other results indicate that this alternate pathway for 1a-dependent RNA recruitment involves 1a interaction with the translating mRNA via the 1a-interactive N-terminal region of the nascent 2a polypeptide. Interaction with nascent 2a also may be involved in 1a recruitment of 2a polymerase to membranes.

Base Sequence↗
PubMed2005

Analysis of HIV-1 subtype B third variable region peptide motifs for induction of neutralizing antibodies against HIV-1 primary isolates.

The HIV-1 gp120 V3 loop is a potent inducer of neutralizing antibodies for T cell line adapted-HIV-1, but less so for primary isolates. We hypothesized that peptides representative of the diversity of natural HIV-1 V3 loop variants might capture elements of conserved higher order structures and so stimulate broadly reactive neutralizing antibodies. We designed a panel of 29 subtype B V3 sequences postulated to reflect the range of V3 diversity. These peptides were used to immunize guinea pigs. The most effective peptide (62.19) clustered around the subtype B consensus sequence and induced antibodies that reproducibly neutralized 31% of the subtype B HIV-1 primary isolates evaluated, but exhibited limited cross-neutralization of non-subtype B HIV-1 strains. Taken together, these data demonstrated that the limited neutralization profile of antibodies induced by optimal subtype B V3 motifs likely represents the maximum breadth of neutralization of subtype B HIV-1 primary isolates attainable by anti-V3 peptide antibodies.

Amino Acid Motifs↗
PubMed1994

V3 region polymorphisms in HIV-1 from Brazil: prevalence of subtype B strains divergent from North American/European prototype and detection of subtype F.

Viral DNA sequences were determined over the V3 region of env from 28 infected individuals living in the high HIV-1 prevalence Brazilian cities of Rio de Janeiro and São Paulo. Twenty-six belonged to envelope sequence subtype B, prevalent in North America and Europe, and one was classified as subtype F, found recently in Brazil and in Romania (one appeared to be a B/F recombinant). Octameric sequences at the tip of the subtype B V3 loops were variable and distinct from those prevalent in North America and Europe. The GPGR motif, prevalent in North American/European strains, was found in only 8 (28.5%) sequences, whereas GWGR was found in 12 (43%) and novel sequences in 8 (28.5%). Brazilian subtype B sequences also diverged from the consensus North American/European strains over the remainder of the V3 loop. These results suggest that Brazilian HIV-1 B strains may have important antigenic differences from prototype subtype B strains currently being evaluated for use in HIV vaccines. These results should be taken into account for future vaccine programs in Brazil.

AIDS Vaccines↗
PubMed1998

Molecular epidemiology of HIV-1 in Brazil: high prevalence of HIV-1 subtype B and identification of an HIV-1 subtype D infection in the city of Rio de Janeiro, Brazil. Evandro Chagas Hospital AIDS Clinical Research Group.

HIV-1-positive individuals were recruited from January 1993 to December 1996 from several cohorts receiving follow-up in the city of Rio de Janeiro, Brazil, to evaluate HIV-1 genetic variability and the potential association with modes of transmission. HIV-1 subtyping was carried out using the heteroduplex mobility assay (HMA), and those samples corresponding to the typical Brazilian subtype B variant were further identified based on the Fok I restriction fragment length polymorphism (RFLP). DNA sequencing was performed to evaluate one case of subtype D infection. From the 131 HIV-1-positive individuals analyzed, 106 (80.9%) could be identified as infected by subtype B and 20 (15.3%) by subtype F. One of the samples (0.8%) was classified as subtype D. DNA samples from 4 patients (3.0%) did not yield polymerase chain reaction (PCR)-amplified products to be typed. Based on the Fok I RFLP, 39 of the 106 subtype B samples (37%) were identified as corresponding to the typical Brazilian subtype B variant containing the GWGR motif at the tip of the V3 loop. No statistically significant association could be detected between HIV-I subtypes and modes of transmission, exposure categories, or gender. This is the first reported case of HIV-1 subtype D infection in Brazil.

Amino Acid Sequence↗
PubMed2002

Expression of transcription factors in the brain of rats with perinatal asphyxia.

No information is available on transcription factors (TF), the main regulators of gene expression, in perinatal asphyxia (PA), and as pathomechanisms in PA are different, data on TFs from ischemia or hypoxia cannot be simply extrapolated to PA, and no studies have been reported to show an expressional pattern or the concerted action of TFs. We, therefore, used a gene-hunting technique, subtractive hybridization, to show sequences different in brains of normoxic and perinatally asphyxiated (10 and 20 min of asphyxia) rats. These subtracted sequences were identified by gene bank and assigned to individual genes. At 10 min of PA the TFs NFI/CAAT-binding protein, NF-kappa-B p65, N-myc, basic helix loop helix protein D82868, and c-myc intron binding protein were upregulated. At 20 min of PA the TFs SOX4 and neuronal death factor were upregulated, whereas the TFs c-maf, PEBP major transcription factor, brn-2, homeodomain protein Af004431, and zinc finger transcriptional factor M65008 were downregulated. The biological meaning of our findings is the demonstration of a pathophysiological pattern of TFs including POU, zinc finger, homeodomain, and basic helix-loop helix motifs in PA, proposing pathomechanisms for brain damage from PA, explaining transcriptional changes in general (as, e.g., NF-kappa-B p65, etc.) or in specific terms (as, e.g., neuronal death factor).

Animals↗
PubMed1999

Mad1 expression in the absence of differentiation: effect of cAMP on the B-lymphoid cell line Reh.

The protein Mad1 heterodimerizes with Max to form an E-box binding complex able to interfere with the transcriptional and transforming activities of c-Myc. Downregulation of c-Myc accompanied by induction of Mad1 upon differentiation has fueled the notion that Mad1 may play a role in the cessation of proliferation associated with the differentiation process. Since studies on Mad1 expression have so far been limited to cells undergoing differentiation, it was of interest to examine Mad1 expression in a cell system unable to differentiate. To do so, we utilized the leukemia-derived B-precursor cell line, Reh, and studied the expressions of Mad1, c-Myc, Mxil, and Max during cAMP-mediated growth inhibition of these cells. Thus, the adenylate cyclase activator forskolin induced growth inhibition of the cells in the G1 phase of the cell cycle. This growth inhibition was associated with transient increased expression of Mad1 concomitant with transient downregulation of c-Myc. The Mad1 protein levels essentially paralleled those of mRNA, with peak levels at 4 h of forskolin treatment. By coimmunoprecipitation we detected increased binding of Mad1 to Max in forskolin-treated cells, indicating that the changes in Mad1 protein levels had functional implications. By continually treating Reh cells with forskolin for 72 h, we observed a sustained elevated expression of Mad1 concomitant with downregulated c-Myc expression, still without changing the differentiation profile of the Reh cells. Interestingly, we showed that other known cell cycle regulatory proteins also were transiently regulated by forskolin. To this extent, following forskolin treatment of Reh cells, cyclin E-cdk2 activity was transiently reduced concomitant with dephosphorylation of pRB. We suggest that the early changes in Mad1 and the cell cycle regulatory proteins initiate a chain of events resulting in permanent growth arrest. Thus, the increased expression of Mad1 in the absence of differentiation indicates that Mad1 expression in Reh cells is linked to growth arrest per se.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1995

CHUK, a new member of the helix-loop-helix and leucine zipper families of interacting proteins, contains a serine-threonine kinase catalytic domain.

We have identified a new member of the helix-loop-helix (H-L-H) and leucine zipper gene families via a reverse transcriptase-polymerase chain reaction based strategy. This new gene, CHUK (conserved helix-loop-helix ubiquitous kinase), may represent the founding member of a new class of interacting chimeric proteins. The nucleotide sequence of a near full-length murine CHUK cDNA clone revealed an encoded polypeptide specifying: a carboxyl-terminal H-L-H domain, an amino terminal serine-threonine kinase catalytic domain, and a leucine zipper-like amphipathic alpha-helix juxtaposed in between the H-L-H and kinase domains. CHUK is highly conserved in evolution and ubiquitously expressed in diverse types of established cell lines, whereas it is differentially expressed in normal murine tissues. The structural features of the CHUK polypeptide suggest that its putative kinase activity may be targetted to H-L-H and/or leucine zipper transcription factors. Alternatively, the dual amphipathic a helices may serve to control its intrinsic kinase activity by interactions with other cellular factors. CHUK may provide new insights into the regulated transmission of cytoplasmic signals to specific nuclear factors manifesting rapid alterations in patterns of cellular gene expression.

Amino Acid Sequence↗
PubMed1994

cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer.

The crystal structure of ternary and binary substrate complexes of the catalytic subunit of cAMP-dependent protein kinase has been refined at 2.2 and 2.25 A resolution, respectively. The ternary complex contains ADP and a 20-residue substrate peptide, whereas the binary complex contains the phosphorylated substrate peptide. These 2 structures were refined to crystallographic R-factors of 17.5 and 18.1%, respectively. In the ternary complex, the hydroxyl oxygen OG of the serine at the P-site is 2.7 A from the OD1 atom of Asp 166. This is the first crystallographic evidence showing the direct interaction of this invariant carboxylate with a peptide substrate, and supports the predicted role of Asp 166 as a catalytic base and as an agent to position the serine -OH for nucleophilic attack. A comparison of the substrate and inhibitor ternary complexes places the hydroxyl oxygen of the serine 2.7 A from the gamma-phosphate of ATP and supports a direct in-line mechanism for phosphotransfer. In the binary complex, the phosphate on the Ser interacts directly with the epsilon N of Lys 168, another conserved residue. In the ternary complex containing ATP and the inhibitor peptide, Lys 168 interacts electrostatically with the gamma-phosphate of ATP (Zheng J, Knighton DR, Ten Eyck LF, Karlsson R, Xuong NH, Taylor SS, Sowadski JM, 1993, Biochemistry 32:2154-2161). Thus, Lys 168 remains closely associated with the phosphate in both complexes. A comparison of this binary complex structure with the recently solved structure of the ternary complex containing ATP and inhibitor peptide also reveals that the phosphate atom traverses a distance of about 1.5 A following nucleophilic attack by serine and transfer to the peptide. No major conformational changes of active site residues are seen when the substrate and product complexes are compared, although the binary complex with the phosphopeptide reveals localized changes in conformation in the region corresponding to the glycine-rich loop. The high B-factors for this loop support the conclusion that this structural motif is a highly mobile segment of the protein.

Adenosine Diphosphate↗
PubMed1999

Oyster IKK-like protein shares structural and functional properties with its mammalian homologues.

In our search for genes involved in oyster immunity we isolated a cDNA encoding a polypeptide closely related to the mammalian IkappaB kinase (IKK) family. IKK proteins play a central role in cell signaling by regulating nuclear factor-kappaB (NF-kappaB) activation. We report here the cloning of an oyster IKK-like protein (oIKK) which possesses the characteristic organization of the mammalian IKK proteins, namely an amino-terminal kinase domain followed by a leucine zipper region and a carboxyl-terminal helix-loop-helix motif. When transfected into human cell lines, oIKK activated the expression of NF-kappaB-controlled reporter gene, whereas transfections with mutants of oIKK deleted within the kinase domain or within the helix-loop-helix motif respectively abolished and greatly reduced reporter gene activation. These results indicate that oIKK can replace the hIKK-alpha in catalyzing NF-kappaB nuclear translocation, and in triggering gene expression. Our results sustain the concept of an evolutionarily conserved signaling machinery in which IKK plays a major role.

Amino Acid Sequence↗
PubMed2005

Helix-loop-helix proteins and lymphocyte development.

Helix-loop-helix (HLH) proteins are transcriptional regulators that control a wide variety of developmental pathways in both invertebrate and vertebrate organisms. Results obtained in the past decade have shown that HLH proteins also contribute to the development of lymphoid lineages. A subset of HLH proteins, the 'E proteins', seems to be particularly important for proper lymphoid development. Members of the E protein family include E12, E47, E2-2 and HEB. The E proteins contribute to B lineage- and T lineage-specific gene expression programs, regulate lymphocyte survival and cellular proliferation, activate the rearrangement of antigen receptor genes and control progression through critical developmental checkpoints. This review discusses HLH proteins in lymphocyte development and homeostasis.

Animals↗
PubMed2006

Structural and functional fingerprint of the mitochondrial ATP-binding cassette transporter Mdl1 from Saccharomyces cerevisiae.

The ATP-binding cassette half-transporter Mdl1 from Saccharomyces cerevisiae has been proposed to be involved in the quality control of misassembled respiratory chain complexes by exporting degradation products generated by the m-AAA proteases from the matrix. Direct functional or structural data of the transport complex are, however, not known so far. After screening expression in various hosts, Mdl1 was overexpressed 100-fold to 1% of total mitochondrial membrane protein in S. cerevisiae. Based on detergent screens, Mdl1 was solubilized and purified to homogeneity. Mdl1 showed a high binding affinity for MgATP (Kd = 0.26 microm) and an ATPase activity with a Km of 0.86 mm (Hill coefficient of 0.98) and a turnover rate of 2.6 ATP/s. Mutagenesis of the conserved glutamate downstream of the Walker B motif (E599Q) or the conserved histidine of the H-loop (H631A) abolished ATP hydrolysis, whereas ATP binding was not affected. Mdl1 reconstituted into liposomes showed an ATPase activity similar to the solubilized complex. By single particle electron microscopy, a first three-dimensional structure of the mitochondrial ATP-binding cassette transporter was derived at 2.3-nm resolution, revealing a homodimeric complex in an open conformation.

ATP-Binding Cassette Transporters↗
PubMed2005

EphB receptor-binding peptides identified by phage display enable design of an antagonist with ephrin-like affinity.

The Eph receptor tyrosine kinases are overexpressed in many pathologic tissues and have therefore emerged as promising drug target candidates. However, there are few molecules available that can selectively bind to a single Eph receptor and not other members of this large receptor family. Here we report the identification by phage display of peptides that bind selectively to different receptors of the EphB class, including EphB1, EphB2, and EphB4. Peptides with the same EphB receptor specificity compete with each other for binding, suggesting that they have partially overlapping binding sites. In addition, several of the peptides contain amino acid motifs found in the G-H loop of the ephrin-B ligands, which is the region that mediates high-affinity interaction with the EphB receptors. Consistent with targeting the ephrin-binding site, the higher affinity peptides antagonize ephrin binding to the EphB receptors. We also designed an optimized EphB4-binding peptide with affinity comparable with that of the natural ligand, ephrin-B2. These peptides should be useful as selective inhibitors of the pathological activities of EphB receptors and as targeting agents for imaging probes and therapeutic drugs.

Amino Acid Motifs↗
PubMed2006

Cloning and characterization of the HPr kinase/phosphorylase gene from Bacillus stearothermophilus No. 236.

The Bacillus stearothermophilus no. 236 gene encoding the bifunctional enzyme HprK/P, the key regulator of carbon catabolite repression/activation (CCR/CCA) in most Gram-positive bacteria, was cloned and the (His)(6)-tagged gene product was characterized in detail. The nucleotide sequence of the hprK/P gene corresponded to an open reading frame of 951 bp that encoded a polypeptide of 316 amino acid residues with a calculated molecular mass of 35,458 Da. The deduced amino acid sequence of the B. stearothermophilus no. 236 HprK/P showed 64.5% identity with the B. subtilis enzyme, allowing us to identify two highly conserved motifs, the nucleotide binding P-loop (Walker motif A) and the HprK/P family signature sequence in the C-terminal half of the protein. Furthermore, complementation experiments showed that the cloned hprK/P gene product was functionally active in the B. subtilis cells. The purified (His)(6)-tagged B. stearothermophilus no. 236 HprK/P migrated on SDS-PAGE gel as a single species with a molecular mass of about 36 kDa, and behaved in gel filtration like a hexameric protein. The recombinant protein catalyzes the pyrophosphate (PPi)-dependent (highest activity at pH 7.0 and 40 degrees C) as well as the ATP-dependent phosphorylation of Ser46 in HPr (maximum activity at pH 8.0 and 45 degrees C). It also catalyzes the inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr, optimally at pH 6.5 and 40 degrees C. BIAcore surface resonance analysis confirmed that a divalent cation, preferentially Mg(2+), was an indispensable cofactor for the three activities of the HprK/P. Fructose-1,6-bisphosphate (FBP) was observed to stimulate ATP-dependent kinase activity, while inorganic phosophate (Pi) inhibited ATP-dependent kinase activity. Mutations in the Walker motif A simultaneously abolished both types of kinase and phosphorylase activities. On the other hand, the conserved signature residues were confirmed to be involved in the PPi-dependent kinase and phosphorylase reactions.

Amino Acid Sequence↗