Exploring the role of large conformational changes in the fidelity of DNA polymerase β

Abstract

The relationships between the conformational landscape, nucleotide insertion catalysis and fidelity of DNA polymerase β are explored by means of computational simulations. The simulations indicate that the transition states for incorporation of right (R) and wrong (W) nucleotides reside in substantially different protein conformations. The protein conformational changes that reproduce the experimentally observed fidelity are significantly larger than the small rearrangements that usually accompany motions from the reactant state to the transition state in common enzymatic reactions. Once substrate binding has occurred, different constraints imposed on the transition states for insertion of R and W nucleotides render it highly unlikely that both transition states can occur in the same closed structure, because the predicted fidelity would then be many orders of magnitude too large. Since the conformational changes reduce the transition state energy of W incorporation drastically they decrease fidelity rather than increase it. Overall, a better agreement with experimental data is attained when the R is incorporated through a transition state in a closed conformation and W is incorporated through a transition state in one or perhaps several partially open conformations. The generation of free energy surfaces for R and W also allow us to analyze proposals about the relationship between induced fit and fidelity.

Why nature really chose phosphate

“…Kamerlin et al 2009b ;Klähn et al 2006), and also, unlike e.g. (Prasad & Warshel, 2011 ;Xiang et al 2008), this study was not properly validated by taking into account the corresponding reaction in water. In contrast, by calculating the PMF for this process using proper relaxation and extensive sampling (Prasad & Warshel, 2011), we obtained a much smaller barrier of 3Á58 kcal mol x1 to the prechemistry step.…”

“…Now one of the main requirements for elucidating the catalytic mechanism has been the ability to obtain free-energy surfaces that are not only sufficiently reliable to capture the correct reaction path, but also to be able to reproduce the proper free-energy surfaces for any possible ' prechemistry ' events that occur prior to the phosphoryl transfer itself. This is an important issue, since, with the exception of (Xiang et al 2008), previous fidelity studies (e.g. Alberts et al 2007 ;Bojin & Schlick, 2007 ;Lin et al 2006Lin et al , 2008Wang & Schlick, 2008) have used a relatively rigid protein model, coupled with energy minimization.…”

“…42. The early conceptual description of this figure has emerged from our previous studies of DNA polymerases (Florián et al 2005 ;Xiang et al 2008), in which we concluded (based on calculations of the barrier for the rate-determining chemical step) that the path along the chemical coordinate passes at a somewhat different value of the ' orthogonal ' coordinate corresponding to the protein structural changes (which is typically considered to correspond to the motion between the open and closed forms). We would like to emphasize that this view is fundamentally different from related assumptions (e.g.…”

“…Unfortunately, these arguments are not based on clear molecular concepts, and lack support from consistent simulations. In fact, even the common suggestion that the fidelity is due to an induced fit effect is not based on clear structure-energy analysis (see Xiang et al 2008). However, if one does want to perform such an analysis, one has to first take into account the actual rates of the conformational transition versus the nucleotide insertion step (which, stopped flow fluorescence analysis has suggested is approximately 5 times slower than the conformational transition Bakhtina et al 2005).…”

“…Apparently, even with a well-defined surface, we must define what the reference state for the induced fit effect is. When this is done (Xiang et al 2008;Prasad and Warshel 2011), we get different results for different definitions. However, none of these results tell us how the induced fit determined the fidelity.…”