Structure

Determining the three-dimensional structures of over a dozen PTPs has facilitated the identification of critical residues involved in catalysis, substrate binding, and regulation. Although PTPs share a low percentage of amino acid sequence identity among all family members, their overall structures are similar. The catalytic domain of PTPs consists of an a/p fold, composed of highly twisted core P-strands flanked by a-helices [2-4]. The PTP signature motif HCXXGXXR(T/S) (and minimally CX5R) defines the active site center, where the catalytic cysteine resides at the base of the active site cleft. Residues in the PTP signature motif form the phosphate-binding loop, where the main-chain N-H groups and the guanidinium side chain of the invariant arginine residue are oriented to coordinate oxygens of the phosphate group during substrate binding and catalysis. The active site is surrounded by intervening loops that are important in providing additional residues for catalysis and substrate specificity. A highly conserved aspartic acid

-v R13D

-v R13D

S43

Figure 1 Crystal structure of C124S mutant VHR bound to bisphosphorylated peptide DDE(Nle)pTGpYVATR [24]. (A) Electrostatic surface of the VHR-peptide complex. Surfaces are shaded according to the local electrostatic potential, ranging from -13 V in gray to +13 V in dark gray. The peptide is represented as a stick, with carbon, nitrogen, oxygen, and phosphate atoms. This figure was generated with GRASP. (B) Close-up view of the phosphotyrosine and phospho-threonine binding sites. The peptide is shown as a stick, and the atoms are colored as in panel (A). This figure was generated with Swiss Pdb Viewer v3.5 and POV-Ray v3.1. A color representation of this figure can be viewed on the CD version of Handbook of Cell Signaling.

residue is required for general acid/base catalysis and is located on a separate loop (general acid loop) near the top of the active site.

Outside the catalytic domain, amino acid sequences vary dramatically among the PTPs. Additional regions may include modular domains such as SH2 (Src homology 2) domains, fibronectin repeats, and immunoglobulin domains [5]. SH2 domains serve as protein interaction modules recognizing specific phosphorylated tyrosines in proteins or peptides. The SH2 domains of SHPs (Src homology phosphatases) target these phosphatases to specific tyrosyl phosphorylated proteins within cells. Also, the N-terminal SH2 domain of SHPs regulate catalytic activity directly. In the absence of an appropriate phosphotyrosine ligand, the N-SH2 binds to and inactivates the PTP domain by blocking substrate access. This restricts SHP activation to particular locations within the cell where the substrates reside [6,7]. Fibronectin repeats and immunoglobulin domains are found in many receptor protein tyrosine phosphatases (RPTPs). Several RPTPs are believed to play a role in the regulation of cell-cell contact and adhesion through homophilic binding interactions between adjacent cells. RPTPs contain one or two intracellular catalytic domains (membrane-proximal D1 and membranedistal D2). D1 domains are catalytically active, but most D2 domains lack several of the critical catalytic residues, resulting in a domain that displays little or no phosphatase activity [8-10].

Despite low sequence identity between the tyrosine-specific phosphatases and DSPs, crystal structures of several DSPs (VHR, Pyst1/MKP3, PTEN, and KAP) show a highly conserved active site core similar to PTPs [11-14].

Figure 1 shows the crystal structure of VHR (Vaccinia H1 related) bound to a bisphosphorylated peptide substrate [15]. VHR represents the minimal catalytic domain among PTPs, which has made VHR a good model in studies of PTP structure and mechanism. The crystal structure of the DSP Cdc25 catalytic domain reveals that Cdc25 has a unique topology [16,17] that identifies Cdc25 as a more distinct family member of the PTPs. Cdc25 upregulates cyclin-dependent serine/threonine protein kinases (Cdks) by dephosphorylating two adjacent phosphothreonine and phosphotyrosine residues, which are inhibitory to Cdk kinase activity. Although Cdc25 appears to use a similar catalytic mechanism [18], sequence homology within the catalytic domains of other PTPs and DSPs is restricted to the CX5R motif. Like Cdc25, low-molecular-weight PTPs (LMW-PTPs) constitute a distinctive class. The crystal structure of bovine LMW-PTPs reveals a unique fold [19,20]. The LMW-PTPs also contain the conserved arginine, aspartate, and cysteine residues within their active sites.

Structural features of PTPs have provided evidence for pep-tide substrate specificity and for selectivity toward the nature of the phosphorylated residue. Peptide specificity appears to be defined largely by residues both N- and C-terminal to the substrate pTyr residue. The structure of PTP1B in a complex with insulin receptor peptides indicates that a second pTyr residue adjacent to the substrate phosphorylation site plays a critical role in specificity [21]. Similarly, the DSP VHR displays a preference for diphosphorylated peptide substrates [15,22]. The peptide-interacting residues in PTP1B and VHR are poorly conserved throughout the entire PTP family, implying that PTPs have distinct protein substrate specificity.

HOnQ-f

SEH cVs

HOPOa^

SER c;s

C-NH

HfK H

AflG

Figure 2 Catalytic mechanism of protein tyrosine phosphatases.

The phosphorylated residue specificity appears to be determined by the depth of the active site pocket, the general acid loop, and the PTP signature motif. Tyrosine-specific PTPs have an « 9-A-deep active site cleft; therefore, only phospho-tyrosine residues can reach the cysteine nucleophile in the active site. For example, the structure of PTP1B in a complex with a peptide derived from epidermal growth factor receptor (EGFR) revealed that Arg221 at the base and Asp48 at the rim of the active site exactly match the length of pTyr residues [23]. The general acid loop also provides some level of substrate specificity. The PTP1B general acid loop (also called WPD loop, where D is Asp181 general acid) closes over the active site upon phosphorylated peptide binding. This allows the Asp181 to be positioned to act as a general acid in the catalytic reaction and for Trp179 and Pro180 to interact with Arg221 in the active site. These interactions stabilize the catalytically competent conformation of the loop [24]. The crystal structure of the Yersinia PTP also shows the ligand-induced conformational change of the general acid loop [25].

In contrast, DSPs have a shallower active site cleft for accommodating both phosphotyrosine and phosphoserine/ threonine residues. Also, the PTP signature motif in the DSPs provides substrate discrimination. Though VHR belongs to the DSP family, VHR prefers phosphotyrosine over phosphoserine/ threonine [15,22]. While most DSPs contain alanine and isoleucine in the X2 and X3 positions of the signature motif HCXjX2GX3X4R(S/T), VHR harbors a glutamate and a tyro-sine, respectively. The crystal structure of VHR bound to a bisphosphorylated peptide (shown in Fig. 1) reveals that the side chains of glutamic acid-126 and tyrosine-128 in the signature motif impart substrate specificity for phosphotyrosine by creating a deep and narrow active site [15]. The smaller residues (isoleucine and alanine, found in many DSPs) allow more efficient phosphothreonine and phosphoserine dephos-phorylation activity [15]. Another putative DSP family member, PTEN (phosphatase and tensin homolog deleted on chromosome 10), is unique among known PTPs, as it has two basic lysine residues within the signature motif. These positive charges are believed to interact with the negative charges of inositol phospholipid PIP3 (phosphatidylinositol 3,4,5-triphosphate), the biological substrate for PTEN [13,26].

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

Get My Free Ebook


Post a comment