MD and QM/MM Study of the Quaternary HutP Homohexamer Complex with mRNA, l-Histidine Ligand, and Mg²

The HutP protein from B. subtilis regulates histidine metabolism by interacting with an antiterminator mRNA hairpin in response to the binding of l-histidine and Mg²⁺. We studied the functional ligand-bound HutP hexamer complexed with two mRNAs using all-atom microsecond-scale explicit-solvent MD simulations performed with the Amber force fields. The experimentally observed protein-RNA interface exhibited good structural stability in the simulations with the exception of some fluctuations in an unusual adenine-threonine interaction involving two closely spaced H-bonds. We further investigated this interaction by comparing QM/MM and MM optimizations, using the QM region comprising almost 350 atoms described at the DFT-D3 level. The QM/MM method clearly improved the adenine-threonine interaction compared to MM, especially when the X-H bond lengths were frozen during the MM optimization to mimic the use of SHAKE in the MD simulations. Thus, both the MM approximation and the use of SHAKE can compromise the description of H-bonds at protein-RNA interfaces. The simulations also revealed a notable Mg²⁺-parameter dependence in the behavior of the ligand-binding pocket (LBP). With the SPC/E water model, the 12-6 Åqvist and Li&Merz parameters provided an entirely stable LBP structure, but the 12-6 Allnér and 12-6-4 Li&Merz parametrizations resulted in a progressive loss of direct nitrogen-Mg²⁺ LBP coordination. The Allnér and Li&Merz 12-6 parametrizations were also tested with the TIP3P water model; the LBP was destabilized in both cases. This illustrates the difficulty of consistently describing different Mg²⁺ interactions using nonpolarizable force fields. Overall, the simulations support the hypothesis that HutP protein becomes fully structured upon ligand binding. Subsequent RNA binding does not affect the protein structure, in keeping with the mechanism inferred from experimental structures.