Bovine mitochondrial ribosomes possess a high affinity binding site for guanine nucleotides

Mammalian mitochondrial ribosomes possess a binding site for guanine nucleotides. GTP binds in unit stoichiometry and with high affinity (Kd = 15.3 +/- 2.8 nM) to the small subunit of bovine mitochondrial ribosomes. This binding activity survives high salt washes, indicating that the nucleotide binds to an integral site within this subunit. GDP also binds to the small subunit with high affinity (Kd = 17 +/- 5.8 nm) and in unit stoichiometry. The GTP binding activity can be competed with GDP but not appreciably by other nucleotides, indicating that both GTP and GDP bind specifically and to the same site. The non-hydrolyzable analogs of GTP, guanylyl-5'-imidophosphate, and guanylyl-(beta,gamma-methylene)- diphosphonate also bind to the small subunit, but with reduced affinity. These results indicate that mammalian mitochondrial ribosomes, unlike other ribosomes, are able to interact directly with guanosine triphosphate, suggesting that the bound GTP may be involved in a novel regulatory mechanism in mitochondrial protein synthesis.     

Presence of nuclear factors bound to both cAMP-responsive element and AP1 factor binding site in the porcine anterior pituitary.

Factors binding to consensus sequences of the cAMP-responsive element (CRE) and the AP1 factor binding site (AP1) were investigated using porcine anterior pituitary nuclear extracts. Each element showed specific gel mobility shifts. By reciprocal competition for the AP1 and CRE binding, CRE prevented AP1 binding completely. On the other hand, AP1 decreased the CRE binding considerably to 20%, suggesting that approximately 80% of the total CRE binding is due to factors which bind to a common site shared by both CRE and AP1, whereas proteins binding to AP1 alone are absent. Relative binding affinities of AP1 against CRE estimated from the reciprocal competition data were 0.17 for CRE binding and 0.56 for AP1 binding. UV cross-linking experiments showed that CRE and AP1 gave different patterns consisting of different molecular size. Inconsistency of the relative binding affinities and the multiple molecular size of binding factors, cannot be explained simply by the presence of two types of binding factor, common CRE/AP1-binding and specific CRE-binding factors. A more likely explanation is that the CRE/AP1-binding factors alter the dimer form by changing each respective partner to bind CRE and/or AP1.     

Identification of a common protein association region in the neuronal Cdk5 activator

Cyclin-dependent protein kinase 5 (Cdk5) depends on the association with neuronal Cdk5 activator (Nck5a) for kinase activity. A variety of cellular proteins have been shown to undergo high affinity association with Nck5a, including three novel proteins, C42, C48, and C53 found by a yeast two-hybrid screen (Ching, Y. P., Qi, Z., and Wang, J. H. (2000) Gene 242, 285-294). The three proteins show competitive binding to Nck5a suggesting that they bind at a common site. The binding site has been mapped to a region of 26 amino acid residues (residues 145 to 170) at the N-terminal boundary of the kinase activation domain of Nck5a. This region of Nck5a contains an amphipathic alpha-helix whose hydrophobic face is involved in Cdk5 activation (Chin, K. T., Ohki, S, Tang, D., Cheng, H. C., Wang, J. H. , and Zhang, M. (1999) J. Biol. Chem. 274, 7120-7127). Several lines of evidence suggest that Nck5a interacts with the binding proteins at the hydrophilic face of the amphipathic alpha-helix. First, the Nck5a-(145-170) peptide can bind Cdk5 and Nck5a-binding proteins simultaneously. Second, the association of Nck5a-(145-170) to C48 can be markedly reduced by high ionic strength whereas the interaction between Nck5a and Cdk5 is not affected. Third, substitution of Glu(157) by glutamine in Nck5a-(145-170) abolishes the peptide's ability to bind to the three Nck5a-binding proteins without diminishing its Cdk5 binding activity.     

Nucleotide binding to the isolated beta subunit of the chloroplast ATP synthase.

The beta subunit isolated from the chloroplast ATP synthase F1 (CF1) has a single dissociable nucleotide binding site, consistent with the proposed function of this subunit in nucleotide binding and catalysis. The beta subunit bound the nucleotide analogs trinitrophenyl-ATP (TNP-ATP) or trinitrophenyl-ADP (TNP-ADP) with nearly equal affinities (Kd = 1-2 microM) but did not bind trinitrophenyl-AMP. Both ATP and ADP effectively competed with TNP-ATP for binding. Other nucleoside triphosphates were also able to compete with TNP-ATP for binding to beta; their order of effectiveness (ATP greater than GTP, ITP greater than CTP) mimicked the normal substrate specificity of CF1. The single nucleotide binding site on the isolated beta subunit very closely resembles the low affinity catalytic site (site 3) of CF1 (Bruist, M.F., and Hammes, G. G. (1981) Biochemistry 20, 6298-6305), suggesting that tight nucleotide binding by other sites on the enzyme involves other CF1 subunits in addition to the beta subunit. The results are inconsistent with an earlier report (Frasch, W.D., Green, J., Caguial, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069), which suggested more than one nucleotide binding site per beta subunit. Binding of nucleotides to the isolated beta subunit was eliminated by a brief heat treatment (40 degrees C for 10 min) of the protein. A small change in the circular dichroism spectrum of beta accompanied the heat treatment indicating that a localized (rather than global) change in the folding of beta, involving at least part of the nucleotide binding domain, had occurred. Also accompanying the loss of nucleotide binding was a loss of the reconstitutive capacity of the beta subunit. ATP protected against the effects of the heat treatment.     

The specific binding of nuclear protein(s) to the cAMP responsive element (CRE) sequence (TGACGTCA) is reduced by the misincorporation of U and increased by the deamination of C.

Point mutations in the cAMP-responsive element (CRE) of the rat somatostatin gene promoter/enhancer sequence (TGACGTCA) were used as a model for assessing the effect of uracil, deriving either from misincorporation during DNA synthesis (T----U) or cytosine deamination (C----U), on the binding of sequence/specific regulatory proteins. The results show that the T----U conversion in both strands of the CRE palindromic sequence reduces its affinity for the CRE binding factor(s), suggesting the crucial role of the methyl group contributed by T for the correct recognition of the sequence. On the other hand, deamination of C in the CpG central dinucleotide (CpG----UpG) causes an increase of binding affinity which is further enhanced by the contemporary deamination in both strands. Then, both uracil misincorporation and cytosine deamination alter the binding to CRE sequence in vitro, suggesting that uracil, if not removed by uracil DNA-glycosylase, could be dangerous for cellular functions.     

TraG-like proteins of type IV secretion systems: functional dissection of the multiple activities of TraG (RP4) and TrwB (R388)

TraG-like proteins are essential components of type IV secretion systems. During secretion, TraG is thought to translocate defined substrates through the inner cell membrane. The energy for this transport is presumably delivered by its potential nucleotide hydrolase (NTPase) activity. TraG of conjugative plasmid RP4 is a membrane-anchored oligomer that binds RP4 relaxase and DNA. TrwB (R388) is a hexameric TraG-like protein that binds ATP. Both proteins, however, lack NTPase activity under in vitro conditions. We characterized derivatives of TraG and TrwB truncated by the N-terminal membrane anchor (TraGdelta2 and TrwBdelta1) and/or containing a point mutation at the putative nucleotide-binding site (TraGdelta2K187T and TraGK187T). Unlike TraG and TrwB, truncated derivatives behaved as monomers without the tendency to form oligomers or aggregates. Surface plasmon resonance analysis with immobilized relaxase showed that mutant TraGK187T was as good a binding partner as the wild-type protein, whereas truncated TraG monomers were unable to bind relaxase. TraGdelta2 and TrwBdelta1 bound ATP and, with similar affinity, ADP. Binding of ATP and ADP was strongly inhibited by the presence of Mg(2+) or single-stranded DNA and was competed for by other nucleotides. Compared to the activity of TraGdelta2, the ATP- and ADP-binding activity of the point mutation derivative TraGdelta2K187T was significantly reduced. Each TraG derivative bound DNA with an affinity similar to that of the native protein. DNA binding was inhibited or competed for by ATP, ADP, and, most prominently, Mg(2+). Thus, both nucleotide binding and DNA binding were sensitive to Mg(2+) and were competitive with respect to each other.     

Divalent cation-nucleotide complex at the exchangeable nucleotide binding site of tubulin

Tubulin was first treated with alkaline phosphatase-agarose to vacate the exchangeable nucleotide binding site and then tested for manganese binding sites by Mn(II) EPR. Buttlaire et al. ((1980) J. Biol. Chem. 255, 2164-2168) have shown that high affinity manganese binding occurs at a single site normally occupied by magnesium. We report that the number of high affinity manganese binding sites per mol of tubulin depends on the number of occupied exchangeable nucleotide binding sites. Thus, removal of nucleotides results in a loss of high affinity manganese binding sites. The EPR spectra of manganese bound to tubulin and to GTP are found to be qualitatively similar. These data indicate that high affinity manganese binding is the result of the formation of a metal-nucleotide complex at the exchangeable nucleotide binding site. In addition it was found that zinc, cobalt, and magnesium bind with approximately equal affinity to this site whereas calcium binds only weakly.