Structural basis for the function and regulation of the receptor protein tyrosine phosphatase CD45

Abstract

CD45 is the prototypic member of transmembrane receptor-like protein tyrosine phosphatases (RPTPs) and has essential roles in immune functions. The cytoplasmic region of CD45, like many other RPTPs, contains two homologous protein tyrosine phosphatase domains, active domain 1 (D1) and catalytically impaired domain 2 (D2). Here, we report crystal structure of the cytoplasmic D1D2 segment of human CD45 in native and phosphotyrosyl peptide-bound forms. The tertiary structures of D1 and D2 are very similar, but doubly phosphorylated CD3zeta immunoreceptor tyrosine-based activation motif peptide binds only the D1 active site. The D2 "active site" deviates from the other active sites significantly to the extent that excludes any possibility of catalytic activity. The relative orientation of D1 and D2 is very similar to that observed in leukocyte common antigen-related protein with both active sites in an open conformation and is restrained through an extensive network of hydrophobic interactions, hydrogen bonds, and salt bridges. This crystal structure is incompatible with the wedge model previously suggested for CD45 regulation.

Figure 1.

The overall structure of the CD45 molecule and the amino acid sequence of the CD45 D1D2 domains. (A) A schematic model of the human CD45 molecule. Numbers in parentheses are amino acid positions. In this work, we refer to the amino acid positions as they appear in the mature protein of the longest isoform (reference 15). FNIII domain, fibronectin III–like domain. (B) The segment of the CD45 cytoplasmic region whose structure is described here is shown as primary sequence. The D1 and D2 sequences are aligned according to their secondary structures. The secondary structure elements are represented as green bars for α helices and red arrows for β strands on top of the amino acid sequence. The wedge region, the KNRY motif, the WPD motif, the PTP signature motif, the Q-rich motif, and the interdomain linker region are marked by yellow overlay. The acidic and basic loops are marked by magenta and blue overlays, respectively.

Figure 2.

Ribbon diagram representations of the CD45 D1D2 domains. Three different views are shown of the CD45 structure. The D1 and D2 domains are shown in yellow and green, respectively. On the top left, the viewer is looking straight into the D1 active site, and the corresponding active site of D2 is facing upward (y direction). For comparison, a corresponding diagram of LAR D1D2 is shown in the boxed inset. Two additional views of CD45 molecule are generated by rotating the molecule by 90° along the horizontal axis in both directions (top right and bottom left). The secondary structure elements in D1 and D2 are labeled, respectively, in the top left and top right panels. The acidic and basic loops are shown in magenta and blue, respectively. The wedge region is also shown.

Figure 3.

Structure of the CD45 D1D2 bound to the CD3 ζITAM-1 phosphopeptide. (A) A stereo view of the D1 active site with the CD3 ζITAM-1 phosphopeptide. Relevant hydrogen bonds between the side chains of the CD45 protein and the ITAM-1 peptide are shown by green lines. (B) The phosphopeptide model is shown with a 2|F_obs|−|F_calc| composite omit map at the 0.9 σ contour level. (C) Molecular surface representation of the D1 active site bound to the CD3 ζITAM-1 peptide. The surfaces are colored according to the electrostatic potential: blue for positive and red for negative. The peptide model is shown using a ball and stick representation.

Figure 4.

Structure of the CD45 D1D2 bound to NPTpYS. (A) A stereo view of the D1 active site with the NPTpYS peptide. Relevant hydrogen bonds between the side chains of the CD45 protein and the NPTpYS peptide are shown by green lines. (B) The phosphopeptide model is shown with a 2|F_obs|−|F_calc|composite omit map at the 0.9 σ contour level.

Figure 5.

Comparisons between the D1 and D2 catalytic centers. (A and B) Ribbon diagrams of active sites in D1 (A) and D2 (B). Side chains of residues implicated in catalysis are shown. (C and D) Molecular surface of the active sites of D1 (C) and D2 (D), as seen from the same direction as in A and B. The surfaces are colored as in Fig. 3 C. Due to the closed conformation of the WPD loop in our structure, the surface charge distribution of the D1 active site pocket could not be easily viewed. To help show a comparison, the WPD loop of the D1 active site is presented in a partially open conformation in this figure, as was seen in the D2 active site.

Figure 6.

Intramolecular interactions between the D1 and D2 domains. The circled region of the small diagram on the left shows the interface between the D1 (yellow) and D2 (green) domains. The detailed view of this area, rotated 60°, along the horizontal axis is shown on the right. Amino acid residues involved in interdomain interactions are shown with carbon in yellow, oxygen in red, and nitrogen in blue. Selected hydrogen bonds are shown by red dotted lines.