The Role of Metal Complexation in the Unfolding Energetics of a Nudix Hydrolase

Dihydroneopterin triphosphate pyrophosphatase (DHNTPase) catalyzes an essential step in bacterial folate biosynthesis. A characteristic of the enzyme is that it can be stabilized by divalent cations. To better characterize the nature of its stabilization, we combine equilibrium denaturation with all-atom adaptive steered molecular dynamics (ASMD) on three forms of E. coli DHNTPase─viz apo (PDB: 5U7E), ${\mathrm{Co}}^{2+}/{\mathrm{SO}}_4^{2-}$-bound (PDB: 5U7F), and ${\mathrm{Ni}}^{2+}/{\mathrm{SO}}_4^{2-}$-bound (PDB: 5U7H)─and identify the structural features that govern the native structure's resistance to unfolding. The metal-liganded forms of the enzyme were seen in experiments to unfold at a higher denaturant midpoint and with a slower rate than apo, indicating increased stability. ASMD yields the potential of mean force (PMF) profiles, and observables─such as native contacts Q, intrapeptide and protein-water H-bonds, residue distances, active-site spread, and site-resolved metal coordination─along a steered coordinate pulling the protein apart. Our findings support a pathway-specific mechanism in which the duration of active-site coherence (compact spread and intact coordination) is the dominant predictor of mechanical/chemical stability. Along the pulling coordinate, residues Glu 117 and Thr40, and metal-sulfate interactions are also seen to be levers for stabilization or disruption.