A novel iron responsive element in the 3'UTR of human MRCKalpha

Human untranslated region (UTR) databases were searched to identify novel proteins potentially regulated by an iron responsive element (IRE), and found two candidates-cell cycle phosphatase Cdc14A variant 1 and myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha), both possessing a putative IRE in their 3'UTR. In further experiments, we focused on MRCKalpha. Biochemical analyses of the MRCKalpha IRE revealed that it was functional and mediated the response to iron level in the same way as transferrin receptor 1 IREs (TfR) did. Similarly to TfR mRNA, MRCKalpha mRNA is stabilized, when iron supply is low, while it is destabilized under iron-rich conditions. The expression of MRCKalpha mRNA was found to be ubiquitous; the highest levels were noted in testes, the lowest in skeletal muscle. The level of MRCKalpha mRNA in various tissues strongly positively correlates with the level of TfR mRNA, indicating its possible role in the transferrin iron uptake pathway.     

A novel RNA structural motif in the selenocysteine insertion element of eukaryotic selenoprotein mRNAs.

In eukaryotes, co-translational insertion of selenocysteine into selenoproteins necessitates the participation of the selenocysteine insertion sequence (SECIS), an element lying in the 3'-untranslated region of selenoprotein mRNAs. We report a detailed experimental study of the secondary structures of the SECIS elements of three selenoprotein mRNAs, the rat and human type I iodothyronine deiodinase (5'DI) and rat glutathione peroxidase (GPx). Based on RNase and chemical probing, a new secondary structure model is established. It is characterized by a stem-loop structure, comprising two helices (I and II) separated by an internal loop, with an apical loop surmounting helix II. Sequence comparisons of 20 SECIS elements, arising from 2 5'DI, 13 GPx, 2 selenoprotein P, and 1 selenoprotein W mRNAs, confirm the secondary structure model. The most striking finding of the experimental study concerns a set of conserved sequences in helix II that interact to form a novel RNA structural motif consisting of a quartet composed of non-Watson-Crick base pairs 5'UGAY3': 5'UGAU3'. The potential for forming the quartet is preserved in 15 SECIS elements, but three consecutive non-Watson-Crick base pairs can nevertheless form in the other five SECIS, the central G.A tandem being invariant in all cases. A 3D model, derived by computer modeling with the use of the solution data, suggests that the base pairing interactions in the G.A tandem are of the type found in GNRA loops. The 3D model displays the quartet lying in an accessible position at the foot of helix II, which is bent at the internal loop, suggesting that the non-Watson-Crick base pair arrangement provides an unusual pattern of chemical groups for putative ligand interaction.     

Identification of a novel phosphatase sequence motif.

We have identified a novel, conserved phosphatase sequence motif, KXXXXXXRP-(X12-54)-PSGH-(X31-54)-SRXXXXX HXXXD, that is shared among several lipid phosphatases, the mammalian glucose-6-phosphatases, and a collection of bacterial nonspecific acid phosphatases. This sequence was also found in the vanadium-containing chloroperoxidase of Curvularia inaequalis. Several lines of evidence support this phosphatase motif identification. Crystal structure data on chloroperoxidase revealed that all three domains are in close proximity and several of the conserved residues are involved in the binding of the cofactor, vanadate, a compound structurally similar to phosphate. Structure-function analysis of the human glucose-6-phosphatase has shown that two of the conserved residues (the first domain arginine and the central domain histidine) are essential for enzyme activity. This conserved sequence motif was used to identify nine additional putative phosphatases from sequence databases, one of which has been determined to be a lipid phosphatase in yeast.     

Identification of a novel Mcl-1 protein binding motif

Recent characterization of Mcl-1 as the primary anti-apoptotic Bcl-2 family member expressed in solid tumors, coupled with its ability to enable therapeutic resistance, has provided the impetus for further study into how Mcl-1 is involved in apoptosis signaling. Here, we employ Sabutoclax, a potent and effective Mcl-1 antagonist, as a competing agent to screen a randomized 12-residue phage display library for peptides that bind strongly to the Bcl-2 homology 3 (BH3) binding groove of Mcl-1. Although the screen identified a number of α-helical peptides with canonical BH3 domain sequences, it also isolated a pair of unique peptide sequences. These sequences exhibit a reverse organization of conserved hydrophobic and acidic residues when compared with canonical BH3 sequences, and we therefore refer to them as reverse BH3 (rBH3) peptides. Furthermore, studies of the rBH3 peptides using NMR spectroscopy, fluorescence polarization displacement assays, and alanine scanning data all suggest that they bind to the BH3 binding groove of Mcl-1 selectively over Bcl-x(L). A search for proteins containing the rBH3 motif has identified a number of interesting Mcl-1 protein partners, some of which have previously been associated with apoptosis regulation involving Mcl-1. These findings provide insights into the development of more specific Mcl-1 antagonists and open the way to the identification of a previously unknown family of apoptosis-regulating and Mcl-1 interacting proteins.     

Identification of a novel ligand binding motif in the transthyretin channel

The design of therapeutic compounds targeting transthyretin (TTR) is challenging due to the low specificity of interaction in the hormone binding site. Such feature is highlighted by the interactions of TTR with diclofenac, a compound with high affinity for TTR, in two dissimilar modes, as evidenced by crystal structure of the complex. We report here structural analysis of the interactions of TTR with two small molecules, 1-amino-5-naphthalene sulfonate (1,5-AmNS) and 1-anilino-8-naphthalene sulfonate (1,8-ANS). Crystal structure of TTR:1,8-ANS complex reveals a peculiar interaction, through the stacking of the naphthalene ring between the side-chain of Lys15 and Leu17. The sulfonate moiety provides additional interaction with Lys15′ and a water-mediated hydrogen bond with Thr119′. The uniqueness of this mode of ligand recognition is corroborated by the crystal structure of TTR in complex with the weak analogue 1,5-AmNS, the binding of which is driven mainly by hydrophobic partition and one electrostatic interaction between the sulfonate group and the Lys15. The ligand binding motif unraveled by 1,8-ANS may open new possibilities to treat TTR amyloid diseases by the elucidation of novel candidates for a more specific pharmacophoric pattern.     

A novel structural motif and structural trees for proteins containing it

In the present study, a novel structural motif that can be represented as a combination of the known βαβ-unit and ψ-motif is described and analyzed. In theory, there are four possible combinations of the motifs since each of them can exist in two forms, left-handed and right-handed. For this study, we have selected 140 nonhomologous proteins in which 158 combinations of such types have been found. The combination of the right-handed ψ-motif and the right-handed βαβ-unit has been shown to occur most often (87 cases out of 158) and the combination of the left-handed βαβ-unit and the left-handed ψ-motif does not occur at all. Three novel structural trees in which the commonly occurring combinations are taken as the root structures have been constructed.     

Covariation of a specificity-determining structural motif in an aminoacyl-tRNA synthetase and a tRNA identity element

Transfer RNA (tRNA) identity determinants help preserve the specificity of aminoacylation in vivo, and prevent cross-species interactions. Here, we investigate covariation between the discriminator base (N73) element in histidine tRNAs and residues in the histidyl-tRNA synthetase (HisRS) motif 2 loop. A model of the Escherichia coli HisRS--tRNA(His) complex predicts an interaction between the prokaryotic conserved glutamine 118 of the motif 2 loop and cytosine 73. The substitution of Gln 118 in motif 2 with glutamate decreased discrimination between cytosine and uracil some 50-fold, but left overall rates of adenylation and aminoacylation unaffected. By contrast, substitutions at neighboring Glu 115 and Arg 121 affected both adenylation and aminoacylation, consistent with their predicted involvement in both half-reactions. Additional evidence for the involvement of the motif 2 loop was provided by functional analysis of a hybrid Saccharomyces cerevisiae-- E. coli HisRS possessing the 11 amino acid motif 2 loop of the yeast enzyme. Despite an overall decreased activity of nearly 1000-fold relative to the E. coli enzyme, the chimera nevertheless exhibited a modest preference for the yeast tRNA(His) over the E. coli tRNA, and preferred wild-type yeast tRNA(His) to a variant with C at the discriminator position. These experiments suggest that part of, but not all of, the specificity is provided by the motif 2 loop. The close interaction between enzyme loop and RNA sequence elements suggested by these experiments reflects a covariation between enzyme and tRNA that may have acted to preserve aminoacylation fidelity over evolutionary time.     

Identification of a novel structural motif in the lipopolysaccharide of the galE/galK double mutant of Haemophilus influenzae strain Eagan

Defined mutants of the galactose biosynthetic (Leloir) pathway were employed to investigate lipopolysaccharide (LPS) oligosaccharide expression in Haemophilus influenzae type b strain Eagan. The structures of the low-molecular-mass LPS glycoforms from strains with mutations in the genes that encode galactose epimerase (galE) and galactose kinase (galK) were determined by NMR spectroscopy on O- and N-deacylated and dephosphorylated LPS-backbone, and O-deacylated oligosaccharide samples in conjunction with electrospray mass spectrometric, glycose and methylation analyses. The structural profile of LPS glycoforms from the galK mutant was found to be identical to that of the galactose and glucose-containing Hex5 glycoform previously identified in the parent strain [Masoud, H.; Moxon, E. R.; Martin, A.; Krajcarski, D.; Richards, J. C. Biochemistry1997, 36, 2091-2103]. LPS from the H. influenzae strain bearing mutations in both galK and galE (galE/galK double mutant) was devoid of galactose. In the double mutant, Hex3 and Hex4 glycoforms containing di- and tri-glucan side chains from the central heptose of the triheptosyl inner-core unit were identified as the major glycoforms. The triglucoside chain extension, β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-α-d-Glcp, identified in the Hex4 glycoform has not been previously reported as a structural element of H. influenzae LPS. In the parent strain, it is the galactose-containing trisaccharide, β-d-Galp-(1→4)-β-d-Glcp-(1→4)-α-d-Glcp, and further extended analogues thereof, that substitute the central heptose. When grown in galactose deficient media, the galE single mutant was found to expresses the same population of LPS glycoforms as the double mutant.     

Identification of a novel homotypic interaction motif required for the phosphorylation of receptor-interacting protein (RIP) by RIP3.

Receptor-interacting protein (RIP), a Ser/Thr kinase component of the tumor necrosis factor (TNF) receptor-1 signaling complex, mediates activation of the nuclear factor kappaB (NF-kappaB) pathway. RIP2 and RIP3 are related kinases that share extensive sequence homology with the kinase domain of RIP. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and recruitment domain, RIP3 possesses a unique C terminus. RIP3 binds RIP through this unique C-terminal segment to inhibit RIP- and TNF receptor-1-mediated NF-kappaB activation. We have identified a unique homotypic interaction motif at the C terminus of both RIP and RIP3 that is required for their association. Sixty-four amino acids within RIP3 and 88 residues within RIP are sufficient for interaction of the two proteins. This interaction is a prerequisite for RIP3-mediated phosphorylation of RIP and subsequent attenuation of TNF-induced NF-kappaB activation.     

Identification and characterization of a novel murine multigene family containing a PHD-finger-like motif

The genes Phf5a and Phf5b-ps are the first two members of a novel murine multigene family that is highly conserved during evolution and belongs to the superfamily of PHD-finger genes. The Phf5 gene family contains an active locus on mouse chromosome 15, region E and several processed pseudogenes on different chromosomes. The active locus, Phf5a, is expressed ubiquitously in pre- and postnatal murine tissues and encodes a protein of 110 amino acids. The protein is localized in the nucleus in a non-homogenous pattern as the nucleolar subcompartment is almost free of Phf5a. The molecular and biological functions of Phf5a are unknown up-to-date, but the systematic deletion of its yeast homolog is lethal, pointing out that the protein is required for cell viability. Interpretation of our data and review of the literature suggest both basic and essential cellular functions of the Phf5a protein, possibly acting as a chromatin-associated protein.     

A novel zinc snap motif conveys structural stability to 3-methyladenine DNA glycosylase I

The Escherichia coli 3-methyladenine DNA glycosylase I (TAG) is a DNA repair enzyme that excises 3-methyladenine in DNA and is the smallest member of the helix-hairpin-helix (HhH) superfamily of DNA glycosylases. Despite many studies over the last 25 years, there has been no suggestion that TAG was a metalloprotein. However, here we establish by heteronuclear NMR and other spectroscopic methods that TAG binds 1 eq of Zn2+ extremely tightly. A family of refined NMR structures shows that 4 conserved residues contributed from the amino- and carboxyl-terminal regions of TAG (Cys4, His17, His175, and Cys179) form a Zn2+ binding site. The Zn2+ ion serves to tether the otherwise unstructured amino- and carboxyl-terminal regions of TAG. We propose that this unexpected "zinc snap" motif in the TAG family (CX(12-17)HX(approximately 150)HX(3)C) serves to stabilize the HhH domain thereby mimicking the functional role of protein-protein interactions in larger HhH superfamily members.