Amino Acid Residues Critical for the Interaction between Bacteriophage T7 DNA Polymerase and Escherichia coli Thioredoxin

Abstract

Upon infection of Escherichia coli, bacteriophage T7 annexes a host protein, thioredoxin, to serve as a processivity factor for its DNA polymerase, T7 gene 5 protein. In a previous communication (Himawan, J., and Richardson, C. C. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9774 -9778), we reported that an E. coli strain encoding a Gly-74 to Asp-74 (G74D) thioredoxin mutation could not support wild-type T7 growth and that in vivo, six mutations in T7 gene 5 could individually suppress this G74D thioredoxin defect. In the present study, we report the purification and biochemical characterization of the G74D thioredoxin mutant and two suppressor gene 5 proteins, a Glu-319 to Lys-319 (E319K) mutant of gene 5 protein and an Ala-45 to Thr-45 (A45T) mutant. The suppressor E319K mutation, positioned within the DNA polymerization domain of gene 5 protein, appears to suppress the parental thioredoxin mutation by compensating for the binding defect that was caused by the G74D alteration. We suggest that the Glu-319 residue of T7 gene 5 protein and the Gly-74 residue of E. coli thioredoxin define a contact point or site of interaction between the two proteins. In contrast, the A45T mutation in gene 5 protein, located within the 3 to 5 exonuclease domain, does not suppress the G74D thioredoxin mutation by simple restoration of binding affinity. Based upon our understanding of the mechanisms of suppression, we propose a model for the T7 gene 5 protein-E. coli thioredoxin interaction.The concept of using genetic or suppressor analysis to investigate protein-protein interaction can be described as follows. If two proteins form a complex, then there must exist a contact point, or more likely, several contact points between them. These contact points would be defined by certain amino acid residues of one protein that must be physically adjacent to certain amino acid residues of the other protein. If a contact point amino acid from one protein is structurally altered significantly by mutation, then complex formation with the second protein would be destroyed. Theoretically, a productive complex could be formed once again by an alteration in the second protein that structurally compensates for the original mutation. Therefore, by mutating one protein of a complex and selecting for extragenic suppressor mutations in the second protein, one should be able to identify the contact points between the two proteins.We (1) have used extragenic suppressor analysis to investigate the interactions between two proteins that are involved in DNA replication in Escherichia coli infected with bacteriophage T7. Similar to our studies, other workers have also used suppressor analysis to study protein-protein interactions in the E. coli DNA replication system (2) and also in the DNA replication system of the yeast Saccharomyces cerevisiae (3). Specifically, we have been investigating by suppressor analysis the interaction between T7 gene 5 protein and E. coli thioredoxin. T7 gene 5 protein, the DNA polymerase of phage T7 (4, 5), has two enzymatic act...

Force-Clamp Spectroscopy Detects Residue Co-evolution in Enzyme Catalysis

“…These results are in agreement with the existence of an activity threshold (21). Because residues Pro 34 and Gly 74 are positioned within the catalytic groove and residue Asp 26 influences the protonation state of the reactive cysteine Cys 32 , we interpret these results in terms of both an evolutionary optimization of the enzyme-substrate binding affinity and the protonation state of the Trx active site. These results represent an important advance in obtaining information on enzyme catalysis by combining the evolutionary analysis of sequences with the experimental study of catalysis at the single molecule level, a method that could be applied to enzyme design.…”

“…This suggests that the rescuing of the activity is due to an allosteric chemical change. The mutation D26E may change the pK a of the reactive Cys 32 by facilitating multiple electrostatic interaction between the residues of the active site (43,48). These mutations are found, in the corresponding positions, in protein-disulfide isomerase and DsbA, oxidase members of the Trx superfamily (49,50).…”

“…The negatively correlated residues 34 and 74 belong to hydrophobic surfaces that interact with different proteins (29,32), in the binding groove of E. coli Trx. Binding residues are essential in mediating the correct positioning of the substrate, and mutations in those regions can alter the enzymatic reaction (47).…”

“…Residues Pro 34 and Asp 26 are thought to play a role in the protonation state of the reactive Cys 32 . Although there is controversy about the exact pK a values of the functional residues in positions 26 and 32 (31,43), it is known that the pK a value of the active site residues is a determinant in the function of Trx, and alterations in those values are responsible for changes in the activity of the different Trx superfamily members (44).…”

A unique loop in the DNA-binding crevice of bacteriophage T7 DNA polymerase influences primer utilization

“…The 3Ј-to-5Ј double-stranded DNA (dsDNA) exonuclease activity of T7 DNA polymerase was assayed essentially as described (39). The 32 P-labeled M13 dsDNA was prepared by extending a 25-mer primer annealed to M13 ssDNA in the presence of 300 mM each of dATP, dTTP, [␣-32 P]dCTP, and [␣-32 P]dGTP, 10 mM MgCl 2 , 1 mM DTT, 50 mM Tris⅐HCl (pH 7.5), and 50 mM NaCl by incubation with 5 nM T7 DNA polymerase for 3 min.…”