The Structure of the Membrane Distal Phosphatase Domain of RPTPR Reveals Interdomain Flexibility and an SH2 Domain Interaction Region † Erica Dutil Sonnenburg, ‡ Alexandrine Bilwes, ‡,§ Tony Hunter, | and Joseph P. Noel* ,‡ Structural Biology Laboratory and Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037 ReceiVed January 13, 2003; ReVised Manuscript ReceiVed April 28, 2003 ABSTRACT: The receptor protein tyrosine phosphatase R (RPTPR) is a transmembrane receptor with two intracellular protein tyrosine phosphatase domains, a catalytically active membrane proximal domain (D1) and a membrane distal phosphatase domain with minimal catalytic activity (D2). Here we elucidate the crystal structure of RPTPR’s D2 domain. Unlike D1, D2 exists as a monomer and lacks the N-terminal inhibitory wedge motif. The N-terminal portion of D2 is disordered, and this region linking D1 to D2 is proteolytically labile in solution whether part of D2 alone or tethered to D1, indicating that the polypeptide backbone of this part of D2 is highly flexible, and therefore accessible to proteases under native conditions. Furthermore, we have crystallized the SH2 domain of the protein tyrosine kinase c-Src, a RPTPR substrate, with a phosphopeptide encompassing the C-terminal phosphorylation site of D2 (pTyr789). The SH2 domain of Src binds RPTPR in an extended conformation. The structural and functional data support a D1-D2 arrangement with significant flexibility between phosphatase domains of RPTPR that is likely to be important for dynamic alterations in intra- and/or intermolecular interactions that are critical for RPTPR function. The receptor protein tyrosine phosphatases (RPTPs) 1 are a family of transmembrane phosphatases responsible for the hydrolysis of the phosphate moiety from a pTyr residue in target phosphoprotein substrates resulting in a variety of intracellular responses, including long-term potentiation, axonal path finding and neural transmission, and transforma- tion (reviewed in refs 1 and 2). The RPTPs consist of an extracellular domain, a single spanning transmembrane region, and two tandem intracellular tyrosine phosphatase domains, with the exception of a small subgroup of RPTPs that contain a single phosphatase domain. The majority of diversity within the RPTP family lies within the extracellular domain and includes protein modules such as immunoglobin- like domains, fibronectin type III-like domains, and heavily glycosylated polypeptide segments. In contrast, the sequences and structures of intracellular tyrosine phosphatase domains are highly homologous not only within the RPTP family but also with those of the nonreceptor tyrosine phosphatases (reviewed in refs 3 and 4). The first, membrane proximal, phosphatase domain, D1, harbors most, or in some cases, all of the catalytic activity. All active tyrosine phosphatases contain an active site cysteine, which forms a cysteinyl phosphate intermediate following nucleophilic attack on the pTyr substrate, as well as a conserved Asp that acts as a general acid-base catalyst to complete the water-mediated hydrolysis of the cysteinyl phosphate intermediate. The membrane distal phosphatase domain, D2, a highly conserved domain within the RPTP family, exhibits little or no phosphatase activity despite the fact that it possesses the catalytic cysteine residue required for nucleophilic attack and a conservative substitution, Glu, in place of the general acid-base Asp catalytic residue. Two independent crystal structures of the membrane proximal phosphatase domain, D1, of RPTPR reveal a crystallographic dimer arrangement in which the active site of one domain is occluded by a helix-turn-helix wedge motif of its dyad related partner (5). The use of engineered disulfide bonds in full-length RPTPR demonstrates that this dimeric configuration renders RPTPR catalytically inactive in ViVo (6). Since the initially determined RPTPR D1 crystal structure, a plethora of evidence supporting dimerization as an important regulatory mechanism in some RPTPs has surfaced. The most compelling evidence for RPTPR dimer- ization is the observation of fluorescence resonance energy transfer (FRET) between cyan and yellow derivatives of the green fluorescent protein fused to RPTPR in ViVo. Trunca- tions of RPTPR reveal that the transmembrane region is necessary and sufficient for dimerization as measured by FRET and chemical cross-linking (7, 8). Therefore, the D1 † This work was supported by NIH/NCI Grant CA54418 to J.P.N. and T.H. and a postdoctoral training grant (T32CA09370) to E.D.S. T.H. is a Frank and Else Schilling American Cancer Society Research Professor. The SSRL Biotechnology Program is supported by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and by the Department of Energy, Office of Biological and Environmental Research. * To whom correspondence should by addressed. Telephone: (858) 453-4100. Fax: (858) 452-3683. E-mail: noel@salk.edu. ‡ Structural Biology Laboratory. § Present address: Department of Chemistry and Chemical Biology, Cornell University, G-60 ST Olin, Ithaca, NY 14853. | Molecular and Cell Biology Laboratory. 1 Abbreviations: RPTP, receptor protein tyrosine phosphatase; PMSF, phenylmethanesulfonyl fluoride; DTT, dithiothreitol; PEG, polyethylene glycol; SDS, sodium dodecyl sulfate; PAGE, polyacry- lamide gel electrophoresis; PVDF, polyvinylidene difluoride; pTyr, phosphotyrosine; rmsd, root-mean-square deviation. 7904 Biochemistry 2003, 42, 7904-7914 10.1021/bi0340503 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/11/2003