On the specificity of aurintricarboxylic acid toward some phosphatases

Protein tyrosine phosphatases (PTPs) play vital roles in the intracellular signaling pathways associated with cell transformation, growth, and proliferation. In a variety of carboxylic acid derivatives, aurintricarboxylic acid (ATA) has been recognized as a binder of the largest affinity to the Yersinia PTP (YopH). Herein, the underlying specificity of ATA, as a reversible and competitive inhibitor of several PTPs (YopH, PTP1B, TCPTP, HePTP, CD45, VHR, and Cdc25A), is explored. By observing each PTP as a primary target receptor and the rest of them as simultaneous secondary receptors, the specificity factor, as a function of ligand concentration, shows at lower [ATA] (between 10-11 and 10-9 M) a 23 to 500-fold specificity in favor of YopH versus the other PTPs, indicating that the ATA-YopH interaction is the most specific. Near [ATA] = 10-8 M, the specificity factor ≈1 is associated with equal occupancy of the primary receptor YopH and the set of secondary receptors. When observing YopH as a primary receptor relative to every other single PTP as a secondary receptor at [ATA] ϵ (10-11, 10-9 M), the specificity to YopH is larger nearly 6, 25, 25, 40, 100, and 120 times than that to PTP1B, HePTP, CD45, TCPTP, Cdc25A, and VHR, respectively. The relative binding free energy contribution of every single YopH residue located in the active site (403-Cys-Arg-Ala-Gly-Val-Gly-Arg-Thr-410) and in the proximal WPD loop (352-Gly-Asn-Trp-Pro-Asp-Gln-Thr-Ala-Val-Ser-361) is evaluated using alanine-scanning mutagenesis. The so-called “hot spots” of binding affinity that dominate the interaction are found to be Trp354 and Thr358 to a greatest extent, as well as Gly352 to a somewhat lesser extent. Ser361 from the WPD loop is established to be inclined toward the destabilization of the ATA:YopH complex. Therefore, some experimental research based on isothermal titration calorimetry could establish which WPD loop residues weaken the interaction, thereby proving whether Ser361 plays a vital role in ATA binding or not.