The zinc finger region of simian virus 40 large T antigen is needed for hexamer assembly and origin melting.

Simian virus 40 large T antigen contains a single sequence element with an arrangement of cysteines and histidines that is characteristic of a zinc finger motif. The finger region maps from amino acids 302 through 320 and has the sequence C-302 L K C-305 I K K E Q P S H Y K Y H-317 E K H-320. Previous genetic analysis has shown that the cysteine and histidine sequences and the contiguous S H Y K Y region in the finger are important for DNA replication in vivo. We show here that representative mutations in either of these elements of the finger prevent the assembly of large T antigen into stable hexamers in vitro. These same mutations have a characteristic effect on the interaction of T antigen with the simian virus 40 core origin of replication. The mutant T antigens bind to the central pentanucleotide domain of the core origin but fail to melt the adjacent inverted repeat domain and to untwist the adenine-thymine domain. These defects would prevent the formation of a replication bubble and the initiation of DNA replication. Finger mutations have lesser effects on the helicase function of T antigen and no observable effect on binding of T antigen to the mouse p53 protein. We propose that the zinc finger region contributes to protein-protein interactions essential for the assembly of stable T-antigen hexamers at the origin of replication and that hexamers are needed for subsequent alterations in the structure of origin DNA. We cannot exclude the possibility that the zinc finger region also makes specific contacts with components of origin DNA.     

A novel zinc finger is required for Mcm10 homocomplex assembly

Mcm10 is a DNA replication factor that interacts with multiple subunits of the MCM2-7 hexameric complex. We report here that Mcm10 self-interacts and assembles into large homocomplexes (approximately 800 kDa). A conserved domain of 210 amino acid residues is sufficient for mediating self-interaction and complex assembly. A novel zinc finger within the conserved domain, CX10CX11CX2H, is essential for the homocomplex formation. Mutant alleles with amino acid substitutions at conserved cysteines and histidine in the zinc finger fail to assemble homocomplexes. A defect in homocomplex assembly correlates with defects in DNA replication and cell growth in the mutants. These observations suggest that homocomplex assembly is essential for Mcm10 function. Multisubunit Mcm10 homocomplexes may provide the structural basis for Mcm10 to interact with multiple subunits of the MCM2-7 hexamer.     

Role of conserved residues of the WRKY domain in the DNA-binding of tobacco WRKY family proteins.

Four cDNA clones of tobacco that could code for polypeptides with two WRKY domains were isolated. Among four NtWRKYs and other WRKY family proteins, sequence similarity was basically limited to the two WRKY domains. Glutathione S-transferase fusion proteins with the C-terminal WRKY domain of four NtWRKYs bound specifically to the W-box (TTGACC), and the N-terminal WRKY domain showed weaker binding activity with the W-box compared to the C-terminal domain. The DNA-binding activity of the WRKY domain was abolished by o-phenanthroline and this inhibition was recovered specifically by Zn2+. Substitution of the conserved cysteine and histidine residues of the plant-specific C2H2-type zinc finger-like motif in the WRKY domain abolished the DNA binding. In addition, mutations in the invariable WRKYGQK sequence at the N-terminal side of the zinc finger-like motif also significantly reduced the DNA-binding activity, suggesting that these residues are required for proper folding of the DNA-binding zinc finger.     

Role of Conserved Residues of the WRKY Domain in the DNA-binding of Tobacco WRKY Family Proteins

Four cDNA clones of tobacco that could code for polypeptides with two WRKY domains were isolated. Among four NtWRKYs and other WRKY family proteins, sequence similarity was basically limited to the two WRKY domains. Glutathione S-transferase fusion proteins with the C-terminal WRKY domain of four NtWRKYs bound specifically to the W-box (TTGACC), and the N-terminal WRKY domain showed weaker binding activity with the W-box compared to the C-terminal domain. The DNA-binding activity of the WRKY domain was abolished by o-phenanthroline and this inhibition was recovered specifically by Zn²⁺. Substitution of the conserved cysteine and histidine residues of the plant-specific C₂H₂-type zinc finger-like motif in the WRKY domain abolished the DNA binding. In addition, mutations in the invariable WRKYGQK sequence at the N-terminal side of the zinc finger-like motif also significantly reduced the DNA-binding activity, suggesting that these residues are required for proper folding of the DNA-binding zinc finger.     

Genetic analysis of a potential zinc-binding domain of the adenovirus E4 34k protein

E4 34k, the product of adenovirus early region 4 (E4) open reading frame 6, modulates viral late gene expression, viral DNA replication, apoptosis, double strand break repair, and transformation through multiple interactions with components in infected and transformed cells. Conservation of several cysteine and histidine residues among E4 34k sequences from a variety of adenovirus serotypes suggests the presence of a zinc binding domain important for function. Consistent with the hypothesis that E4 34k is a zinc metalloprotein, zinc binding by baculovirus-expressed E4 34k protein was demonstrated in a zinc blotting assay. To investigate the relationship between the potential zinc-binding region and E4 34k function, a series of mutant genes containing single amino acid substitutions at each of the conserved cysteine and histidine residues in E4 34k were constructed. The mutant proteins were examined for the ability to complement the late protein synthetic defect of an E4 deletion mutant, to physically interact with the viral E1b 55-kDa protein (E1b 55k) and cellular p53 protein, to relocalize E1b 55k, and to destabilize the p53 protein. These analyses identified a subset of cysteine and histidine residues required for stimulation of late gene expression, physical interaction with E1b 55k, and p53 destabilization. These data suggest that a zinc-binding domain participates in the formation of the E4 34k-E1b 55k physical complex and that the complex is required in late gene expression and for p53 destabilization.     

Zinc binding by the methylation signaling domain of the Escherichia coli Ada protein.

The Escherichia coli Ada protein repairs O6-methylguanine residues and methyl phosphotriesters in DNA by direct transfer of the methyl group to a cysteine residue located in its C- or N-terminal domain, respectively. Methyl transfer to the N-terminal domain causes it to acquire a sequence-specific DNA binding activity, which directs binding to the regulatory region of several methylation-resistance genes. In this paper we show that the N-terminal domain of Ada contains a high-affinity binding site for a single zinc atom, whereas the C-terminal domain is free of zinc. The metal-binding domain is apparently located within the first 92 amino acids of Ada, which contains four conserved cysteine residues. We propose that these four cysteines serve as the zinc ligand residues, coordinating the metal in a tetrahedral arrangement. One of the putative ligand residues, namely, Cys69, also serves as the acceptor site for a phosphotriester-derived methyl group. This raises the possibility that methylation-dependent ligand reorganization about the metal plays a role in the conformational switching mechanism that converts Ada from a non-sequence-specific to a sequence-specific DNA-binding protein.     

Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme

Succinate dehydrogenase consists of two protein subunits and contains one FAD and three iron-sulfur clusters. The flavin is covalently bound to a histidine in the larger, Fp, subunit. The reduction oxidation midpoint potentials of the clusters designated S-1, S-2, and S-3 in Bacillus subtilis wild-type membrane-bound enzyme were determined as +80, -240, and -25 mV, respectively. Magnetic spin interactions between clusters S-1 and S-2 and between S-1 and S-3 were detected by using EPR spectroscopy. The point mutations of four B. subtilis mutants with defective Fp subunits were mapped. The gene of the mutant specifically lacking covalently bound flavin in the enzyme was cloned. The mutation was determined from the DNA sequence as a glycine to aspartate substitution at a conserved site seven residues downstream from the histidine that binds the flavin in wild-type enzyme. The redox midpoint potential of the iron-sulfur clusters and the magnetic spin interactions in mutated succinate dehydrogenases were indistinguishable from the those of the wild type. This shows that flavin has no role in the measured magnetic spin interactions or in the structure and stability of the iron-sulfur clusters. It is concluded from sequence and mutant studies that conserved amino acid residues around the histidyl-FAD are important for FAD binding; however, amino acids located more than 100 residues downstream from the histidyl in the Fp subunit can also effect flavinylation.     

Insights into the Initiation of JC Virus DNA Replication Derived from the Crystal Structure of the T-Antigen Origin Binding Domain

JC virus is a member of the Polyomavirus family of DNA tumor viruses and the causative agent of progressive multifocal leukoencephalopathy (PML). PML is a disease that occurs primarily in people who are immunocompromised and is usually fatal. As with other Polyomavirus family members, the replication of JC virus (JCV) DNA is dependent upon the virally encoded protein T-antigen. To further our understanding of JCV replication, we have determined the crystal structure of the origin-binding domain (OBD) of JCV T-antigen. This structure provides the first molecular understanding of JCV T-ag replication functions; for example, it suggests how the JCV T-ag OBD site-specifically binds to the major groove of GAGGC sequences in the origin. Furthermore, these studies suggest how the JCV OBDs interact during subsequent oligomerization events. We also report that the OBD contains a novel “pocket”; which sequesters the A1 & B2 loops of neighboring molecules. Mutagenesis of a residue in the pocket associated with the JCV T-ag OBD interfered with viral replication. Finally, we report that relative to the SV40 OBD, the surface of the JCV OBD contains one hemisphere that is highly conserved and one that is highly variable.     

Histidine ligands in bacterial metallothionein enhance cluster stability

The cyanobacterial metallothionein (MT) SmtA is the prototype for bacterial MTs and protects against elevated levels of zinc. In contrast to mammalian MTs, bacterial MTs coordinate to metal ions not only via cysteine sulfurs, but unusually for MTs, also via histidine nitrogens. To investigate whether histidine coordination in these metal-sulfur clusters provides advantages over S-coordination only, we mutated the two metal-binding histidine residues in the cyanobacterial MT SmtA from Synechococcus PCC7942 to cysteines. We show that the mutant proteins are still capable of binding up to four zinc ions as is the wild-type protein. However, the mutations perturb protein folding and metal-binding dynamics. Interestingly, several homologues of SmtA also show variations in these two residues. We conclude that histidine residues in Synechococcus PCC7942 SmtA have a stabilising effect due to electrostatic interactions that impact on protein folding and metal cluster charge, and are involved in fine-tuning the reactivity of the bound metal ions.     

Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants.

We constructed insertion and deletion mutants with mutations within the adeno-associated virus (AAV) sequences of the infectious recombinant plasmid pSM620. Studies of these mutants revealed at least three AAV phenotypes. Mutants with mutations between 11 and 42 map units were partially or completely defective for rescue and replication of the AAV sequences from the recombinant plasmids (rep mutants). The mutants could be complemented by mutants with replication-positive phenotypes. The protein(s) that is affected in rep mutants has not been identified, but the existence of the rep mutants proves that at least one AAV-coded protein is required for viral DNA replication. Also, the fact that one of the rep mutant mutations maps within the AAV intron suggests that the intron sequences code for part of a functional AAV protein. Mutants with mutations between 63 and 91 map units synthesized normal amounts of AAV duplex DNA but could not generate single-stranded virion DNA (cap mutants). The cap phenotype could be complemented by rep mutants and is probably due to a defect in the major AAV capsid protein, VP3. This suggests that a preformed capsid or precursor is required for the accumulation of single-stranded AAV progeny DNA. Mutants with mutations between 48 and 55 map units synthesized normal amounts of AAV single-stranded and duplex DNA but produced substantially lower yields of infectious virus particles than wild-type AAV (lip mutants). The lip phenotype is probably due to a defect in the minor capsid protein, VPI, and suggests the existence of an additional (as yet undiscovered) AAV mRNA. Evidence is also presented for recombination between mutant AAV genomes during lytic growth.     

Identification by mutagenesis of conserved arginine and tryptophan residues in rat liver carnitine palmitoyltransferase I important for catalytic activity

Carnitine palmitoyltransferase I catalyzes the conversion of long-chain acyl-CoA to acylcarnitines in the presence of l-carnitine. To determine the role of the conserved arginine and tryptophan residues on catalytic activity in the liver isoform of carnitine palmitoyltransferase I (L-CPTI), we separately mutated five conserved arginines and two tryptophans to alanine. Substitution of arginine residues 388, 451, and 606 with alanine resulted in loss of 88, 82, and 93% of L-CPTI activity, respectively. Mutants R601A and R655A showed less than 2% of the wild type L-CPTI activity. A change of tryptophan 391 and 452 to alanine resulted in 50 and 93% loss in carnitine palmitoyltransferase activity, respectively. The mutations caused decreases in catalytic efficiency of 80-98%. The residual activity in the mutant L-CPTIs was sensitive to malonyl-CoA inhibition. Mutants R388A, R451A, R606A, W391A, and W452A had no effect on the K(m) values for carnitine or palmitoyl-CoA. However, these mutations decreased the V(max) values for both substrates by 10-40-fold, suggesting that the main effect of the mutations was to decrease the stability of the enzyme-substrate complex. We suggest that conserved arginine and tryptophan residues in L-CPTI contribute to the stabilization of the enzyme-substrate complex by charge neutralization and hydrophobic interactions. The predicted secondary structure of the 100-amino acid residue region of L-CPTI, containing arginines 388 and 451 and tryptophans 391 and 452, consists of four alpha-helices similar to the known three-dimensional structure of the acyl-CoA-binding protein. We predict that this 100-amino acid residue region constitutes the putative palmitoyl-CoA-binding site in L-CPTI.     

Disease-associated mutations inactivate AMP-lysine hydrolase activity of Aprataxin

Ataxia-oculomotor apraxia syndrome 1 is an early onset cerebellar ataxia that results from loss of function mutations in the APTX gene, encoding Aprataxin, which contains three conserved domains. The forkhead-associated domain of Aprataxin mediates protein-protein interactions with molecules that respond to DNA damage, but the cellular phenotype of the disease does not appear to be consistent with a major loss in DNA damage responses. Disease-associated mutations in Aprataxin target a histidine triad domain that is similar to Hint, a universally conserved AMP-lysine hydrolase, or truncate the protein NH2-terminal to a zinc finger. With novel fluorigenic substrates, we demonstrate that Aprataxin possesses an active-site-dependent AMP-lysine and GMP-lysine hydrolase activity that depends additionally on the zinc finger for protein stability and on the forkhead associated domain for enzymatic activity. Alleles carrying any of eight recessive mutations associated with ataxia and oculomotor apraxia encode proteins with huge losses in protein stability and enzymatic activity, consistent with a null phenotype. The mild presentation allele, APTX-K197Q, associated with ataxia but not oculomotor apraxia, encodes a protein with a mild defect in stability and activity, while enzyme encoded by the atypical presentation allele, APTX-R199H, retained substantial function, consistent with altered and not loss of activity. The data suggest that the essential function of Aprataxin is reversal of nucleotidylylated protein modifications, that all three domains contribute to formation of a stable enzyme, and that the in vitro behavior of cloned APTX alleles can score disease-associated mutations.     

The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase

The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function.     

The zinc finger region of simian virus 40 large T antigen

Simian virus 40 large T antigen contains a single sequence element with an arrangement of cysteines and histidines that is characteristic of a zinc finger motif. The finger region maps from amino acids 302 through 320 and has the sequence Cys-302LeuLysCys-305IleLysLysGluGlnProSerHisTyrLysTyrHis- 317GluLysHis-320. In a conventional representation, the binding of zinc to the cysteines and histidines at positions 302, 305, 317, and 320 would form two minor loops and one major loop from the intervening amino acids. We made single amino acid substitutions at every position in the finger to identify possible functional elements within the putative metal-binding domain. Amino acids in the zinc finger could be divided into three classes characterized by distinct roles in DNA replication and transformation. Class 1 consisted of amino acids in the two minor loops of the finger and in the amino-terminal part of the major loop. Mutations here did not affect either replication or transformation. Class 2 consisted of the SerHisTyrLysTyr amino acids located in the carboxy terminus of the major loop of the finger. Mutations in this contiguous region reduced replication of the mutant viruses to different degrees. This clustering suggested that the region is an active site important for a specific function in DNA replication. With the exception of a mutation in the histidine at position 313, these mutations had no effect on transformation. Class 3 consisted of the proposed zinc-binding amino acids at positions 302, 305, 317, and 320 and the histidine at position 313 in the major loop of the finger. Mutations in these amino acids abolished the viability of the virus completely and had a distinctive effect on the transforming functions of the protein. Thus, the five cysteines and histidines of class 3 may play an important role in determining the overall structure of the protein. The histidine at position 313 may function both in the active site where it is located and in cooperation with the proposed zinc-binding ligands.     

Functional equivalency and diversity of cis-acting elements among yeast replication origins.

The DNA replication origins of the yeast Saccharomyces cerevisiae require several short functional elements, most of which are not conserved in sequence. To better characterize ARS305, a replicator from a chromosomal origin, we swapped functional DNA elements of ARS305 with defined elements of ARS1. ARS305 contains elements that are functionally exchangeable with ARS1 A and B1 elements, which are known to bind the origin recognition complex; however, the ARS1 A element differs in that it does not require a 3' box adjacent to the essential autonomously replicating sequence consensus. At the position corresponding to ARS1 B3, ARS305 has a novel element, B4, that can functionally substitute for every type of short element (B1, B2, and B3) in the B domain. Unexpectedly, the replacement of element B4 by ARS1 B3, which binds ABF1p and is known as a replication enhancer, inhibited ARS305 function. ARS305 has no short functional element at or near positions corresponding to the B2 elements in ARS1 and ARS307 but contains an easily unwound region whose functional importance was supported by a broad G+C-rich substitution mutation. Surprisingly, the easily unwound region can functionally substitute for the ARS1 B2 element, even though ARS1 B2 was found to possess a distinct DNA sequence requirement. The functionally conserved B2 element in ARS307 contains a known sequence requirement, and helical stability analysis of linker and minilinker mutations suggested that B2 also contains a DNA unwinding element (DUE). Our findings suggest that yeast replication origins employ a B2 element or a DUE to mediate a common function, DNA unwinding during initiation, although not necessarily through a common mechanism.