Residue 259 is a key determinant of substrate specificity of protein-tyrosine phosphatases 1B and alpha.
The aim of this study was to define the structural elements that determine the differences in substrate recognition capacity of two protein-tyrosine phosphatases (PTPs), PTP1B and PTPalpha, both suggested to be negative regulators of insulin signaling. Since the Ac-DADE(pY)L-NH(2) peptide is well recognized by PTP1B, but less efficiently by PTPalpha, it was chosen as a tool for these analyses. Calpha regiovariation analyses and primary sequence alignments indicate that residues 47, 48, 258, and 259 (PTP1B numbering) define a selectivity-determining region. By analyzing a set of DADE(pY)L analogs with a series of PTP mutants in which these four residues were exchanged between PTP1B and PTPalpha, either in combination or alone, we here demonstrate that the key selectivity-determining residue is 259. In PTPalpha, this residue is a glutamine causing steric hindrance and in PTP1B a glycine allowing broad substrate recognition. Significantly, replacing Gln(259) with a glycine almost turns PTPalpha into a PTP1B-like enzyme. By using a novel set of PTP inhibitors and x-ray crystallography, we further provide evidence that Gln(259) in PTPalpha plays a dual role leading to restricted substrate recognition (directly via steric hindrance) and reduced catalytic activity (indirectly via Gln(262)). Both effects may indicate that PTPalpha regulates highly selective signal transduction processes.
Peters
G H
GH
Technical University of Denmark, Department of Chemistry, Membrane and Statistical Physics Group (MEMPHYS), DK-2800 Lyngby, Denmark. ghp@kemi.dtu.dk
Iversen
L F
LF
Branner
S
S
Andersen
H S
HS
Mortensen
S B
SB
Olsen
O H
OH
Moller
K B
KB
Moller
N P
NP
D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005973:Glutamine / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D009842:Oligopeptides / Q000378:metabolism; D011487:Protein Conformation; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4; D011956:Receptors, Cell Surface; D013379:Substrate Specificity; D018909:src Homology Domains
D009842:Oligopeptides; D011956:Receptors, Cell Surface; C410740:acetyl-aspartyl-alanyl-aspartyl-glutamyl-phosphotyrosyl-leucinamide; D005973:Glutamine; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; C516609:Ptpn1 protein, mouse; C516843:Ptpra protein, mouse; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4; D005998:Glycine