Protein Tyrosine Phosphatase 1B
Silva and David Marcey
CLU Biology Department
©David Marcey, 2001
II. Structural Features
III. Dephophorylation of Tyrosine Residue
IV. Dephosphorylation of Cysteinyl-Phosphate Intermediate
V. The PTP Domain of a FERM-PTP
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.
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.
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,
starts when a phosphorylated tyrosine residue enters
the deep, active site cleft of PTP1B molecule, the base of which is the PTP
loop . Tyr
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 .
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
intermediate . Mutation of either Gln
262 or Asp 181 blocks this step of the reaction.
W2 breaks the bond between
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
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.
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 .
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.