Theoretical Development of DnaG Primase as a Novel Narrow-Spectrum Antibiotic Target

Abstract

The widespread use of antibiotics to treat infections is one of the reasons that global mortality rates have fallen over the past 80 years. However, antibiotic use is also responsible for the concomitant rise in antibiotic resistance because it results in dysbiosis in which commensal and pathogenic bacteria are both greatly reduced. Therefore, narrow-range antibiotics are a promising direction for reducing antibiotic resistance because they are more discriminate. As a step toward addressing this problem, the goal of this study was to identify sites on DnaG primase that are conserved within Gram-positive bacteria and different from the equivalent sites in Gram-negative bacteria. Based on sequence and structural analysis, the primase C-terminal helicase-binding domain (CTD) was identified as most promising. Although the primase CTD sequences are very poorly conserved, they have highly conserved protein folds, and Gram-positive bacterial primases fold into a compact state that creates a small molecule binding site adjacent to a groove. The small molecule would stabilize the protein in its compact state, which would interfere with the helicase binding. This is important because primase CTD must be in its open conformation to bind to its cognate helicase at the replication fork.

Figure 1

Phylogenetic full-length protein sequence trees for (A) DnaG primase and (B) DnaB helicase.

Figure 2

Domain organization of the DnaG–DnaB complex. The structures of (A) three individual domains and (B) three multidomain proteins and complexes have been determined. The organisms are abbreviated as follows: Gstea, G. stearothermophilus; Ecoli, E. coli; Saure, S. aureus; Mtube, M. tuberculosis and Aaeol, A. aeolicus. The structures from the PDB are 1D0Q;13TW;2LZN;1Z8S;2R5U;2AU3;2R6A; and 2R6C.

Figure 3

Primase zinc-binding domain. (A) The ZBD sequence and secondary structure alignment showing the zinc-binding residues in gray and the residues responsible for initiation specificity in purple (for Gram-positive organisms) and pink (for Gram-negative organisms). The numbers along the top relate to the residues in the S. aureus sequence. The five beta strands in the 1D0Q.pdb secondary structure from G. stearothermophilus are shown next. The identical (*), highly conserved (:), and conserved (.) residues are shown separately for the Gram-positive and Gram-negative organisms. (B) Primase ZBD from G. stearothermophilus showing the specificity residues, Ile58 and Phe59, in purple.

Figure 4

Primase CTD sequence alignment. The primase CTD from the organisms in Table 1 were aligned with numbers corresponding to the S. aureus sequence and with two arrows showing the location of two critical G. stearothermophilus residues. Along the top are the secondary structures from Gram-positive organisms and, along the bottom, from Gram-negative organisms. Among Gram-positives, there are two closed structures and one open. The red box and delta sign show the major structural differences between the open and closed conformations. In the center of the sequences are the few conserved residues (: is highly conserved;. is conserved). Along the bottom are the four open conformations from Gram-negative organisms. The green highlighted residues fill the small molecule binding pocket of the S. aureus closed conformation. The orange highlighted residues are at interface B. The purple highlighted residues are buried at interface A. The gray highlighted residues are conserved helix-stabilizing residues, two of which are not conserved in Gram-positive sequences, where the conformational change occurs.

Figure 5

Bacterial primase CTD conformational changes and interfaces. (A) Diagram of the S. aureus primase CTD in its open and closed conformations. Interfaces A and B are between the two DnaB NTD and the “open” conformation of a single primase CTD. Interface A residues are in purple and interface B in orange. Small molecules, such as acyclovir, bind to the groove formed by the closed conformation to form interface C in green. (B) Key interface residues are shown in the open conformation of S. aureus primase CTD, a homology model using 2R6C (G. stearothermophilus) as a template structure. (C) Key interface residues are shown in the closed conformation of S. aureus primase CTD (2LZN).

Figure 6

G. stearothermophilus DnaG CTD key interface A residues—E572, K585, and K592—on the terminal helical hairpin in blue as determined by virtual mutation.

Figure 7

DnaB helicase NTD sequence alignment. The DnaB NTD from the organisms in Table 1 were aligned with numbers corresponding to the S. aureus sequence. Along the top are the secondary structures from Gram-positive organisms and, along the bottom, from Gram-negative organisms. The secondary structure for M. tuberculosis is shown below the main alignments. The Gram-negative organisms and M. tuberculosis have a linker structure (red box and delta symbol) that differs from Gram-positives. In the center of the sequences are the identical (*), highly conserved (:) and conserved (.) residues. The purple highlighted residues are at interface A. The yellow highlighted residues are at interface B.

Figure 8

Colocalization of key residues at interface C. Homology model of the closed conformation of C. difficile primase CTD that was created using 2LZN (S. aureus) as a template structure. The green residues are equivalent to those that form the small molecule binding pocket in S. aureus primase. The red residues are the equivalent residues that are predicted to most perturb the DnaB interaction in G. stearothermophilus as described above.