Elucidating the Phosphate Binding Mode of Phosphate-Binding Protein: The Critical Effect of Buffer Solution

Abstract

Phosphate is an essential component of cell functions, and the specific transport of phosphorus into a cell is mediated by phosphate-binding protein (PBP). The mechanism of PBP-phosphate recognition remains controversial: on the basis of similar binding affinities at acidic and basic pHs, it is believed that the hydrogen network in the binding site is flexible to adapt to different protonation states of phosphates. However, only hydrogen (1H) phosphate was observed in the sub-angstrom X-ray structures. To address this inconsistency, we performed molecular dynamics simulations using the AMOEBA polarizable force field. Structural and free energy data from simulations suggested that 1H phosphate was the preferred bound form at both pHs. The binding of dihydrogen (2H) phosphate disrupted the hydrogen-bond network in the PBP pocket, and the computed affinity was much weaker than that of 1H phosphate. Furthermore, we showed that the discrepancy in the studies described above is resolved if the interaction between phosphate and the buffer agent is taken into account. The calculated apparent binding affinities are in excellent agreement with experimental measurements. Our results suggest the high specificity of PBP for 1H phosphate and highlight the importance of the buffer solution for the binding of highly charged ligands.

Accurate Biomolecular Simulations Account for Electronic Polarization

“…Moreover, accelerated sampling methods start to be applied also to polarizable approaches (Célerse et al, 2019) offering improved simulation capabilities and access to accurate and fast evaluation of free energies of binding thanks to GPUs (Harger et al, 2017). Such capabilities allow to tackle hard systems as in the case of the Phosphate binding mode of the Phosphate-binding protein where it was possible to highlight the critical effect of the buffer solution ending a long standing controversy thanks to free energy computations (Qi et al, 2018).…”

“…The representation of electronic polarization in classical MD simulations can vary largely with Drude and induced point dipoles approaches on one side and continuum approximations on the other (Cieplak et al, 2009; Lopes et al, 2009; Leontyev and Stuchebrukhov, 2011; Schröder, 2012; Baker, 2015; Shi et al, 2015; Lemkul et al, 2016; Bedrov et al, 2019; Jing et al, 2019). With the advances in both computational power together with theory and algorithms it is practically achievable to perform simulations with explicit polarizable dipoles on systems with relevant sizes and complexity (Qi et al, 2018; Bedrov et al, 2019; Lagardère et al, 2019; Loco et al, 2019). In particular, it is currently realistic to perform simulations with explicit polarization at time scales, which are competitive to the standard fixed-charge simulations (Lemkul et al, 2016; Lagardère et al, 2018; Célerse et al, 2019).…”

Control of the phoBR Regulon in Escherichia coli

“…PstS binds P i under physiological conditions with a K d (dissociation constant) of ∼10 μM (108). Molecular dynamics analysis suggest that PstS binds the 1H form of P i , not the 2H form (109), although this issue is not entirely settled (110). PstC and PstA compose the membrane-spanning components of the transporter (111,112).…”

Computational insights into the binding of IN17 inhibitors to MELK

“…The positions of substitution groups R1, R2, and R3 are labeled. Image generated using Ligplot+ [56] Correlation between experimental binding affinity and computational prediction. All values in kcal mol −1 Fig.…”

“…In addition, the experimental measurement was performed at very high buffer concentration (50 mM vs 10 nM for protein), which can affect interaction of this pair due to the buffer agent (HEPES) being able to bind in the protein pocket [56,57]. Note that the HEPES or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, also contains the same piperazine moiety.…”