Building better enzymes: Molecular basis of improved non‐natural nucleobase incorporation by an evolved DNA polymerase

Abstract

Obtaining semisynthetic microorganisms that exploit the information density of “hachimoji” DNA requires access to engineered DNA polymerases. A KlenTaq variant has been reported that incorporates the “hachimoji” P:Z nucleobase pair with a similar efficiency to that seen for Watson–Crick nucleobase incorporation by the wild type (WT) KlenTaq DNA polymerase. The variant polymerase differs from WT KlenTaq by only four amino acid substitutions, none of which are located within the active site. We now report molecular dynamics (MD) simulations on a series of binary complexes aimed at elucidating the contributions of the four amino acid substitutions to altered catalytic activity. These simulations suggest that WT KlenTaq is insufficiently flexible to be able to bind AEGIS DNA correctly, leading to the loss of key protein/DNA interactions needed to position the binary complex for efficient incorporation of the “hachimoji” Z nucleobase. In addition, we test literature hypotheses about the functional roles of each amino acid substitution and provide a molecular description of how individual residue changes contribute to the improved activity of the KlenTaq variant. We demonstrate that MD simulations have a clear role to play in systematically screening DNA polymerase variants capable of incorporating different types of nonnatural nucleobases thereby limiting the number that need to be characterized by experiment. It is now possible to build DNA molecules containing nonnatural nucleobase pairs in addition to A:T and G:C. Exploiting this development in synthetic biology requires engineered DNA polymerases that can replicate nonnatural nucleobase pairs. Computational studies on a DNA polymerase variant reveal how amino acid substitutions outside of the active site yield an enzyme that replicates nonnatural nucleobase pairs with high efficiency. This work will facilitate efforts to obtain bacteria possessing an expanded genetic alphabet.

Building better polymerases: Engineering the replication of expanded genetic alphabets

“…Molecular dynamics (MD) simulations are a well-validated method of studying protein dynamics (95) and have yielded important information about conformational changes undergone by DNA polymerases (96,97). Our recent work also demonstrates that MD simulations can be used to understand how substitutions far from the active site might alter substrate selectivity (98). For example, microsecond trajectories have shown how the dynamic motions of ZP Klentaq differ from those of WT Klentaq in the binary complex containing template-primer DNA duplexes (Fig.…”

“…They can also highlight correlated motions in networks of residues that are critical to altered domain motions in the variant DNA polymerase (Fig. 6B) (98), which permit the engineered enzyme to bind WC and Z:P-containing template-primer duplexes in an equivalent fashion, thereby increasing the efficiency of UBP incorporation. This seems to be a consequence of replacing Met-444 and Asp-551 at the base of the thumb domain by valine and glutamate, respectively, which allows the fingers, palm, and thumb domains in ZP Klentaq to move into position about the AEGIS template-primer duplex.…”

“…Thus, replacing Met-444 by valine in the hydrophobic core changes the populated interactions with adjacent residues (Phe-564, Met-779, and Leu-780) in the hydrophobic core (Fig. 6C) with the consequence that the G helix becomes more flexible in the variant compared with the WT polymerase (98).…”

“…As well as modeling how amino acid substitutions might impact long time-scale motions, such as domain reorientations, MD simulations can illuminate specific molecular changes that have the greatest impact on the altered catalytic biophysical and catalytic properties of the DNA polymerase variant. In the case of ZP Klentaq, the role of each of the substitutions (M444V, P527A, D551E, or E832V) was determined using MD simulations of the four single-point Klentaq variants (98). These calculations showed that replacing Met-444 in the hydrophobic core of the palm domain with valine makes a significant contribution to altering dynamic motions in ZP Klentaq.…”

“…In addition, changing Asp-551 to glutamate alters the interactions of the H and I helices at the base of the thumb domain, which affects G helix flexibility (Fig. 6B) (98).…”