Protein Tyrosine Phosphatase 1B

Nathan Silva and David Marcey
CLU Biology Department
©David Marcey, 2001

I. Introduction
II. Structural Features
III. Dephophorylation of Tyrosine Residue
IV. Dephosphorylation of Cysteinyl-Phosphate Intermediate
V. The PTP Domain of a FERM-PTP
VI. References

Note:  This exhibit is best viewed if the cue buttons ( ) are pressed in sequence and if the viewer does not independently manipulate the molecule on the left.

   Protein Tyrosine Phophatases (PTPs) represent a large family of enzymes. They play a very important role in cellular signaling within and between cells. PTPs work antagonistically with Protein Tyrosine Kinases (PTKs) to regulate signal transduction in a cell. PTKs phosphorylate tyrosine residues on a substrate protein and PTPs remove these phosphates from substrate tyrosines (dephosphorylation). Since the phosphorylation status of a protein can modulate its function, PTKs and PTPs work together to regulate protein function in response to a variety of signals, including hormones, mitogens, and oncogenes.
 

 Protein Tyrosine Phosphatase 1B (PTP1B) is a well studied non-receptor PTP. This exhibit focuses on recent structural work on this enzyme (Jia, et al., 1995; Pannifer, et al., 1998). PTP1B secondary structure includes 9 alpha helicies and 1 main beta sheet composed of 8 strands  . A structural feature that is highly conserved among PTPs is the catalytic, or PTP loop (also known as the signature motif)  . This PTP loop comprises 11 residues: (I/V)HCXAGXXR(S/T)G. Cys 215 and Arg 221 residues in the PTP loop are those most vital for catalysis  . Another conserved loop, the recognition loop, plays an important role in substrate recognition. The residues Val 49 and Tyr 46 assist the substrate's insertion into catalytic site . Ser 216 of the PTP loop forms a hydrogen bond with the the recognition loop, stabilizing the active site cleft. A third conserved loop is the WPD loop, the function of which will be described later. The WPD loop here is shown in the "closed" conformation. On and near the WPD loop are key residues that function in PTP1B catalysis. Asp 181 and Gln 262 become especially important in the second part of the reaction . These structural features of PTP1B provide for the chemistry of dephosphorylation, detailed below.

The reaction starts when a phosphorylated tyrosine residue enters the deep, active site cleft of PTP1B molecule, the base of which is the PTP loop . Tyr 46 and Val49 of the recognition loop facilitates this entry. Phosphotyrosine is an amphipathic molecule. The phosphorylated end of the tyrosine is polar, but the phenol ring is non-polar and would normally be repelled from a polar catalytic site. Tyr46 and Val49 provide a non-polar pocket for the phenol ring of the phosphotyrosine substrate while the phosphorylated end is securely placed in the catalytic cleft. When the substrate enters the catalytic site, a major conformational change occurs in the WPD loop. The loop closes over the phenyl ring of the tyrosine residue, holding it in place and further positioning it so that a subsequent nucleophilic attack may occur. At this same time, Asp 181 is moved in close to the tyrosine phosphate so it can act as an acid during the reaction. Binding also occurs within the PTP loop; Arg 221 shifts to optimize its connection with the phosphate attached to the tyrosine residue. The slight shift of Arg 221 increases binding with Pro 180, Trp 179, and Phe 182. All of these interactions lead to a stabile, closed conformation for the WPD loop  . The phosphorylated tyrosine residue is situated in such a way that the phosphorus atom and the gamma sulfur atom of Cys 215 are juxtaposed. This is essential for catalysis because the Cys 215 residue of the PTP loop will remove the tyrosine's phosphate and store it briefly as an intermediate  . First, Asp 181 adds a proton (hydrogen) to the oxygen of tyrosine. This neutralizes the tyrosine and it is then free to diffuse away from the catalytic cleft. The captured phosphate then binds to the sulfur of Cys 215, thereby forming the cysteinyl-phosphate intermediate  .

   The WPD loop retains its closed conformation after the Tyrosine residue diffuses away from the enzyme because amino acids Arg 221, Pro180, Trp179, and Phe 182 maintain interactions with the phosphate group bound to Cys 215  .  The phosphate is removed from the cysteine via a nucleophilic attack of a water molecule. Although this attack is kinetically unfavorable because of steric repulsion, Gln 262 and Asp 181 (the amino acid that protonated the oxygen of the tyrosine residue) neutralize and position the H₂O (W2) for attacking the cysteinyl-phosphate intermediate . Mutation of either Gln 262 or Asp 181 blocks this step of the reaction. W2 breaks the bond between the phosphate and the cysteine. The phosphate then binds to W2 forming a water phosphate complex . The enzyme then returns to a standard conformation, ready to accept another phosphorylated tyrosine into the active site.

V. The PTP Domain of a FERM-PTP   

Whereas the PTP1B protein consists solely of a PTP domain, there are many PTPs that contain auxilliary domains connected to a catalytic PTP domain. FERM-PTPs are family of proteins that contain a PTP domain connected to a Band 4.1/ezrin (FERM) domain implicated in membrane-cytoskeletal interactions. Pez, a subfamily of FERM-PTPs, has members with a FERM domain linked to a catalytic PTP domain through a central linker domain with conserved motifs that are likely phosphorylation and protein interaction sites (Edwards, et al., 2001). We have modeled the PTP domain of Drosophila Pez using the homology modeling program Swiss-Model (Guex and Peitsch, 1997), and conclude that DPez is likely a functional phosphatase despite some unusual features of the active site cleft (Edwards, et al., 2001). The following features are worthy of note.

The DPez and PTP1B backbones are largely congruent. However, note the deletion in DPez of an 8 amino acid b-hairpin (between b5-b6) relative to PTP1B . The residues brought together by this deletion are adjacent in other PTP structures, including PTP1B, so our homology model predicts that the deletion does not disrupt tertiary structure.

Like PTP1B, the active site cleft of DPez is constructed by the WPD, recognition, and PTP loops, but there are some important amino acid substitutions .
In PTP1B, Y⁴⁶ in the recognition loop is hydrogen bonded to S²¹⁶ in the PTP loop. Note that the loss of a bulky tyrosine caused by the Y⁴⁶>>D⁴⁶ substitution in the recognition loop may be compensated by the addition of a bulky leucine sidechain caused by the S²¹⁶>>L²¹⁶ substitution in the catalytic loop . The A²¹⁷>>E²¹⁷ substitution in the catalytic loop may allow E²¹⁷ to H-bond to the recognition loop, compensating for the loss of the Y⁴⁶-S²¹⁶ H-bond .

VI. References

Edwards, K., T. Davis, D. Marcey, J. Kurihara, D. Yamamoto. 2001. Comparative Analysis of the Band 4.1/ezrin-related Protein Tyrosine Phosphatase Pez from Two Drosophila Species: Implication for Structure and Function. Gene 275: 195-205.

Guex, N., and Peitsch, M.C. (1997). SWISS-MODEL and the Swiss-PdbViewer: An environment for comarative protein modelling. Electrophoresis 18: 2714-2723.

Jia, Z., Barford, d., Flint, A.J., and N.K.Tonks (1995). Structural Basis for Phosphotyrosine Peptide Recognition by PTP1B. Science 268: 1754-1758.

Pannifer A., Flint A., Tonks N., and Barford D.(1998). Visualization of the Cysteinyl-phosphate Intermediate of a Protein-tyrosine Phosphatase by X-ray Crystallography. The Journal of Biological Chemistry 273: 10454-10462.

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