Human APOBEC3A (A3A) is a single-stranded DNA (ssDNA) cytidine deaminase that restricts viral pathogens and endogenous retrotransposons and plays a role in the innate immune response. Furthermore, its potential to act as a genomic DNA mutator has implications for a role in carcinogenesis. A deeper understanding of A3A’s deaminase and nucleic acid binding properties, which is central to its biological activities, has been limited by the lack of structural information. Here, we report the NMR solution structure of A3A and show that the critical interface for interaction with ssDNA substrates includes residues extending beyond the catalytic center. Importantly, by monitoring deaminase activity in real time, we find that A3A displays similar catalytic activity on A3A-specific TTCA- or A3G-specific CCCA-containing substrates, involving key determinants immediately 5′ of the reactive C. Our results afford novel mechanistic insights into A3A-mediated deamination and provide the structural basis for further molecular studies.

Cytochrome P450s (CYPs) exhibit a large plasticity and flexibility in the active site allowing for the binding of a large variety of substrates. The impact of plasticity and flexibility on ligand binding is investigated by docking 65 known CYP2D6 substrates to an ensemble of 2500 protein structures. The ensemble was generated by molecular dynamics simulations of CYP2D6 in complex with five representative substrates. The effect of induced fit, the conformation of Phe483, and thermal motion on the accuracy of site of metabolism (SOM) predictions is analyzed. For future predictions, the three most essential CYP2D6 structures were selected which are suitable for different kinds of ligands. We have developed a binary decision tree to decide which protein structure to dock the ligand into, such that each ligand needs to be docked only once, leading to successful SOM prediction in 80% of the substrates.

To overcome the problem of insufficient conformational sampling within biomolecular simulations, we have developed a novel Hamiltonian replica exchange molecular dynamics ͑H-REMD͒ scheme that uses soft-core interactions between those parts of the system that contribute most to high energy barriers. The advantage of this approach over other H-REMD schemes is the possibility to use a relatively small number of replicas with locally larger differences between the individual Hamiltonians. Because soft-core potentials are almost the same as regular ones at longer distances, most of the interactions between atoms of perturbed parts will only be slightly changed. Rather, the strong repulsion between atoms that are close in space, which in many cases results in high energy barriers, is weakened within higher replicas of our proposed scheme. In addition to the soft-core interactions, we proposed to include multiple replicas using the same Hamiltonian/level of softness. We have tested the new protocol on the GTP and 8-Br-GTP molecules, which are known to have high energy barriers between the anti and syn conformation of the base with respect to the sugar moiety. During two 25 ns MD simulations of both systems the transition from the more stable to the less stable ͑but still experimentally observed͒ conformation is not seen at all. Also temperature REMD over 50 replicas for 1 ns did not show any transition at room temperature. On the other hand, more than 20 of such transitions are observed in H-REMD using six replicas ͑at three different Hamiltonians͒ during 6.8 ns per replica for GTP and 12 replicas ͑at six different Hamiltonians͒ during 8.7 ns per replica for 8-Br-GTP. The large increase in sampling efficiency was obtained from an optimized H-REMD scheme involving soft-core potentials, with multiple simulations using the same level of softness. The optimization of the scheme was performed by fast mimicking ͓J. Hritz and C. Oostenbrink, J. Chem. Phys. 127, 204104 ͑2007͔͒.