Vital function for PRELI and essential requirement for its LEA motif

Proteins containing the late embryogenesis abundant (LEA) motif comprise an evolutionarily conserved family, long postulated to protect plant embryos from stress and death. However, the significance of LEA-containing proteins and the mechanisms behind their function remain undetermined. Here we show that PRELI, a mammalian protein that possesses tandem repeats of the LEA motif, can protect cells against staurosporine, TNF-α or UV irradiation-induced apoptosis. We found that key to PRELI-dependent mechanisms that promote cell resistance to death are the stabilization of the respiratory chain, upholding of mitochondrial membrane potential and retention of apoptogenic molecules. By in vitro and in vivo studies, we also show that the expression of mutant PRELI/LEA- proteins lacking the LEA motif, results in the complete loss of PRELI's anti-apoptotic functions. Collectively, our data uncover a new molecular player in the control of apoptosis and support the hypothesis that LEA-containing proteins are evolutionarily conserved cell protectors against stress and death. ^

Interactions and structural analysis of the zinc -binding motif in decorin, a small leucine rich proteoglycan in extracellular matrix

Decorin, a dermatan/chondroitin sulfate proteoglycan, is ubiquitously distributed in the extracellular matrix (ECM) of mammals. Decorin belongs to the small leucine rich proteoglycan (SLRP) family, a proteoglycan family characterized by a core protein dominated by Leucine Rich Repeat motifs. The decorin core protein appears to mediate the binding of decorin to ECM molecules, such as collagens and fibronectin. It is believed that the interactions of decorin with these ECM molecules contribute to the regulation of ECM assembly, cell adhesions, and cell proliferation. These basic biological processes play critical roles during embryonic development and wound healing and are altered in pathological conditions such as fibrosis and tumorgenesis. ^ In this dissertation, we discover that decorin core protein can bind to Zn2+ ions with high affinity. Zinc is an essential trace element in mammals. Zn2+ ions play a catalytic role in the activation of many enzymes and a structural role in the stabilization of protein conformation. By examining purified recombinant decorin and its core protein fragments for Zn2+ binding activity using Zn2+-chelating column chromatography and Zn2+-equilibrium dialysis approaches, we have located the Zn2+ binding domain to the N-terminal sequence of the decorin core protein. The decorin N-terminal domain appears to contain two Zn2+ binding sites with similar high binding affinity. The sequence of the decorin N-terminal domain does not resemble any other reported zinc-binding motifs and, therefore, represents a novel Zn 2+ binding motif. By investigating the influence of Zn2+ ions on decorin binding interactions, we found a novel Zn2+ dependent interaction with fibrinogen, the major plasma protein in blood clots. Furthermore, a recombinant peptide (MD4) consisting of a 41 amino acid sequence of mouse decorin N-terminal domain can prolong thrombin induced fibrinogen/fibrin clot formation. This suggests that in the presence of Zn2+ the decorin N-terminal domain has an anticoagulation activity. The changed Zn2+-binding activities of the truncated MD4 peptides and site-directed mutagenesis generated mutant peptides revealed that the functional MD4 peptide might contain both a structural zinc-binding site in the cysteine cluster region and a catalytic zinc site that could be created by the flanking sequences of the cysteine cluster region. A model of a loop-like structure for MD4 peptide is proposed. ^

Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome

The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

Light-activated rhodopsin induces structural binding motif in G protein α subunit

A large superfamily of transmembrane receptors control cellular responses to diverse extracellular signals by catalyzing activation of specific types of heterotrimeric GTP-binding proteins. How these receptors recognize and promote nucleotide exchange on G protein α subunits to initiate signal amplification is unknown. The three-dimensional structure of the transducin (Gt) α subunit C-terminal undecapeptide Gtα(340–350) IKENLKDCGLF was determined by transferred nuclear Overhauser effect spectroscopy while it was bound to photoexcited rhodopsin. Light activation of rhodopsin causes a dramatic shift from a disordered conformation of Gtα(340–350) to a binding motif with a helical turn followed by an open reverse turn centered at Gly-348, a helix-terminating C capping motif of an αL type. Docking of the NMR structure to the GDP-bound x-ray structure of Gt reveals that photoexcited rhodopsin promotes the formation of a continuous helix over residues 325–346 terminated by the C-terminal helical cap with a unique cluster of crucial hydrophobic side chains. A molecular mechanism by which activated receptors can control G proteins through reversible conformational changes at the receptor–G protein interface is demonstrated.

Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif

Surface proteins of Staphylococcus aureus are linked to the bacterial cell wall by sortase, an enzyme that cleaves polypeptides at the threonine of the LPXTG motif. Surface proteins can be released from staphylococci by treatment with hydroxylamine, resulting in the formation of threonine hydroxamate. Staphylococcal extracts, as well as purified sortase, catalyze the hydroxylaminolysis of peptides bearing an LPXTG motif, a reaction that can be inhibited with sulfhydryl-modifying reagents. Replacement of the single conserved cysteine at position 184 of sortase with alanine abolishes enzyme activity. Thus, sortase appears to catalyze surface-protein anchoring by means of a transpeptidation reaction that captures cleaved polypeptides as thioester enzyme intermediates.

Crystal structures of the copper and nickel complexes of RNase A: Metal-induced interprotein interactions and identification of a novel copper binding motif

We report the crystal structures of the copper and nickel complexes of RNase A. The overall topology of these two complexes is similar to that of other RNase A structures. However, there are significant differences in the mode of binding of copper and nickel. There are two copper ions per molecule of the protein, but there is only one nickel ion per molecule of the protein. Significant changes occur in the interprotein interactions as a result of differences in the coordinating groups at the common binding site around His-105. Consequently, the copper- and nickel-ion-bound dimers of RNase A act as nucleation sites for generating different crystal lattices for the two complexes. A second copper ion is present at an active site residue His-119 for which all the ligands are from one molecule of the protein. At this second site, His-119 adopts an inactive conformation (B) induced by the copper. We have identified a novel copper binding motif involving the α-amino group and the N-terminal residues.

Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization

Recent advances in our understanding of the immune response are allowing for the logical design of new approaches to cancer immunization. One area of interest is the development of new immune adjuvants. Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can induce production of a wide variety of cytokines and activate B cells, monocytes, dendritic cells, and NK cells. Using the 38C13 B cell lymphoma model, we assessed whether CpG ODN can function as immune adjuvants in tumor antigen immunization. The idiotype served as the tumor antigen. Select CpG ODN were as effective as complete Freund’s adjuvant at inducing an antigen-specific antibody response but were associated with less toxicity. These CpG ODN induced a higher titer of antigen-specific IgG2a than did complete Freund’s adjuvant, suggesting an enhanced TH1 response. Mice immunized with CpG ODN as an adjuvant were protected from tumor challenge to a degree similar to that seen in mice immunized with complete Freund’s adjuvant. We conclude that CpG ODN are effective as immune adjuvants and are attractive as part of a tumor immunization strategy.

High-affinity binding of the Drosophila Numb phosphotyrosine-binding domain to peptides containing a Gly-Pro-(p)Tyr motif

The phosphotyrosine-binding (PTB) domain is a recently identified protein module that has been characterized as binding to phosphopeptides containing an NPXpY motif (X = any amino acid). We describe here a novel peptide sequence recognized by the PTB domain from Drosophila Numb (dNumb), a protein involved in cell fate determination and asymmetric cell division during the development of the Drosophila nervous system. Using a Tyr-oriented peptide library to screen for ligands, the dNumb PTB domain was found to bind selectively to peptides containing a YIGPYφ motif (φ represents a hydrophobic residue). A synthetic peptide containing this sequence bound specifically to the isolated dNumb PTB domain in solution with a dissociation constant (Kd) of 5.78 ± 0.74 μM. Interestingly, the affinity of this peptide for the dNumb PTB domain was increased (Kd = 1.41 ± 0.10 μM) when the second tyrosine in the sequence was phosphorylated. Amino acid substitution studies of the phosphopeptide demonstrated that a core motif of sequence GP(p)Y is required for high-affinity binding to the dNumb PTB domain. Nuclear magnetic resonance experiments performed on isotopically labeled protein complexed with either Tyr- or pTyr-containing peptides suggest that the same set of amino acids in the dNumb PTB domain is involved in binding both phosphorylated and nonphosphorylated forms of the peptide. The in vitro selectivity of the dNumb PTB domain is therefore markedly different from those of the Shc and IRS-1 PTB domains, in that it interacts preferentially with a GP(p)Y motif, rather than NPXpY, and does not absolutely require ligand phosphorylation for binding. Our results suggest that the PTB domain is a versatile protein module, capable of exhibiting varied binding specificities.

Involvement of unique leucine-zipper motif of PSD-Zip45 (Homer 1c/vesl-1L) in group 1 metabotropic glutamate receptor clustering

Several scaffold proteins for neurotransmitter receptors have been identified as candidates for receptor targeting. However, the molecular mechanism underlying such receptor clustering and targeting to postsynaptic specializations remains unknown. PSD-Zip45 (also named Homer 1c/vesl-1L) consists of the NH2 terminus containing the enabled/VASP homology 1 domain and the COOH terminus containing the leucine zipper. Here, we demonstrate immunohistochemically that metabotropic glutamate receptor 1α (mGluR1α) and PSD-Zip45/Homer 1c are colocalized to synapses in the cerebellar molecular layer but not in the hippocampus. In cultured hippocampal neurons, PSD-Zip45/Homer1c and N-methyl-d-aspartate receptors are preferentially colocalized to dendritic spines. Cotransfection of mGluR1α or mGluR5 and PSD-Zip45/Homer 1c into COS-7 cells results in mGluR clustering induced by PSD-Zip45/Homer 1c. An in vitro multimerization assay shows that the extreme COOH-terminal leucine zipper is involved in self-multimerization of PSD-Zip45/Homer 1c. A clustering assay of mGluRs in COS-7 cells also reveals a critical role of this leucine-zipper motif of PSD-Zip45/Homer 1c in mGluR clustering. These results suggest that the leucine zipper of subsynaptic scaffold protein is a candidate motif involved in neurotransmitter receptor clustering at the central synapse.

Nucleosomes, linker DNA, and linker histone form a unique structural motif that directs the higher-order folding and compaction of chromatin

The compaction level of arrays of nucleosomes may be understood in terms of the balance between the self-repulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes ≈1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed ≈8 nm from the nucleosome center and remain apposed for 3–5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique three-dimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.

Identification of a proline-binding motif regulating CD2-triggered T lymphocyte activation

An intracellular protein termed CD2 binding protein 2 (CD2BP2), which binds to a site containing two PPPGHR segments within the cytoplasmic region of CD2, was identified. Mutagenesis and NMR analysis demonstrated that the CD2 binding region of CD2BP2 includes a 17-aa motif (GPY[orF]xxxxM[orV]xxWxxx GYF), also found in several yeast and Caenorhabditis elegans proteins of unknown function. In Jurkat T cells, over-expression of the isolated CD2BP2 domain binding to CD2 enhances the production of interleukin 2 on crosslinking of CD2 but not the T cell receptor. Hence, a proline-binding module distinct from SH3 and WW domains regulates protein–protein interactions.

The α-helical FXXΦΦ motif in p53: TAF interaction and discrimination by MDM2

Transcriptional activation domains share little sequence homology and generally lack folded structures in the absence of their targets, aspects that have rendered activation domains difficult to characterize. Here, a combination of biochemical and nuclear magnetic resonance experiments demonstrates that the activation domain of the tumor suppressor p53 has an FXXΦΦ motif (F, Phe; X, any amino acids; Φ, hydrophobic residues) that folds into an α-helix upon binding to one of its targets, hTAFII31 (a human TFIID TATA box-binding protein-associated factor). MDM2, the cellular attenuator of p53, discriminates the FXXΦΦ motif of p53 from those of NF-κB p65 and VP16 and specifically inhibits p53 activity. Our studies support the notion that the FXXΦΦ sequence is a general α-helical recognition motif for hTAFII31 and provide insights into the mechanistic basis for regulation of p53 function.

Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: Finding of a lipase motif and the induction by methyl jasmonate

Chlorophyllase (Chlase) is the first enzyme involved in chlorophyll (Chl) degradation and catalyzes the hydrolysis of ester bond to yield chlorophyllide and phytol. In the present study, we isolated the Chlase cDNA. We synthesized degenerate oligo DNA probes based on the internal amino acid sequences of purified Chlase from Chenopodium album, screened the C. album cDNA library, and cloned a cDNA (CaCLH, C. album chlorophyll-chlorophyllido hydrolase). The deduced amino acid sequence (347 aa residues) had a lipase motif overlapping with an ATP/GTP-binding motif (P-loop). CaCLH possibly was localized in the extraplastidic part of the cell, because a putative signal sequence for endoplasmic reticulum is at the N terminus. The amino acid sequence shared 37% identity with a function-unknown gene whose mRNA is inducible by coronatine and methyl jasmonate (MeJA) in Arabidopsis thaliana (AtCLH1). We expressed the gene products of AtCLH1 and of CaCLH in Escherichia coli, and they similarly exhibited Chlase activity. Moreover, we isolated another full-length cDNA based on an Arabidopsis genomic fragment and expressed it in E. coli, demonstrating the presence of the second Arabidopsis CLH gene (AtCLH2). No typical feature of signal sequence was identified in AtCLH1, whereas AtCLH2 had a typical signal sequence for chloroplast. AtCLH1 mRNA was induced rapidly by a treatment of MeJA, which is known to promote senescence and Chl degradation in plants, and a high mRNA level was maintained up to 9 h. AtCLH2, however, did not respond to MeJA.

A dileucine motif in the C terminus of the β2-adrenergic receptor is involved in receptor internalization

The cytoplasmic C terminus of the β2-adrenergic receptor and many other G protein-coupled receptors contains a dileucine sequence that has been implicated in endosome/lysosome targeting of diverse proteins. In the present study, we provide evidence for an essential role of this motif in the agonist-induced internalization of the β2-adrenergic receptor. Mutation of Leu-339 and/or Leu-340 to Ala caused little changes in surface expression, ligand binding, G protein coupling, and signaling to adenylyl cyclase, when these receptors were transiently or stably expressed in CHO or HEK-293 cells. However, agonist-induced receptor internalization was markedly impaired in the L339,340A double mutant and reduced in the two single mutants. This impairment in receptor internalization was seen by using various approaches to determine internalization: binding of hydrophobic vs. hydrophilic ligands, loss of surface β2-adrenergic receptor immunoreactivity, and immunofluorescence microscopy. The selective effects of these mutations suggest that the C-terminal dileucine motif is involved in agonist-induced internalization of the β2-adrenergic receptor.