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), Co2+/SO42−-bound (PDB: 5U7F), and Ni2+/SO42−-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.
Alt et al. (Fri,) studied this question.
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