Identification of the Magnesium-binding Domain of the High-affinity ATP-binding Site of the Bacillus subtilis and Escherichia coli SecA Protein

The hepatitis C virus nonstructural 5B protein (NS5B) protein has been shown to require either magnesium or manganese for its RNA-dependent RNA polymerase activity. As a first step toward elucidating the nature and the role(s) of the metal ions in the reaction chemistry, we have utilized endogenous tryptophan fluorescence to quantitate the interactions of magnesium and manganese ions with this protein. The association of either Mg 2؉ or Mn 2؉ ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was used to determine the apparent dissociation constants for both ions. The apparent K d values for the binding of Mg 2؉ and Mn 2؉ ions to the free enzyme were 3.1 and 0.3 mM, respectively. Dual ligand titration experiments demonstrated that both ions bind to a single common site, for which they compete. The kinetics of real time metal ion binding to the NS5B protein were also investigated. Based on the results of our fluorescence and near-UV circular dichroism experiments, we show that NS5B undergoes conformational changes upon the binding of metal ions. However, this process does not significantly stimulate the binding to the RNA or NTP substrates. We envisage that the ioninduced conformational change is a prerequisite for catalytic activity by both correctly positioning the side chains of the residues located in the active site of the enzyme and also contributing to the stabilization of the intermediate transition state. Currently, hepatitis C virus (HCV)1 is the leading etiological agent of non-A non-B hepatitis, with more than 170 million people worldwide being infected with HCV (1). About 80% of patients with acute HCV infection will progress to chronic hepatitis. Of these, 20% will develop cirrhosis, and 1-5% will develop hepatocellular carcinoma (2-5). HCV is a positive, single-stranded RNA virus of the Flaviviridae family. The genome is ϳ10,000 nucleotides long and encodes a single polyprotein of about 3,010 amino acids (6). The polyprotein is processed by both host cell and viral proteases into three major structural proteins and several nonstructural proteins necessary for viral replication (6).One key enzyme encoded by HCV is the nonstructural 5B protein (NS5B), which has been shown to be an RNA-dependent RNA polymerase (7-12). The HCV NS5B protein contains characteristic motifs, such as the GDD motif, shared by RNAdependent RNA polymerases (13). The NS5B protein is thus believed to be responsible for the genome replication of HCV. Indeed, polymerase activity has been demonstrated with recombinant NS5B expressed in both insect cells and Escherichia coli (8, 14 -26). The activity has been extensively studied because it is one of the major targets for the development of antiviral drugs. The NS5B protein can utilize a wide range of RNA molecules as template, although it appears to prefer certain homopolyribonucleotides (27). By itself, NS5B appears to lack specificity for HCV RNA and displays activity on heterologous nonviral RNA (8). This lack of spec...

Section: Fig 2 Titration Of Ns5b With Mgmentioning

confidence: 82%

Characterization of the Metal Ion Binding Properties of the Hepatitis C Virus RNA Polymerase

Bougie

Charpentier

Bisaillon

2003

“…Specifically, the broadening of resonances arising from both mobile segments upon addition of Mn 2ϩ indicates that both mobile regions 1 and 2 are close to the Mg 2ϩ -binding site, which is proposed to involve residues Asp 209 and Asp 217 (35). The proximity of mobile region 1 to these residues is not surprising, given the observed direct interactions between these segments in the structure (21), but these data provide the first direct evidence that the extreme C-terminal region of SecA (unresolvable in the crystal structure) approaches closely to the interface between NBF-I and -II.…”

Section: Locations Of Assigned Arginines Indicated By Open Circles)mentioning

confidence: 99%

Functionally Significant Mobile Regions of Escherichia coli SecA ATPase Identified by NMR

Chou¹,

Swain²,

Gierasch³

2002

SecA, a 204-kDa homodimeric protein, is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane. SecA promotes translocation by nucleotide-modulated insertion and deinsertion into the cytoplasmic membrane once bound to both the signal sequence and portions of the mature domain of the preprotein. SecA is proposed to undergo major conformational changes during translocation. These conformational changes are accompanied by major rearrangements of SecA structural domains. To understand the interdomain rearrangements, we have examined SecA by NMR and identified regions that display narrow resonances indicating high mobility. The mobile regions of SecA have been assigned to a sequence from the second of two domains with nucleotide-binding folds (NBF-II; residues 564 -579) and to the extreme C-terminal segment of SecA (residues 864 -901), both of which are essential for preprotein translocation activity. Interactions with ligands suggest that the mobile regions are involved in functionally critical regulatory steps in SecA.

“…The specific activity of these mutant proteins was less than onetenth that of the wild-type protein. DnaA A184V, DnaA46, and DnaA5 required longer incubation periods for expression of replication activity; the time lag for DNA replication reaction for these mutant DnaA proteins has been reported (16,17,27). In the case of DnaA K178I and DnaA D235N, the time course of DNA replication is approximately linear, as is the case for the wild-type protein (Fig.…”

Section: Table Imentioning

confidence: 76%

Site-directed Mutational Analysis for the ATP Binding of DnaA Protein

Mizushima¹,

Takaki²,

Kubota³

et al. 1998

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, is activated by binding to ATP in vitro. We introduced site-directed mutations into two amino acids of the protein conserved among various ATP-binding proteins and examined functions of the mutated DnaA proteins, in vitro and in vivo. Both mutated DnaA proteins (Lys-178 3 Ile or Asp-235 3 Asn) lost the affinity for both ATP and ADP but did maintain binding activity for oriC. Specific activities in an oriC DNA replication system in vitro were less than one-tenth those of the wild-type protein. Assay of the generation of oriC sites sensitive to P1 nuclease, using the mutated DnaA proteins, revealed a defect in induction of the duplex opening at oriC. On the other hand, expression of each mutated DnaA protein in the temperature-sensitive dnaA46 mutant did not complement the temperature sensitivity. We suggest that Lys-178 and Asp-235 of DnaA protein are essential for the activity needed to initiate oriC DNA replication in vitro and in vivo and that ATP binding to DnaA protein is required for DNA replication-related functions.Initiation of DNA replication, a key step in the regulation of cell proliferation, seems to be regulated by the control of activity of initiator proteins. DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli (1-4), has a high affinity for adenine nucleotides; the ATP-binding form of DnaA protein is active, whereas the ADP-binding form is inactive in an oriC DNA replication system in vitro (5). Synthesized organic compounds designed to block the ATP binding to DnaA protein specifically inhibited oriC DNA replication in vitro (6). These observations mean that adenine nucleotide binding to DnaA protein may regulate the activity of the protein in vivo.There is genetic evidence to support the notion that initiation of DNA replication is regulated by adenine nucleotide binding to DnaA protein in vivo; (i) spontaneous dnaA46 and dnaA5 mutants show recessive lethality and DnaA46 and DnaA5 proteins have a decreased affinity for ATP and ADP (7, 8); (ii) a spontaneous dnaAcos mutant shows dominant lethality due to overinitiation of DNA replication and DnaAcos protein loses the affinity for ATP and ADP (9, 10); (iii) site-directed mutation (Glu-204 3 Gln) also causes dominant lethality, and the mutated protein, DnaA E204Q, has decreased intrinsic ATPase activity (11).DnaA protein has Walker A and B motifs that are common to a number of nucleotide-binding proteins (12). Analysis by sitedirected mutation for conserved amino acids located in Walker A and B motifs of these nucleotide-binding proteins revealed the necessity of these amino acids for nucleotide binding and also for functions related to nucleotide-binding activity. Substitution of conserved amino acids located in Walker A and B motifs of SecA, an essential protein for protein translocation across cytoplasmic membranes in E. coli (13), showed that ATP binding and ATPase activity of this protein are essential for protein translocation, in vitro ...