PDBsum entry 1d4w

Signaling protein

PDB id: 1d4w

Contents

Protein chain

104 a.a. *

Ligands

SER-LEU-THR-ILE-PTR-ALA-GLN-VAL-GLN-LYS ×2

Waters ×413

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Crystal structures of the xlp protein sap reveal a class of sh2 domains with extended, Phosphotyrosine-Independent sequence recognition.

Authors

F.Poy, M.B.Yaffe, J.Sayos, K.Saxena, M.Morra, J.Sumegi, L.C.Cantley, C.Terhorst, M.J.Eck.

Ref.

Mol Cell, 1999, 4, 555-561. [DOI no: 10.1016/S1097-2765(00)80206-3]

PubMed id

10549287

Abstract

SAP, the product of the gene mutated in X-linked lymphoproliferative syndrome (XLP), consists of a single SH2 domain that has been shown to bind the cytoplasmic tail of the lymphocyte coreceptor SLAM. Here we describe structures that show that SAP binds phosphorylated and nonphosphorylated SLAM peptides in a similar mode, with the tyrosine or phosphotyrosine residue inserted into the phosphotyrosine-binding pocket. We find that specific interactions with residues N-terminal to the tyrosine, in addition to more characteristic C-terminal interactions, stabilize the complexes. A phosphopeptide library screen and analysis of mutations identified in XLP patients confirm that these extended interactions are required for SAP function. Further, we show that SAP and the similar protein EAT-2 recognize the sequence motif TIpYXX(V/I).

Figure 1.
Figure 1. Structure of SAP and the Location of Missense Mutations Identified in XLP Patients(A) Ribbon diagram showing the SAP/SLAM pY281 complex. The bound phosphopeptide is shown in a stick representation (yellow). Selected SAP residues that form the binding site are shown in blue. Elements of secondary structure are labeled using the standard SH2 domain nomenclature ([8]). Note that the pY −3 to pY −1 residues of the peptide make a parallel β sheet interaction with strand βD; the side chains of these peptide residues make hydrophobic contacts with Tyr-50, Ile-51, and Tyr-52 in strand βD, and with Leu-21. Thr (pY −2) in the peptide hydrogen bonds with Glu-17 and with a buried water molecule. The phosphotyrosine is coordinated in a manner similar to that observed in the N-terminal domain of SHP-2, and as in SHP-2, the phosphate group is rotated “above” the plane of the phosphotyrosine ring. Interestingly, arginine 13 (at position αA2), which is conserved in almost all SH2 domains and usually contributes to phosphotyrosine coordination, does not participate in phosphate binding in the SAP complex. Instead, arginine 55 (βD6) hydrogen bonds with the phosphate group. C-terminal to phosphotyrosine, Val(pY +3) binds in a mostly hydrophobic cleft.(B) Point mutations identified in XLP patients cluster along the peptide-binding site and at the back of the domain. Mutations that would be expected to directly disrupt the phosphotyrosine-binding pocket are shown in green, and those that would disrupt C-terminal interactions in magenta. The remaining mutations (gold) are remote from the peptide-binding surface and may destabilize the folded protein (see text).(C) Structure-based sequence comparisons of human SAP, murine EAT-2, and other SH2 domains. Elements of secondary structure are indicated above the alignment. Numbering corresponds to human SAP. The black diamonds indicate the mutations illustrated in (B).

Figure 2.
Figure 2. Structure and Comparisons of the SAP/SLAM Y281 Complex(A) Surface representation of the SAP domain with the bound nonphosphorylated peptide shown in green. Hydrophobic residues at the −1 and −3 positions of the peptide intercalate with hydrophobic and aromatic residues on the surface of the domain (see also [D] and Figure 1A). C-terminal to phosphotyrosine, Val+3 is buried in a mostly hydrophobic groove.(B) Superposition of the phosphorylated and nonphosphorylated peptides shows that they adopt an essentially identical conformation. An alpha-carbon trace of the domain is shown in gray.(C) Superposition of the unliganded domain (blue) and the phosphopeptide (yellow) and nonphosphorylated peptide complexes (green). In the absence of bound peptide, the EF and BG loops fold inward to close the hydrophobic +3 binding groove. The conformation of the phosphotyrosine-binding pocket is essentially the same in all structures. In the unliganded structure, a sulfate ion occupies the position of the phosphate group in the phosphopeptide complex.(D) Detail of the phosphotyrosine-binding pocket in the SLAM/pY281 complex. Red spheres represent ordered water molecules. The pY281 peptide is shown in yellow. Thin cyan lines indicate potential hydrogen bonds. Note that Arg-13 is poorly ordered and does not participate in phosphotyrosine coordination.(E) Detail of the phosphotyrosine-binding pocket in the nonphosphorylated SLAM/Y281 complex. Red spheres represent ordered water molecules. The Y281 peptide is shown in green. Thin cyan lines indicate potential hydrogen bonds. Note that Arg-32 organizes an extensive network of hydrogen bonds in spite of the lack of phosphorylation of Tyr-281.

The above figures are reprinted by permission from Cell Press: Mol Cell (1999, 4, 555-561) copyright 1999.

Secondary reference #1

Title

The X-Linked lymphoproliferative-Disease gene product sap regulates signals induced through the co-Receptor slam.

Authors

J.Sayos, C.Wu, M.Morra, N.Wang, X.Zhang, D.Allen, S.Van schaik, L.Notarangelo, R.Geha, M.G.Roncarolo, H.Oettgen, J.E.De vries, G.Aversa, C.Terhorst.

Ref.

Nature, 1998, 395, 462-469. [DOI no: 10.1038/26683]

PubMed id

9774102

Figure 6.
Figure 6 Genomic analysis of patients with XLP. a, Location of the point mutation near exon 2 of patient A1; a mechanism is shown to explain the generation of the variant from hSAP 55 found in all healthy individuals. b, Exon 2 sequence was amplified by PCR from genomic DNA from patient A1 and from three healthy individuals (CT-1, CT-2 and B3) and from two cell lines (Raji and Jurkat). In addition, PCR products from 78 healthy women and 30 healthy men were analysed (data not shown). DNA products were digested with Mnl I and resolved on a 10% polyacrylamide gel by standard methods. c, hSAP exon 1, exon 2, exon 3 and exon 4 and BRCA1 exon 2 were amplified by PCR from genomic DNA from patients A1, B1, B2 and B3, and from the cell line Raji. DNA products were resolved on a 2% agarose gel.

Figure 9.
Figure 9 SAP has a positive effect in the SLAM co-stimulatory pathway. Jurkat cells were transfected with SLAM, SAP and SLAM+SAP constructs or vector (pCDNA3), as described in Methods. Luciferase activity was measured after stimulation with the indicated antibodies.

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd