The Elp2 subunit of elongator and elongating RNA polymerase II holoenzyme is a WD40 repeat protein

A novel yeast gene, ELP2, is shown to encode the 90-kDa subunit of the Elongator complex and elongating RNA polymerase II holoenzyme. ELP2 encodes a protein with eight WD40 repeats, and cells lacking the gene display typical elp phenotypes, such as temperature and salt sensitivity. Generally, different combinations of double and triple ELP gene deletions cause the same phenotypes as single ELP1, ELP2, or ELP3 deletion, providing genetic evidence that the ELP gene products work together in a complex.     

The Elongator subunit Elp3 contains a Fe4S4 cluster and binds S-adenosylmethionine

The Elp3 subunit of the Elongator complex is highly conserved from archaea to humans and contains a well-characterized C-terminal histone acetyltransferase (HAT) domain. The central region of Elp3 shares significant sequence homology to the Radical SAM superfamily. Members of this large family of bacterial proteins contain a FeS cluster and use S-adenosylmethionine (SAM) to catalyse a variety of radical reactions. To biochemically characterize this domain we have expressed and purified the corresponding fragment of the Methanocaldococcus jannaschii Elp3 protein. The presence of a Fe4S4 cluster has been confirmed by UV-visible spectroscopy and electron paramagnetic resonance (EPR) spectroscopy and the Fe content determined by both a colorimetric assay and atomic absorption spectroscopy. The cysteine residues involved in cluster formation have been identified by site-directed mutagenesis. The protein binds SAM and the binding alters the EPR spectrum of the FeS cluster. Our results provide biochemical support to the hypothesis that Elp3 does indeed contain the Fe4S4 cluster which characterizes the Radical SAM superfamily and binds SAM, suggesting that Elp3, in addition to its HAT activity, has a second as yet uncharacterized catalytic function. We also present preliminary data to show that the protein cleaves SAM.     

Molecular architecture and ligand recognition determinants for T4 RNA ligase

RNA ligase type 1 from bacteriophage T4 (Rnl1) is involved in countering a host defense mechanism by repairing 5'-PO4 and 3'-OH groups in tRNA(Lys). Rnl1 is widely used as a reagent in molecular biology. Although many structures for DNA ligases are available, only fragments of RNA ligases such as Rnl2 are known. We report the first crystal structure of a complete RNA ligase, Rnl1, in complex with adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPcPP). The N-terminal domain is related to the equivalent region of DNA ligases and Rnl2 and binds AMPcPP but with further interactions from the additional N-terminal 70 amino acids in Rnl1 (via Tyr37 and Arg54) and the C-terminal domain (Gly269 and Asp272). The active site contains two metal ions, consistent with the two-magnesium ion catalytic mechanism. The C-terminal domain represents a new all alpha-helical fold and has a charge distribution and architecture for helix-nucleic acid groove interaction compatible with tRNA binding.     

Assembly architecture and DNA binding of the bacteriophage P22 terminase small subunit

Morphogenesis of bacteriophage P22 involves the packaging of double-stranded DNA into a preassembled procapsid. DNA is translocated by a powerful virally encoded molecular motor called terminase, which comprises large (gp2, 499 residues) and small (gp3, 162 residues) subunits. While gp2 contains the phosphohydrolase and endonuclease activities of terminase, the function of gp3 may be to regulate specific and nonspecific modes of DNA recognition as well as the enzymatic activities of gp2. Electron microscopy shows that wild-type gp3 self-assembles into a stable and monodisperse nonameric ring. A three-dimensional reconstruction at 18 Å resolution provides the first glimpse of P22 terminase architecture and implies two distinct modes of interaction with DNA—involving a central channel of 20 Å diameter and radial spikes separated by 34 Å. Electromobility shift assays indicate that the gp3 ring binds double-stranded DNA nonspecifically in vitro via electrostatic interactions between the positively charged C-terminus of gp3 (residues 143-152) and phosphates of the DNA backbone. Raman spectra show that nonameric rings formed by subunits truncated at residue 142 retain the subunit fold despite the loss of DNA-binding activity. Difference density maps between gp3 rings containing full-length and C-terminally truncated subunits are consistent with localization of residues 143-152 along the central channel of the nonameric ring. The results suggest a plausible molecular mechanism for gp3 function in DNA recognition and translocation.     

On the structure-function relationship of peroxidases and peroxygenase

The structure-function relationship of two kinds of hemoproteins, peroxidases and peroxygenase, is discussed and a tentative model for the active site (heme vicinity) structure of each hemoprotein is proposed. The mechanism of Compound I formation from peroxidases is presumed to involve an electrophilic attack of hydroperoxide, the electrophilicity of which is increased by forming a hydrogen bond to a distal acid group (with β-equatorial arrangement) on the heme iron, the basicity of which is being increased by electron donation from the anionic fifth ligand. On the other hand, the mechanism for peroxygenase is presumed to involve a nucleophilic attack of hydroperoxide, the nucleophilicity of which is increased by forming a hydrogen bond to a distal base group (with α-axial arrangement) to the heme iron ligating the neutral fifth ligand. It is presumed that Compound I of peroxidases, which consists of porphyrin π cation radical and ferryl iron, is stabilized by a π-π type charge transfer interaction between the radical, and stacking imidazolate group (not necessarily different from the distal group) which then ionizes, and by electron donation from the anionic fifth ligand. On the other hand, Compound I of peroxygenase, which is postulated to be an oxene complex, is presumed to be stabilized by an electrostatic interaction with a strongly negative environment, and by ionization of the fifth ligand, if such can happen.     

Ion transport and ligand binding by the Na-K-Cl cotransporter, structure-function studies

The cation-Cl cotransporters (CCCs) mediate the coupled movement of Na and/or K to that of Cl across the plasmalemma of animal cells. Eight CCCs have been identified to date: two Na-K-Cl cotransporters (NKCC), four K-Cl cotransporters (KCCs), one Na-Cl cotransporter (NCC) and one CCC interacting protein (CIP). All of the NKCCs and KCCs are inhibited by loop diuretics; mercury and other modifying agents are also known to block NKCC-mediated transport. In this work, we have utilized a mutational approach to study the interaction between different substrates and the NKCCs. We relied on the strategy of exchanging domains between functionally distinct carriers (the shark NKCCl and the human NKCCl) to identify residues or group of residues that are involved in the interaction with ions, loop diuretics and Hg. Our results show that the N- and C-termini have no role in determining the species differences in ion transport and bumetanide binding. On the other hand, the interaction between Hg and the NKCCs is found to partially involve the C-terminus through residues that contain available sulfhydryl groups. Within the transmembrane segments, variant residues in the 2nd, 4th and 7th predicted alpha-helices are shown to encode the differences in ion transport between the shark and the human cotransporters. For loop diuretic binding, several regions throughout the central domain appear to be involved. Interestingly, these regions are not the same as those involved in cation or anion transport, and in Hg binding.     

RNA binding proteins of the large subunit of bovine mitochondrial ribosomes.

RNA binding properties of proteins from the large subunit of bovine mitochondrial ribosomes were studied using four different approaches: binding of radiolabeled RNA to western blotted proteins; disassembly of the intact 39 S ribosomal subunits with urea; binding of ribosomal proteins to RNA in the presence of urea; and binding of proteins extracted with lithium chloride to RNA. Results from these four approaches allowed us to identify a set of six proteins (L7, L13, L14, L21, L26, and L44) which appear to be strong RNA binding proteins. Seven additional proteins (L8, L11, L28, L35, L40, L49, and L50) were identified as secondary RNA binding proteins. RNA binding properties of the proteins in both of these sets were compared with the topographic disposition and susceptibility towards lithium chloride extraction of the individual proteins. Proteins from the first set are good candidates for early assembly proteins since they have a high affinity for RNA, are generally found in 4M lithium chloride core particles, and are among the most buried proteins in the 39 S subunit.     

Casein kinase-2 structure-function relationship: creation of a set of mutants of the beta subunit that variably surrogate the wildtype beta subunit function.

Nine mutants of human casein kinase-2 beta subunit have been created and assayed for their ability to assemble with the catalytic alpha subunit to give, at a 1:1 molar ratio, a fully competent CK-2 holoenzyme as judged by the following criteria: 1) the generation of an active heterotetrameric form of CK-2 exhibiting the expected sedimentation coefficient and 2) the enhancement of catalytic activity of CK-2 alpha. Extended deletions of 71 and 44 residues from the C-terminal end, but not a 7 residue deletion (including the cdc2 phosphorylation site) prevent both reconstitution of the holoenzyme and, consequently, stimulation of activity. This indicates that residue(s) located in the 171-209 sequence is essential for reconstitution. Also a four residue's N-terminal deletion (removing the autophosphorylation site) and single to quintuple substitutions of alanine for the acidic residues clustered in the 55-70 sequence give rise to mutants that still assemble with the alpha subunit to give a tetrameric holoenzyme. However, in the case of the mutants A57,59, A63,64, A59-61,63,64 in vitro assembly with the CK-2 alpha subunit was not complete. There were also intermediate complexes, free alpha-subunit and beta-mutants found to sediment at various positions in the sucrose density gradient. In comparison to CK-2 beta +, mutants A57,59, A59-61 and A59-61,63,64 show an increased stimulation of the catalytic activity supporting the view that these residues play a crucial role in determining the basal activity of reconstituted CK-2 holoenzyme.     

Probing the structure-function relationship of nerve growth factor

We compared the receptor binding, antigenicity, biological activation, and cell-mediated proteolytic degradation properties of mouse nerve growth factor (mNGF) and human NGF (hNGF). The affinity of hNGF toward human NGF-receptor is greater than that of mNGF, but the affinity of mNGF toward rat NGF-receptor is greater than that of hNGF. Thus, the specificity of the interaction between NGF and its receptor resides both on the NGF and on its receptor. Using a group of anti-NGF monoclonal antibodies that competitively inhibit the binding of NGF to receptor, sites differing between mNGF and hNGF were detected. Together, these results indicate that the sites on hNGF and mNGF, responsible for binding to NGF-receptor, are similar but not identical. In comparing the relative abilities of mNGF and hNGF to stimulate a biological response in PC12 cells, we observed that mNGF was better at stimulating neurite outgrowth than was hNGF, consistent with the differences observed for receptor binding affinity. However, the ED₅₀ for biological activation is approximately 100-fold lower than theK _d for receptor occupancy, and, thus, the dose-response curve is not consistent with a simple activation proportional to receptor occupancy. The data are consistent with a model requiring a low-level threshold occupancy of NGF-receptor (K _d=10^–9 M) in order to stimulate full biological activity. Finally, we observed the degradation of NGF by PC12 cells. We found that the NGF molecule is significantly degraded via a receptor-mediated uptake mechanism. Together, the data provide insight into regions of the NGF molecule involved in contacts with the receptor leading to formation of the NGF: NGF-receptor complex. Additionally, they establish the link between occupancy of receptor and biological activation and the requirement for receptor-mediated uptake in order to degrade NGF proteolytically in cultured PC12 cells.     

The structure-function relationship of a signaling-competent,dimeric Reelin fragment

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