Examining Both Sides of a Janus PTB Domain

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Figure 1. Recognition of the 3⬘ Splice Site SF1 and the U2AF heterodimer recognize the branchpoint sequence (BPS), polypyrimidine tract (Py), and 3⬘ splice site (AG) by forming a network of protein-RNA and protein-protein interactions mediated by conserved KH and RRM domains.

C against the ␤ sheet surface in U2AF65 RRM3 could act as a conformational lock preventing RNA recognition. This new structure proposes an interesting new twist on the structure and function of the most common RNA binding protein family. Further, it identifies a new interaction motif that is used by different components of spliceosomal regulatory complexes and provides much needed insight into the network of protein interactions responsible for spliceosome assembly.

Gabriele Varani1 and Andres Ramos2 1 Department of Biochemistry and Department of Chemistry University of Washington Seattle, Washington 98195 2 Molecular Structure Division National Institute for Medical Research London NW7 1AA United Kingdom

Selected Reading 1. Zhou, Z., Licklider, L.J., Gygi, S.P., and Reed, R. (2002). Nature 419, 182–185. 2. Selenko, P., Gregorovic, G., Sprangers, R., Stier, G., Rhani, Z., Kra¨mer, A., and Sattler, M. (2003). Mol. Cell 11, 965–976. 3. Liu, Z., Luyten, I., Bottomley, M.J., Messias, A.C., Houningnou, S.M., Sprangers, R., Zanier, K., Kra¨mer, A., and Sattler, M. (2001). Science 294, 1098–1102. 4. Kielkopf, C.L., Rodionova, N.A., Green, M.R., and Burley, S.K. (2001). Cell 106, 595–605. 5. Banerjee, H., Rahn, A., Davis, W., and Singh, R. (2003). RNA 9, 88–99. 6. Gozani, O., Potashkin, J., and Reed, R. (1998). Mol. Cell. Biol. 18, 4752–4760. 7. Varani, G., and Nagai, K. (1998). Annu. Rev. Biophys. Biomol. Struct. 27, 407–445. 8. Allain, F.-H.T., Gubser, C.C., Howe, P.W.A., Nagai, K., Neuhaus, D., and Varani, G. (1996). Nature 380, 646–650.

Structure, Vol. 11, May, 2003, 2003 Elsevier Science Ltd. All rights reserved.

Examining Both Sides of a Janus PTB Domain The crystal structure of the Disabled-1 PTB domain in a ternary complex with the phosphoinositide PI-4,5P2 and an NPVY peptide of the apolipoprotein E receptor-2 reveals how this conserved scaffold simultaneously recognizes two distinct biological ligands required for signaling through this family of receptors. Phosphotyrosine binding (PTB) domains are structurally conserved modules known to be important mediators of protein-protein interactions [1, 2]. In the original models of PTB domain function, protein-protein association could be triggered by tyrosine phosphorylation, providing an easily regulated anchor for docking proteins containing this module. Specifically, the first identified PTB

DOI 10.1016/S0969-2126(03)00077-7

domains were observed to bind to ligands containing a canonical NPXpY motif (where pY represents phosphotyrosine and X is any amino acid), with hydrophobic amino acids N-terminal to this sequence conferring additional specificity [3–5]. Whereas their original discovery is attributed to phosphotyrosine recognition in the context of very specific consensus sequences, more recent advances have demonstrated a much broader range of ligand binding activities for the PTB domain, including to peptides independent of tyrosine phosphorylation [1, 2]. In this issue of Structure, Stolt and coworkers describe the crystal structures of the murine Disabled-1 (Dab1) PTB domain (Figure 1), which reveal the first glimpse of this resourceful protein domain bound to two distinct ligands: to an unphosphorylated NPVY-containing peptide bound to one side of the molecule, and to a phosphoinositide in an electropositive pocket spatially remote from the peptide binding site [6]. These structures highlight the two sides of this PTB domain, empha-

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Figure 1. Engagement of Two Distinct Binding Sites in the Dab1 PTB Domain The conserved PTB/PH domain superfold found in mouse Disabled-1 PTB domain is colored blue. The peptide binding pocket typical of a PTB domain is occupied by an unphosphorylated NPVY-containing peptide derived from ApoER2 depicted in orange. The phosphoinositide binding pocket characteristic of a PH domain is found on the opposite side of the scaffold and is occupied by PI4,5P2 colored red.

sizing the structural plasticity required by this single conserved scaffold to recognize such a diverse set of biological ligands. Whereas the PTB domain is best known for binding to NPXY-related peptide sequences (whether tyrosine phosphorylated or not), the pleckstrin homology (PH) domain is mainly recognized for binding to phospholipids in order to localize proteins to the plasma membrane [7, 8]. Despite differences in amino acid sequence and ligand binding specificities, all known PTB and PH domain structures adopt a remarkably similar structural fold [1, 2, 7, 8]. The similarity of these modules, however, can only be defined at the level of three-dimensional structure due to very limited sequence homology among members of the PTB/PH domain superfamily. The minimal PTB/PH domain superfold is comprised of a ␤ sandwich of two nearly orthogonal, antiparallel ␤ sheets capped on one side by a C-terminal ␣ helix (Figure 1) [9, 10]. Additional secondary structure elements are often built onto the basic core to endow these domains with both specificity and functional versatility. Because of their similar three-dimensional fold, the functional distinction between a PTB and a PH domain has largely been based on which type of ligand occupies which binding site. Although the term PTB does not accurately reflect the activities of the PTB domain, its mode of peptide recognition may be described by the presence of several unifying features. First, the ligand is always accommodated in a cleft formed by ␤5 of the second ␤ sheet and the C-terminal ␣ helix. Second, the peptide always adopts an extended conformation to form intermolecular antiparallel ␤ strand interactions with the protein, thereby extending the second ␤ sheet by one strand. Third, the peptide often contains an NPX(p)Y or related sequence with a propensity for forming a type I ␤ turn to position key C-terminal peptide residues for stabilizing intermolecular interactions. All three of these structural features are found in the Dab1 PTB domain in complex with the apolipoprotein E receptor-2 (ApoER2) peptide [6], indicating a mode of molecular recognition consistent with that of other PTB domains [1, 2]. Finally, small changes in and around the peptide binding site can account for large differences in ligand selectivity. The Dab1 PTB domain exhibits an unusual, strong prefer-

ence for unphosphorylated tyrosine over phosphorylated tyrosine within the NPXY motif due to a cleft formed by the ␤4-␤5 and ␤6-␤7 loops (Figure 1). These segments are shorter than the corresponding loops in other PTB domains such as those from the adaptor protein Shc [9] and insulin receptor substrate 1 (IRS-1) [11, 12]. The Shc and IRS-1 PTB domains only recognize the NPXY motif in a tyrosine phosphorylation-dependent manner. The tight tyrosine binding pocket in the Dab1 PTB domain positions the hydroxyl group of the unphosphorylated tyrosine to form a hydrogen bond with the backbone carboxyl group of Gly 131, and also places the His 136 imidazole ring within van der Waals contact of the tyrosine hydroxyl. So, the length, rigidity, and amino acid composition of the loops surrounding the peptide binding groove help dictate a PTB domain’s selection of target sequence and aid in discriminating the phosphorylation state of tyrosine. More interestingly, when Stolt and colleagues calculated the electrostatic potential of the Dab1/ApoER2 binary complex, they noted a distinct electrostatic polarization reminiscent of a PH domain. The PH domain characteristically exhibits electrostatic polarization on its molecular surface, separating the molecule into acidic and basic regions [7, 8]. A variety of different phospholipids have been found to bind to the basic region of the PH domain, which is located away from the PTB domain peptide binding groove, on the other side of the scaffold [7, 8]. Whereas previous structures have shown that this superfold is able to accommodate one type of ligand or the other, Stolt and colleagues show us that it can accommodate both. The ternary complex of Dab1 PTB domain/ApoER2 peptide/PI-4,5P2 reported here by Stolt et al. represents the first structure showing simultaneous occupancy of both the PTB and PH domain ligand binding sites on the same protein molecule (Figure 1). Dab1 serves as a hybrid domain that possesses a PH domain-like phosphoinositide binding site localized on one side of the scaffold and a PTB domain-like peptide binding surface on the opposite side. The authors suggest a role for Dab1 phosphoinositide binding in recruiting the adaptor molecule to the membrane, as a functionally relevant aspect of transducing signals from the low-density lipoprotein (LDL) receptor family.

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Notably, many cytoplasmic signaling adaptor proteins that bind activated cell surface receptors also associate with the plasma membrane through phospholipid interactions. Such a multivalent approach is advantageous in positioning the adaptor protein as well as the receptor for optimal association in specific locations at the membrane. Various mechanisms have evolved to accomplish membrane localization. Some signaling proteins contain both PH and PTB/SH2 domains in combination to achieve phospholipid and receptor binding. Additionally, some PTB domain-containing adaptor proteins such as fibroblast growth factor receptor substrate 2 (also known as SNT) contain an N-terminal myristoylation sequence, which serves to tether the proteins to the plasma membrane. In the case of Dab1, membrane association could be achieved through the built-in phosphoinositide binding activity of a single PTB/PH hybrid domain that also interacts with the cell surface receptor. Given the role of Dab1 as an adaptor for LDL receptor signaling, membrane localization is perhaps an even more essential component of its function. This pathway results in multiple membrane-associated processes, including downstream activation of the phosphatidylinositol-3 kinase pathway and receptor-mediated endocytosis [6]. The three-dimensional crystal structure of the Dab1 PTB domain/ApoER2 peptide/PI-4,5P2 ternary complex reported here provides a structural realization of a dual-mode molecular recognition by this important adaptor. It remains to be seen whether these two distinct binding modes are functionally coupled in vivo, and whether this seemingly efficient use of a single protein domain for two biological interactions is advantageous for a transient and reversible event in receptor signaling.

Kelley S. Yan and Ming-Ming Zhou Structural Biology Program Department of Physiology and Biophysics Mount Sinai School of Medicine New York University New York, New York 10029 Selected Reading 1. Forman-Kay, J.D., and Pawson, T. (1999). Curr. Opin. Struct. Biol. 9, 690–695. 2. Yan, K.S., Kuti, M., and Zhou, M.-M. (2002). FEBS Lett. 513, 67–70. 3. Kavanaugh, W.M., and Williams, L.T. (1994). Science 266, 1862– 1865. 4. Blaikie, P., Immanuel, D., Wu, J., Li, N., Yajnik, V., and Margolis, B. (1994). J. Biol. Chem. 269, 32031–32034. 5. Gustafson, T.A., He, W., Craparo, A., Schaub, C.D., and O’Meill, T.J. (1995). Mol. Cell. Biol. 15, 2500–2508. 6. Stolt, P.C., Jeon, H., Song, H.K., Herz, J., Eck, M.J., and Blacklow, S.C. (2003). Structure 11, this issue, 569–579. 7. Blomberg, N., Baraldi, E., Nilges, M., and Saraste, M. (1999). Trends Biochem. Sci. 24, 441–445. 8. Lemmon, M.A., Ferguson, K.M., and Schlessinger, J. (1996). Cell 85, 621–624. 9. Zhou, M.-M., Ravichandran, K.S., Olejniczak, E.T., Petros, A.P., Meadows, R.P., Sattler, M., Harlan, J.E., Wade, W., Burakoff, S.J., and Fesik, S.W. (1995). Nature 378, 584–592. 10. Yoon, H.S., Hajduk, P.J., Petros, A.M., Olejniczak, E.T., Meadows, R.P., and Fesik, S.W. (1994). Nature 369, 672–675. 11. Zhou, M.-M., Huang, B., Olejniczak, E.T., Meadows, R.P., Shuker, S.B., Miyazak, M., Tru¨b, T., Shoelson, S.E., and Fesik, S.W. (1996). Nat. Struct. Biol. 3, 388–393. 12. Eck, M.J., Dhe-pagnon, S., Tru¨b, T., Nolte, R., and Shoelson, S.E. (1996). Cell 85, 695–705.

Structure, Vol. 11, May, 2003, 2003 Elsevier Science Ltd. All rights reserved.

Pushing Induced Fit to Its Limits: tRNA-Dependent Active Site Assembly in Class I Aminoacyl-tRNA Synthetases Aminoacyl-tRNA synthetases are responsible for aminoacylating their cognate tRNAs with a unique amino acid. Recent structural work shows how binding of substrates is coupled to assembly of the active site. Aminoacyl-tRNA synthetases (aaRSs) constitute a family of RNA binding proteins that play a central role in translation by forming the aminoacyl-tRNAs used in protein biosynthesis. In most organisms, there are 20 distinct aaRSs, each of them being responsible for aminoacylating its cognate tRNA(s) with a unique amino acid in a two-step catalytic reaction. The first step leads to

DOI 10.1016/S0969-2126(03)00080-7

the formation of an enzyme-bound intermediate called aminoacyl-adenylate from amino acids, ATP, and magnesium ions. At the second step, the aminoacyl moiety is transferred to one of the hydroxyl groups of the 3⬘terminal adenosine of the tRNA to form an aminoacyltRNA. Due to their fundamental importance, the aaRSs are likely to be one of the most ancient families of proteins and have therefore been analyzed extensively (for a recent review of the field, see [1]). The aaRSs are divided into two classes, which correspond to two architectures of the active site core characterized by conserved amino acid residues. Prodigious efforts spanning more than 25 years led to the determination of the crystal structures of mainly all aaRSs, either in the free state or in complexes with the other partners of the aminoacylation reaction. aaRSs now constitute the best textbook example of multidomain proteins, including insertion and terminal functional modules, appended to one of the two class-specific active site domains. In several cases, the specificity