The metal face of protein tyrosine phosphatase 1B

Abstract

A new paradigm in metallobiochemistry describes the activation of inactive metalloenzymes by metal ion removal. Protein tyrosine phosphatases (PTPs) do not seem to require a metal ion for enzymatic activity. However, both metal cations and metal anions modulate their enzymatic activity. One binding site is the phosphate binding site at the catalytic cysteine residue. Oxyanions with structural similarity to phosphate, such as vanadate, inhibit the enzyme with nanomolar to micromolar affinities. In addition, zinc ions (Zn²⁺) inhibit with picomolar to nanomolar affinities. We mapped the cation binding site close to the anion binding site and established a specific mechanism of inhibition occurring only in the closed conformation of the enzyme when the catalytic cysteine is phosphorylated and the catalytic aspartate moves into the active site. We discuss this dual inhibition by anions and cations here for PTP1B, the most thoroughly investigated protein tyrosine phosphatase. The significance of the inhibition in phosphorylation signaling is becoming apparent only from the functions of PTP1B in the biological context of metal cations as cellular signaling ions. Zinc ion signals complement redox signals but provide a different type of control and longer lasting inhibition on a biological time scale owing to the specificity and affinity of zinc ions for coordination environments. Inhibitor design for PTP1B and other PTPs is a major area of research activity and interest owing to their prominent roles in metabolic regulation in health and disease, in particular cancer and diabetes. Our results explain the apparent dichotomy of both cations (Zn²⁺) and oxyanions such as vanadate inhibiting PTP1B and having insulin-enhancing ("anti-diabetic") effects and suggest different approaches, namely targeting PTPs in the cell by affecting their physiological modulators and considering a metallodrug approach that builds on the knowledge of the insulin-enhancing effects of both zinc and vanadium compounds.

Fig. 1

Protein tyrosine phosphatases catalyze the dephosphorylation of the substrate by a ping-pong mechanism. The enzyme initially is in a resting state with the WPD loop open (PDB id: 2CM2). Upon substrate (in orange) entry into the catalytic pocket, the WPD loop closes (PDB id: 1PTU, Michaelis complex). The enzyme goes through the first transition state (PDB id: 3I7Z) and the first product is released with the enzyme left in a phospho-cysteine intermediate (PDB id: 1A5Y, Q262A mutant). A water molecule is subsequently activated in the second transition state (PDB id: 3I80), before the WPD loop opens again (PDB id: 2HNQ, Michaelis complex) and the inorganic phosphate is released. Modified from Brandao et al., ; reproduced with permission.

Fig. 2

Docking simulation performed on different PTP1B crystal structures. (A) Closed form (PDB id: 3I80) and (B) phospho-cysteine intermediate (PDP id: 1A5Y). Since the position of the zinc ion in the two structures differs by about 4 Å Gln266 in (A) is too far for coordination. Gln²⁶² is not shown in (B) as the structure was obtained on a Gln262Ala mutant to avoid hydrolysis of the phospho-cysteine intermediate. Protein structure is represented in pale cyan, zinc ion as pink sphere, and relevant amino acids as sticks .

Fig. 3

X-ray crystal structure of (A) first transition state complex between PTP1B, metavanadate, and the Tyr in the peptide DADEYL (PDB id: 3I7Z) and (B) second transition state complex between PTP1B and orthovanadate (PDB id: 3I80) . Protein structure is represented in pale cyan, peptide DADEYL in orange, and vanadate and relevant amino acids as sticks. H-bonds are depicted as yellow dot lines.

Fig. 4

X-ray crystal structure of Yersinia enterocolitica PTP (R235C, W354F) complexed with divanadate (PDB id: 3F9B) . Protein structure is represented in pale green, and vanadate and relevant amino acids as sticks. H-bonds are depicted as yellow dot lines.

Fig. 5

X-ray crystal structures of (A) Yersinia PTP (R235C) (PDB id: 1YTW) and (B) human PTP1B (PDB id: 2HNQ) complexed with tungstate . Protein structures are represented in pink and pale cyan respectively, tungstate and relevant amino acids as sticks. H-bonds are depicted as yellow dot lines, distances are in Å.

Fig. 6

X-ray crystal structure of bovine low molecular weight PTP complexed with molybdate (PDB id: 1Z13) . Protein structure is represented in green, molybdate and relevant amino acids as sticks. H-bonds are depicted as yellow dot lines, distances are in Å.

Fig. 7

X-ray crystal structure of human PTP1B complexed with nitrate (PDB id: 4BJO): (A) open form and (B) closed form . Protein structure is represented in pale cyan, and nitrate and relevant amino acids as sticks. H-bonds are depicted as yellow dot lines, distances are in Å. (C) X-ray crystal structure of Yersinia PTP (R235C) complexed with nitrate (PDB id: 1YTN) . Protein structure is represented in pink, nitrate and relevant amino acids as sticks. H-bonds are depicted as yellow dot lines, distances are in Å.

Fig. 8

Mechanisms involved in increasing cytosolic zinc after insulin stimulation. Insulin binding to its receptor induces zinc increase through the zinc transporter Zip7 located on the endoplasmic reticulum (ER) membrane or through other Zips located on the plasma membrane. Insulin stimulates the insulin receptor (IR), which triggers an increase in ROS production. Oxidation of metallothioneins (MT) releases the bound zinc ions. Zinc then inhibits PTP1B, hence blocking the dephosphorylation of the insulin receptor. The figure was made using Servier Medical Art (www.servier.com).

Fig. 9

Redox chemistry of the active site cysteine in PTPs.

Fig. 10

Modulation of PTP1B by oxidation of the catalytic cysteine and zinc binding at different stages of the catalytic cycle. While oxidation targets the enzyme in the open conformation, zinc modulates the enzyme after the WPD loop has closed. Modified from Brandao et al. .