Challenges in Protein QM/MM Simulations with Intra-Backbone Link Atoms.

Hybrid quantum mechanical/molecular mechanical (QM/MM) simulations fuel discoveries in many fields of science including computational biochemistry and enzymology. Development of more convenient tools leads to an increase in the number of works in which mechanical insights into enzymes' mode of operation are obtained. Most commonly, these tools feature hydrogen-capping (link atom) approach to provide coupling between QM and MM subsystems across a covalent bond. Extensive studies were conducted to provide a solid foundation for the correctness of such an approach when a bond to a nonpolar MM atom is considered. However, not every task may be accomplished this way. Certain scenarios of using QM/MM in computational enzymology encourage or even necessitate the incorporation of backbone atoms into the QM region. Two out of three backbone atoms are polar, and in QM/MM with electrostatic embedding, a neighboring link atom will be hyperpolarized. Several schemes to mitigate this effect were previously proposed alongside a rigorous assessment of quantitative effects on model systems. However, it was not clear whether they may translate into qualitatively different results and how link atom hyperpolarization may manifest itself in a real-life enzymological scenario. Here, we show that the consequences of such an artifact may be severe and may completely overturn the conclusions drawn from the simulations. Our case advocates for the use of charge redistribution schemes whenever intra-backbone QM/MM boundaries are considered. Moreover, we addressed how different boundary types and charge redistribution schemes influence backbone dynamics. We showed that the results are heavily dependent on which boundary MM terms are retained, with charge alteration being of secondary importance. In the worst case, only three intra-backbone boundaries may be used with relative confidence in the adequacy of resulting simulations, irrespective of the hyperpolarization mitigation scheme. Thus, advances in the field are certainly needed to fuel new discoveries. As of now, we believe that issues raised in this work might encourage authors in the field to report what boundaries, boundary MM terms, and charge redistribution schemes they are using, so their results may be correctly interpreted.