Atomic Scale Determination of Enzyme Flexibility and Active Site Stability through Static Modes: Case of Dihydrofolate Reductase

Abstract

A Static Mode approach is used to screen the biomechanical properties of DHFR. In this approach, a specific external stimulus may be designed at the atomic scale granularity to arrive at a proper molecular mechanism. In this frame, we address the issues related to the overall molecular flexibility versus loop motions and versus enzymatic activity. We show that backbone motions are particularly important to ensure DHFR domain communication and notably highlight the role of a α-helix in Met20 loop motion. We also investigate the active site flexibility in different bound states. Whereas in the occluded conformation the Met20 loop is highly flexible, in the closed conformation backbone motions are no longer significant, the Met20 loop is rigidified by new intra- and intermolecular weak bonds, which stabilizes the complex and promotes the hydride transfer. Finally, while various simulations, including I14 V and I14A mutations, confirm that Ile14 is a key residue in catalytic activity, we isolate and characterize at the atomic scale how a specific intraresidue chemical group makes it possible to assist ligand positioning, to direct the nicotinamide ring toward the folate ring.

DHFR Inhibitors: Reading the Past for Discovering Novel Anticancer Agents

“…It is interesting to note that DHFR has no disulfide bridges and it does not need to be coordinated by metal ions to exercise its biochemical activity [8]. An important structural element of the enzyme is the presence of the “Met20” or “loop 1” consisting of residues 9-24 [17,18,19,20,21]. It helps to stabilize the nicotinamide ring of NADPH to promote the passage of hydride from NADPH to dihydrofolate and it is able to open, close or occlude, the active site of the enzyme [22,23].…”

Toward in Silico Biomolecular Manipulation through Static Modes: Atomic Scale Characterization of HIV-1 Protease Flexibility

“…35−41 In particular, we have successfully quantitatively correlated results to experiments, 36 and probed active site stability and mutation effects on enzymatic activity. 38 The static mode method allows calculating all the deformations of a molecule submitted to elementary external excitations. Each mode contains the deformation field induced by the specific excitation of a specific molecular site.…”

“…To this end, we systematically solicit the static modes while solving a constrained optimization problem: while conserving the amplitude of the force applied on each atom, we optimize its direction to get the maximum distance variation between two atoms. The calculation details have already been described in Brut et al 38 For each atomic constraint, we report the resulting Cα(Asp25)−Cα(Asp25′) distance variation in Figure 2A. This graph allows localizing the residues that impact Asp25−Asp25′ distance stability and potentially enzymatic activity.…”

“…Anharmonic effects could be introduced using matrices of higher order. Details about the method have already been published and partial results on several biomolecules reported. − In particular, we have successfully quantitatively correlated results to experiments, and probed active site stability and mutation effects on enzymatic activity . The static mode method allows calculating all the deformations of a molecule submitted to elementary external excitations.…”

“…For this reason, HIV-1 PR constitutes a good model system making it possible for us to validate the predictive nature of our method as well as to illustrate its potential beyond state of the art. In particular, we show how our static modes − can be post-treated in an interactive manner to design complex excitations. Through manipulation tools, an operator can design and evaluate an infinite set of single to multiple excitations, constraints, or interactions with no further computational cost.…”