Investigating the trade-off between folding and function in a multidomain Y-family DNA polymerase

Abstract

The way in which multidomain proteins fold has been a puzzling question for decades. Until now, the mechanisms and functions of domain interactions involved in multidomain protein folding have been obscure. Here, we develop structure-based models to investigate the folding and DNA-binding processes of the multidomain Y-family DNA polymerase IV (DPO4). We uncover shifts in folding mechanism among ordered domain-wise folding, backtracking folding, and cooperative folding, modulated by interdomain interactions. These lead to "U-shaped' folding kinetics. We characterize the effects of interdomain flexibility on the promotion of DPO4-DNA (un)binding, which probably contributes to the ability of DPO4 to bypass DNA lesions, a known biological role of Y-family polymerases. We suggest that the native topology of DPO4 leads to a trade-off between fast, stable folding and tight functional DNA binding. Our approach provides an effective way to quantitatively correlate the roles of protein interactions in conformational dynamics at the multidomain level.

Investigating the Conformational Dynamics of a Y-Family DNA Polymerase during Its Folding and Binding to DNA and a Nucleotide

“… 13 It is worth noting that our previous simulations with single-basin one-bead SBMs generated only one peak on the heat capacity curve and resulted in sigmoidal-like melting curves. 14 − 16 The results suggest that the presence of the side chain bead and electrostatics in the SBM is critical to recapture the global folding behaviors of DPO4. 31 Due to the simplified interactions and the coarse-grained nature in the SBM, the simulation temperatures cannot directly correspond to the experimental ones.…”

“…The multiple intermediate unfolding states of DPO4 were observed in our previous studies as a result of “divided-and-conquer” domain-wise folding. 14 , 16 …”

“… 39 Thus, Q DNA # can provide a more precise description at both unbound states and binding states, compared to our previous studies. 16 , 22 We further calibrated our model to the binding affinity by modulating the strengths of the DPO4–DNA interchain native contacts. From the quantified binding free energy landscape ( Figure 2 A), we identified four free energy minima that separate the DNA-binding process into three stages: from the completely dissociative unbound state (DNA US ) to the initially anchoring encounter complex (DNA EC ), then to the partially bound intermediate state (DNA IS ), and finally to the fully bound state (DNA BS ).…”

“…DPO4 has been observed to undergo stepwise unfolding with the intermediate observed in both the experiment 13 and the simulations. 14 − 16 The unfolding intermediate, which shows an extended linker and unstable LF domain interfaces with well-folded individual domains in DPO4, was further hypothesized to benefit the formation of multiple DPO4 conformations during its binding to DNA or proliferating cell nuclear antigen (PCNA). 13 This fact indicates a positive role of the DPO4 unfolding in facilitating the functional binding processes.…”

Highly Dynamic Polynuclear Metal Cluster Revealed in a Single Metallothionein Molecule

“…Using its ultrasharp AFM tip, it is able not only to image nanoscale surface but also to manipulate a single molecule mechanically as single-molecule force spectroscopy (SMFS) [8][9][10][11][12]. By stretching one molecule under defined coordination, SMFS can induce the conformational change of a molecule such as protein (un)folding [13][14][15], breaking a stable chemical bond as well as capturing important transient states [16][17][18][19][20][21]. For example, several metal-binding proteins have been studied by AFM-SMFS, in which the metal cluster is ruptured during protein unfolding [22][23][24][25][26][27][28][29].…”