Mechanistic insights into the phosphoryl transfer reaction in cyclin-dependent kinase 2: A QM/MM study

Abstract

Cyclin-dependent kinase 2 (CDK2) is an important member of the CDK family exerting its most important function in the regulation of the cell cycle. It catalyzes the transfer of the gamma phosphate group from an ATP (adenosine triphosphate) molecule to a Serine/Threonine residue of a peptide substrate. Due to the importance of this enzyme, and protein kinases in general, a detailed understanding of the reaction mechanism is desired. Thus, in this work the phosphoryl transfer reaction catalyzed by CDK2 was revisited and studied by means of hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. Our results suggest that the base-assisted mechanism is preferred over the substrate-assisted pathway when one Mg 2+ is present in the active site, in agreement with a previous theoretical study. The base-assisted mechanism resulted to be dissociative, with a potential energy barrier of 14.3 kcal/mol, very close to the experimental derived value. An interesting feature of the mechanism is the proton transfer from Lys129 to the phosphoryl group at the second transition state, event that could be helping in neutralizing the charge on the phosphoryl group upon the absence of a second Mg 2+ ion. Furthermore, important insights into the mechanisms in terms of bond order and charge analysis were provided. These descriptors helped to characterize the synchronicity of bond forming and breaking events, and to characterize charge transfer effects. Local interactions at the active site are key to modulate the charge distribution on the phosphoryl group and therefore alter its reactivity.

How a Second Mg2+ Ion Affects the Phosphoryl-Transfer Mechanism in a Protein Kinase: A Computational Study

“…In this mechanism, Asp127 acts as a base activating the nucleophilic hydroxyl group through a HB interaction and receiving the proton once the phosphoryl transfer is almost completed. This late proton transfer reaction to Asp127 was also detected in previous computational studies in CDK2 with one Mg 2+ ion 35,36 and in computational studies in PKA with two metal ions 40,41,48,49 to its homologous residue (Asp166). We explored through the adaptive string method the complete base-assisted mechanism, which involves, in a second step, a proton transfer from Asp127 to one of the γ-phosphate oxygens (O2γ in this case), leaving the transferred phosphoryl group protonated.…”

“…A more recent QM/MM study analyzing the potential energy surface (PES) of the reaction performed in our group also reaffirmed that the base-assisted mechanism is more favorable than the substrate-assisted one. 36 These last results agreed with computational studies in PKA [40][41][42]48,49 and in other kinases, 50,51 which also point to a base-assisted mechanism with a strong dissociative character as the operating one, though the substrate-assisted mechanism has been proposed as the most favorable in other kinases. 52,53 Despite the numerous experimental and computational studies performed in CDK2, there is no clear understanding of how much a second Mg 2+ ion would affect the phosphoryltransfer mechanism and its associated free energy barrier.…”

“…The binding site for this Mg 2+ ion is the one that had been found occupied previously in CDK2 crystals, 4,13,32 and therefore, until just recently, the phosphoryl transfer mechanism in CDK2 had been described involving only that single Mg 2+ ion (1-Mg system). [33][34][35][36] However, Bao et al 6 obtained a crystallographic structure of a pCDK2/Cyclin A transition state complex mimic with a second Mg 2+ ion (Mg1) bound within the active site, showing that CDK2 most probably works with two Mg 2+ ions as cofactors (2-Mg system). In this structure, the second ion is coordinated by a β-phosphate oxygen from ADP and a fluorine atom from the transition state (TS) mimic MgF3 -, which would be representing a γ-phosphate oxygen if ATP was present in the active site; in a bidentate coordination by Asp145, and two water molecules (Fig.…”

How a Second Mg2+ Ion Affects the Phosphoryl Transfer Mechanism in a Protein Kinase: A Computational Study

“…Therefore, it is very clear that the TSs described at the DFTB3/ff99SB level exhibit a high dissociative character, which is expected for the base-assisted mechanism and agrees with other computational studies. 35,36,40 protonated making a HB with the Oγ(Thr) oxygen (Fig. S4B).…”

How a Second Mg2+ Ion Affects the Phosphoryl Transfer Mechanism in a Protein Kinase: A Computational Study

“…The binding site for this Mg 2+ ion is the one that had been found occupied previously in CDK2 crystals, 4,13,32 and therefore, until just recently, the phosphoryl transfer mechanism in CDK2 had been described involving only that single Mg 2+ ion (1-Mg system). [33][34][35][36] However, Bao et al 6 obtained a crystallographic structure of a pCDK2/Cyclin A transition state complex mimic with a second Mg 2+ ion (Mg1) bound within the active site, showing that CDK2 most probably works with two Mg 2+ ions as cofactors (2-Mg system). In this structure, the second ion is coordinated by a β-phosphate oxygen from ADP and a fluorine atom from the transition state (TS) mimic MgF3 -, which would be representing a γ-phosphate oxygen if ATP was present in the active site; in a bidentate coordination by Asp145, and two water molecules (Fig.…”