The energetic network of hotspot residues between Cdc25B phosphatase and its protein substrate

Abstract

We have investigated the functional network of hotspot residues at the remote docking site of two cell cycle regulators, namely Cdc25B phosphatase and its native protein substrate Cdk2-pTpY/CycA. Specifically, we have studied the roles of energetically important residues (Arg488, Arg492, Tyr497 on Cdc25B and Asp206 and Asp210 on Cdk2-pTpY/CycA) by generating a diverse set of substitutions and performing double and triple mutant cycle analyses. This transient protein-protein interaction is particularly well-suited for this mutagenic approach because various control experiments ensure that the effect of each mutation is limited to the interaction of interest. We find binary coupling energies for ion pairs and hydrogen bonds ranging from 0.7 kcal/mol to 3.9 kcal/mol and ternary coupling energies of 1.9 kcal/mol and 2.8 kcal/mol. Overall our biochemical analyses are in good agreement with the docked structure of the complex and suggest the following roles for the individual hotspot residues on Cdc25B. The most important contributor, Arg492, forms a specific and tight bidentate interaction with Asp206 and a weaker interaction with Asp210 that cannot be replaced by a Lys. Although Tyr497 does not directly participate in this ionic network, it is important for buttressing Arg492 using both its hydrophobic (aromatic ring) and hydrophilic characteristics (hydrogen bonding). Arg488 participates less specifically in the electrostatic network with Asp206 and Asp210 of the protein substrate as it can partially be replaced by Lys. Our data provide insight how a cluster of residues in a docking site remote from the site of the chemical reaction can bring about efficient and specific substrate recognition.

Figure 1

Snapshot of the Cdc25B – Cdk2-pTpY/CycA complex highlighting the network of hotspot residues at the remote docking site. Cdc25B, Cdk2, and CycA are shown in cartoon in purple, blue, and gray, respectively. The active site region is denoted by a dashed circle and pThr14 and pTyr15 of Cdk2 and the active site loop of Cdc25 (Cys473 to Arg478) are shown in licorice. The remote hotspot is denoted by a dotted circle, with a blow-up on the right showing the average distances (heavy atom to heavy atom) observed from the molecular dynamics trajectory at 305K from 200 – 1000 ps . Residues from Cdk2 are underlined.

Figure 2

Double mutant cycle analysis with alternative hotspot mutants. The pair-wise coupling energies (ΔΔG_int) were determined according to Equation 1 and 2. The listed numbers are mean values of at least three separate determinations (± 0.7) and in units of kcal/mol.

Figure 3

Construction of a cube for triple mutant cycle analysis. MT_i represents each mutant residue whereas WT denotes a native residue. Each face of the cube represents a complete double mutant cycle, and opposing faces are identical except one cycle includes the third mutant residue. The difference between ΔΔG_ints obtained from the opposing faces of cube represents the ternary coupling energy of the entire system.

Figure 4

Calculations of pair-wise coupling energies for triple mutant cycle analyses of R492L/Y497A/D206A. Two horizontally juxtaposed boxes are opposing faces of the triple mutant cycle cube described in Figure 3. Each three-digit number represents the absence of the particular wild-type residue (i.e. R492L, Y497A, D206A) whereas WT stands for the presence of the native residue. Each value next to an arrow represents the observed (normal text) or calculated (italicized text) change in free energy upon mutation. The ΔΔG_int and the set of three WT and mutant residues in the center of each cycle stands for the determined coupling energy for the two mutant residues in the presence or absence of the third residue (panel A vs. B, etc.). A positive ΔΔG_int represents a favorable pair-wise interaction, a negative value represents an unfavorable interaction, and zero is neutral. The difference of ΔΔG_ints between two side-by-side boxes is the ternary coupling energy and is identical for all three pairs of double mutant cycles. All the values are in units of kcal/mol.

Figure 5

Calculations of pair-wise coupling energies for triple mutant cycle analyses of R492L/Y497A/D210A. As for Figure 4, but replacing D206A with D210A.