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Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including cancer, autoimmunity, and neurological disorders. A high degree of structural similarity between their catalytic domains, however, has hindered the development of selective pharmacological agents. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over T-cell PTP (TCPTP), two PTPs with high sequence conservation. Here, we use molecular modeling, with supporting experimental validation, to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations suggest that PTP1B and TCPTP share a h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically essential WPD loop, which it links to the L–11 loop and neighboring α3 and α7 helices on the other side of the catalytic domain. Terpenoid binding to either of two proximal C-terminal sites─an α site and a β site─can disrupt the allosteric network; however, binding to the α site forms a stable complex only in PTP1B. In TCPTP, two charged residues disfavor binding at the α site in favor of binding at the β site, which is conserved between the two proteins. Our findings thus indicate that minor amino acid differences at the poorly conserved α site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate more broadly how minor differences in the conservation of neighboring─yet functionally similar─allosteric sites can affect the selectivity of inhibitory scaffolds (e.g., fragments).
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This content will become publicly available on December 10, 2026
Conformational Dynamics and Catalytic Backups in a Hyper-thermostable Engineered Archaeal Protein Tyrosine Phosphatase
Protein tyrosine phosphatases (PTPs) are a family of enzymes that play important roles in regulating cellular signaling pathways. The activity of these enzymes is regulated by the motion of a catalytic loop that places a critical conserved aspartic acid side chain into the active site for acid–base catalysis upon loop closure. These enzymes also have a conserved phosphate-binding loop that is typically highly rigid and forms a well-defined anion-binding nest. The intimate links between loop dynamics and chemistry in these enzymes make PTPs an excellent model system for understanding the role of loop dynamics in protein function and evolution. In this context, archaeal PTPs, which have often evolved in extremophilic organisms, are highly understudied, despite their unusual biophysical properties. We present here an engineered chimeric PTP (ShufPTP) generated by shuffling the amino acid sequence of five extant hyperthermophilic archaeal PTPs. Despite ShufPTP’s high sequence similarity to its natural counterparts, it presents a suite of unique properties, including high flexibility of the phosphate binding P-loop, facile oxidation of the active-site cysteine, mechanistic promiscuity, and, most notably, hyperthermostability, with a denaturation temperature likely >130 °C (>8 °C higher than the highest recorded growth temperature of any archaeal strain). Our combined structural, biochemical, biophysical, and computational analysis provides insight both into how small steps in evolutionary space can radically modulate the biophysical properties of an enzyme and showcases the tremendous potential of archaeal enzymes for biotechnology, to generate novel enzymes capable of operating under extreme conditions.
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- PAR ID:
- 10675284
- Publisher / Repository:
- ACS Publications
- Date Published:
- Journal Name:
- JACS Au
- Volume:
- 6
- Issue:
- 1
- ISSN:
- 2691-3704
- Page Range / eLocation ID:
- 59-81
- Subject(s) / Keyword(s):
- Archaea Protein Tyrosine Phosphatases Sequence Shuffling Catalytic Backups Protein Conformational Dynamics
- Format(s):
- Medium: X
- Associated Dataset(s):
-
View Associated Dataset(s) >>
- Conformational Dynamics and Catalytic Backups in a Hyper-Thermostable Engineered Archaeal Protein Tyrosine Phosphatase
- Ligand Free Putative Ancestral Protein Tyrosine Phosphatase ShufPTP - C93-Cyclic Sulfenamide - Intermediate p-loop Conformation: 9e9m
- Ligand Free Putative Ancestral Protein Tyrosine Phosphatase ShufPTP - Lowest p-loop Conformation: 9e9n
- Vanadate-bound Putative Ancestral Protein Tyrosine Phosphatase ShufPTP - Highest p-loop Conformation: 9e9u
- Vanadate-bound Putative Ancestral Protein Tyrosine Phosphatase ShufPTP - Intermediate p-loop Conformation: 9e9l
- Sponsoring Org:
- National Science Foundation
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