Insights into the importance of WPD-loop sequence for activity and structure in protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) possess a conserved mobile catalytic loop, the WPD-loop, which brings an aspartic acid into the active site where it acts as an acid/base catalyst. Prior experimental and computational studies, focused on the human enzyme PTP1B and the PTP from Yersinia pestis, YopH, suggested that loop conformational dynamics are important in regulating both catalysis and evolvability. We have generated a chimeric protein in which the WPD-loop of YopH is transposed into PTP1B, and eight chimeras that systematically restored the loop sequence back to native PTP1B. Of these, four chimeras were soluble and were subjected to detailed biochemical and structural characterization, and a computational analysis of their WPD-loop dynamics. The chimeras maintain backbone structural integrity, with somewhat slower rates than either wild-type parent, and show differences in the pH dependency of catalysis, and changes in the effect of Mg2+. The chimeric proteins' WPD-loops differ significantly in their relative stability and rigidity. The time required for interconversion, coupled with electrostatic effects revealed by simulations, likely accounts for the activity differences between chimeras, and relative to the native enzymes. Our results further the understanding of connections between enzyme activity and the dynamics of catalytically important groups, particularly the effects of non-catalytic residues on key conformational equilibria.


S4 generate Markov state models (MSMs) describing WPD-loop and E-loop motion (described in the main text Materials and Methods).
In order to equilibrate each system the following protocol was used: Hydrogens atoms and solvent molecules were energy minimized (using 500 steps of steepest descent followed by 500 steps of conjugate gradient minimization). To prevent the movement of non-hydrogen and nonsolvent atoms during the minimization, 10 kcal mol −1 Å −1 positional restraints were used to keep all (non-solvent) heavy atoms fixed. The solvent was then heated rapidly from 50 K to 298 K (NVT ensemble, 1 fs timestep) over the course of 200 ps, with the previously described restraints still maintained. These positional restraints were then replaced with 5 kcal mol −1 Å −1 positional restraints on only the Cα carbon atoms of each residue and subjected to another round of energy minimization (500 steps of steepest descent followed by 500 steps of conjugate gradient).
Retaining these positional restraints, the system was heated from 25 K to 298 K over the course of 50 ps (NVT ensemble, 1 fs time step). Simulations were then performed in the NPT ensemble (1 atm, 298 K, 2 fs time step) by first gradually reducing the 5 kcal mol −1 Å −1 Cα carbon restraints over the course of 50 ps. This was done by reducing the restraint weight by 1 kcal mol −1 Å −1 every 10 ps. A final 1 ns long NVT MD simulation with no restraints placed on the system was then performed, with the final structure produced after this run, used as the starting point for production MD simulations.
Production MD simulations were run with a 2 fs time step (with the SHAKE 6 algorithm applied) and an 8 Å direct space non-bonded cut-off, with long range electrostatics evaluated using the particle-mesh Ewald 7 method. Temperature and pressure were regulated using Langevin temperature control (collision frequency of 1 ps −1 ) and a Berendsen barostat (pressure relaxation time of 1 ps). Figure S1. Top, the substrate p-nitrophenyl phosphate (pNPP) showing the positions where kinetic isotope effects were measured: the bridge oxygen atom, the position of bond cleavage, 18 (V/K)bridge, and the nitrogen atom in the leaving group, 15 (V/K). Below, the isotopic isomers used for KIE measurements. Natural abundance pNPP consists of a mixture of A and B and was used for measurement of 15 (V/K). The isotopic isomers A and C were synthesized as described in the manuscript and mixed to prepare the substrate used for measuring the bridging-18 (V/K)bridge. Figure S2. Implied timescales estimated at a range of lag times for WT PTP1B and each Chimera simulated. The shaded region of each line plot depicts the 95% confidence intervals by a Bayesian MSM. This data was used to select an appropriate lag time to build the Markov state models. The number of clusters used to discretise the datasets prior to the implied timescale calculation is given.   loop-closed conformation. The substituted residue Gln182 is oriented on the top of the active site and hydrogen bonds to water molecule W1, which also coordinates to an oxygen atom of Hepes, the side chain of Gln266, and the backbone amide proton of Gln182. This water molecule is common to other X-ray structures in the catalytic pathway of PTP1B wildtype and is also observed in other members of the PTP family (e.g. Yersinia PTP and PTPβ). 9, 10 HEPES is commonly used as a buffer in the crystallization of PTP1B. It is a poor competitive inhibitor of PTPs and binding at the active site is not commonly observed in X-ray structures. The alkyl ring of HEPES is oriented in a position that could clash with the side chain of F182 in the native enzyme, so the F182Q mutation likely allows HEPES binding.   Figure 5B are indicated with a red asterix and were selected because in at least one Chimera the difference in electrostatic contributions (relative to the WT) was ≥ |0.25| kcal mol −1 . This data is provided in tabular form in Table S8.  Table S9.   Tables   Table S1. List of forward and reverse primers used to construct the different PTP1B chimeras studied in this work. Chimera 7 was made using WT PTP1B as a template.  None required.

Mutagenesis of WPD-loop from Chimera 4
sequence to Chimera 1.
Mutagenesis of WPD-loop from Chimera 4 sequence to Chimera 3.
None required.

His-d 214
a Glu186 is Ser186 in Chimera 1. All other residues were kept in their neutral forms as they fell outside of the explicit simulation sphere used in the surface constrained all-atom solvent (SCAAS) model, 11 as described in our prior work. 1 a Data obtained from fits of the pH-rate profiles shown in Figure 3 to Eq. 1. and activation free energies (∆G ‡ exp) derived from the experimentally observed rates using the Eyring equation. Note that, as described in the main text, Chimera 1 is not active, and is presented here only for comparison.   (Figure 1). SCys-P denotes the distance between the cysteine side chain and the phosphorus atom of the phosphate group, P-OH2O denotes the distance between the phosphorus atom and the nucleophilic water molecule in the hydrolysis step, OH2O-H denotes the distance between the nucleophilic water molecule and the proton being transferred back to the aspartic acid side chain, and H-OAsp denotes the distance between the proton and the relevant oxygen atom of the aspartic acid side chain. All distances are shown in Å. Data is presented as average values and standard error of the mean over 30 individual EVB trajectories per system.