transmembrane signaling protocols

Peptide Recognition Mechanisms of Eukaryotic Signaling Modules Chi-Hon Lee, David Cowburn, and John Kuriyan 1. Introduction The formation of specific protem-protein interactions is one of the key mechanisms for signal transduction mediated by tyrosme phosphorylation. These intermolecular mteracttons target signaling proteins to particular cellu- lar locations and modulate the enzymatic activities that further propagate the signal. A dtstmctive characteristic of the pathways that are mitiated by tyrosme phosphorylation is that target recognition and catalytic activity are usually functions of separate domains within the signaling molecules that participate m these pathways. Each of the signalmg molecules contains one or more of a set of modular peptide-bmdmg domains that are responsible for generating protein-protein interactions. Such peptide-recognition domains are modular in both structural and functtonal respects: They are capable of folding correctly when removed from the parent protein, and they can usually recognize their targets even when isolated. The first peptide-recognition modules to be identified were the Src homol- ogy 2 and 3 domains (SH2 and SH3 domains), so named because they share sequence similarity with two separate noncatalytic regions of the Src family tyrosme kmases (1,2). SH2 and SH3 domains are now well-known for their crittcal roles m eukaryotic signal transduction, and they function by recogmz- ing sites that contam phosphotyrosyl residues (for SH2) and prolme-rich sequences (for SH3) (reviewed in refs. 3-5). Several other peptide-bmdmg domains have been discovered recently, and the determinatton of their three-dimensional structures have provided some surprtses. The phosphotyrosme bindmg/phosphotyrosine interaction (PTB/PI) From Methods m Molecular Bfology, Vol 84 Transmembrane Slgnahng Protocols Edlted by D Bar-Sag1 0 Humana Press Inc , Totowa, NJ 3 4 Lee, Cowburn, and Kuriyan domain bmds to phosphopeptides containmg NPXY* motifs (Y*, phosphoty- rosme) (6,7). The architecture and mode of peptide recogmtton of the PTB domains is unrelated to that of the SH2 domains, although both recognize phosphotyrosme. Most strikmgly, the architecture and topology of the PTB domams resemble closely that of another signalmg module, the plekstrin homology (PH) domain, although there IS no sequence similartty between these domams (8-10). Furthermore, the newly discovered PDZ domains, which rec- ognize non-phosphorylated peptide sequences at the carboxyl-termmus of ron- channel proteins, have a core topology and peptide-binding mechanism with elements m common with the PTB domams (II). The WW domains, whose structure has been determined recently, represent an alternative mode of recog- nizing prolme-containing motifs when compared to the well-known SH3 domains (12) Again, the SH3 and WW domains are unrelated m sequence or structure. In this chapter, we focus on the structural aspects of these peptide-bmdmg domains, with emphasis on the sequence-specific recogmtton of targets. Much of the discussion is focused on the SH2 and SH3 domains, because more is known about them. The PTB and PDZ domains are discussed briefly m the context of their structural resemblance to PH domains. Newly characterized domams, such as the WW domam and the 14-3-3 protein, are not discussed. 2. SH2 Domains The SH2 domain was first recognized as a phosphotyrosme-binding module during studies of the mechanisms of viral oncogenes that interfere with cellular signaling (1,13,14). Subsequent experiments demonstrated that an individual SH2 domain binds to specific regions of tyrosme-phosphorylated proteins, such as particular sequences m the cytoplasmic regions of activated receptor tyro- sme kinases (reviewed m ref. 15). The first three-dimensional structures of SH2 domains confirmed that the module corresponds to a well-folded domain with a defined peptide-binding surface (16-18). In addition, the crystal struc- ture of the Src tyrosme kmase SH2 domain complexed with low-affinity phosphotyrosyl peptides revealed the mechamsm of phosphotyrosme recogm- tion that has subsequently been found to be conserved in general terms among all SH2 domains of known structure (18). Compartsons of SH2-target sequences m tyrosme-phosphorylated proteins such as platelet-derived growth-factor (PDGF) receptor and the polyoma-virus middle-T antigen indicated that residues immediately surrounding the phosphotyrosme determme the binding specificity of SH2 domains (19-22). However, a general picture of SH2-target specificity did not emerge until an exhaustive investigation was carried out using a peptide library approach Peptide Recognition Mechamsms 5 (23,24). This established that the three residues immediately C-terminal to the phosphotyrosme are the key determinants of specificity. The determination of the structures of high-affinity peptide complexes of Src and the closely related Lck-SH2 domains provided the fu-st view of sequence-specific peptide recog- rutron (25,26) By combmmg the structural information with selecttvity data from the pepttde-library study, the sequence preference can be correlated with particular residues in the SH2 domain (23,27). Subsequently, the structures of peptrde complexes of the SH2 domams of the tyrosme phosphatase SH-PTP2 (28), phospholipase C-~(29) and the adapter protems GRB2 (30) and She (31) have further clarified the mechanism of peptide recognition and have extended our understanding of SH2 specificity. An additional level of complextty was added when the brochemtcal and structural analysts extended toward larger components of signaling molecules, containing more than one domain. Structures of the adapter-protein GRB2 (32) and the regulatory unit of Abl tyrosme kinase (33) have provided insights into spatial arrangements of multiple domains. Furthermore, structural analysis of multi-domain constructs of ZAP-70 (34), Lck tyrosine kmase (35), and the tyrosine phosphatase SH-PTP2 (36) revealed the cooperatrve recogmtton of peptides by larger-signaling molecules of which these domains are compo- nent parts. 2.1. General Architecture The SH2 domain is a compact a-@-structure comprised of around 100 residues (see Fig. 1 for a sequence alignment). The central scaffold is an anti- parallel P-sheet formed by strands A, B, C, D, and G. Two a-helices, aA and aB, flank the central P-sheet (see Fig. 2 for a schematic diagram and the nota- tion used). This P-sheet runs perpendicular to the peptide-binding surface, and divides the domain mto two functronally distinct regions. One region, com- prrsmg helix aA, loop BC (the phosphate-binding loop), and the adjacent face of the central P-sheet, provides resrdues that interact with the phosphotyrosme. The other region includes helix aB, loops EF, BG, and the other face of the central P-sheet, and interacts with pepttde resrdues immediately followmg the phosphotyrosme; this regton accounts for the sequence-specific recognition. The peptide ligand lies across the surface of the domain approximately orthogonal to the central P-sheet (Fig. 2). The peptide ligands are usually in an extended conformatron and do not participate m secondary-structure forma- tion with the domain. The phosphotyrosine residue appears to be the main anchor point of the SH2-peptlde complex, allowing the domain to read out the three to six residues immediately followmg the phosphotyrosme. The peptide residues N-terminal to the phosphotyrosine make limited and nonspectftc mter- NJ AB pB BC PC CD PD PD’ EG3 BG4 II HGQLKE KNGDVI WC G IDVYIIGG IRRFIS lsLsDLIGYVsHVl SCLL KGE KLL IYP I Fig. 1. Alignment of SH2 sequences and defmmon of the residue notation The sequences of different SH2 domains are aligned, based on the secondary-structure definitions of Src and Lck (26). The boundaries of the secondary structural elements of Src are shown by solid boxes, and the notation for these elements is shown sche- matically at the bottom. The important residues are mdrcated by vertical lines at the top (Adapted with permrssron from ref. 28.) 6 Peptide Recognition Mechamsms 7 actions with the domain, and therefore most likely contribute little to the bmd- mg specificity. The N- and C-termmi of the SH2 domain are located on the side of the domain opposite to the peptide-bmdmg surface. For this reason, the domain can be readily inserted into different molecular contexts without affecting the peptide-binding ability. 2.2. Peptide-Binding Specificity and Affinity Several lines of evidence indicate that different SH2 domains bind to distinct phosphotyrosme contammg sites of their target proteins m vivo, and that the linear sequence surrounding the specific phosphotyrosine determines the binding specificity (19-22). To illustrate, a point mutation (Tyr 739 to Phe) in the PDGF receptor selectively elimmates the binding of the Ras GTPase activating protem (GAP) to the activated receptors, but the bmdmg of other SH2-containing proteins (such as PLC-y and PI-3 kmase) remains intact (37). It appears that the local sequence, rather than the ter- tiary structure, of the SH2-targets dominates the binding specificity. Tyrosine-phosphorylated peptides that contain sequences resembling the local sequence of the target protein (the Tyr 739 of PDGF receptor in this case) compete efficiently for the bmdmg of the target protein (PDGFR) to a particular SH2 domain (GAP) (37). In addition, the observation that a mutant PDGF receptor contammg a deletion near the GAP-SH2 binding site binds to the GAP-SH2 domain with nearly the same affinity as the wild- type PDGF receptor suggested that the tertiary structure is not a primary factor m determmmg bmdmg affmlty (38). These observations establish the relevance of studies usmg isolated peptides. A systematic search for optimal peptide sequences for SH2 domains had been carried out by screening a random phosphopeptide library (23,24). Of over 20 different SH2 domains tested, each showed distinct selectivity m the three residues immediately C-terminal to phosphotyrosme in the peptide ligand. Such sequence preference could be correlated with the side-chains of residues at several critical positions of the SH2 domain (24). The clearest example of this correlation is provided for the residue at the PDS position of the SH2 domain, which contacts the peptide side chains at position +l and +3. Certam SH2 domains, including Src-family tyrosme kmases as well as GAP and the adapter proteins GRB2 and Nck, have aromatic residues at pD5, and preferen- tially bmd to pepttdes contammg polar side chains at +l. In contrast, other SH2 domains (~85, phosphohpase C-y, the tyrosme phosphatases) contam hydro- phobic side chains at pD5, and select for hydrophobic residues at +l. Quantitative analysis using isothermal-titration calorimetry and surface- plasma resonance (39) mdicated that the SH2-peptide mteraction is of only moderate strength (Kd -0.1-3 0 pM> compared with strong mteractions Lee, Cowburn, and Kuriyan A Fig. 2. (see also facing page) Schematic diagram of two SH2-peptide complexes (A) The Src-YEEI complex and (B) the N-terminal SH2 domain of SH-PTP2 complexed with a peptide derived from Tyr 895 of IRS-l, The view is from the pep- tide-binding surface and illustrates the secondary-structure elements and the notation used. The peptide is shown in a ball-and-stick representation and comprises phosphotyrosine (p-Tyr), residue +l, residue +2, and so on. ol-helices and P-strands are shown as ribbons and arrows, respectively. such as those between transcription factors and their specific DNA targets (Kd ~1 nil4). The phosphotyrosine is absolutely required for binding to SH2 domains (40). Peptide Recognition Mechanisms Fig. 2. Peptide residues immediately following the phosphotyrosine (+ 1 to +6) are the critical determinants for binding to individual SH2 domains; however, a varying range of amino acids are tolerated at each site. Although the selectivity of individual SH2 domains is not sharply defined, the specificity and affinity can increase dramatically when cooperative binding interactions occur (see discussion Subheading 5.1. for tandem SH2 domains of ZAP-70). Kinetic analysis of SH2-peptide interaction has shown that the association and disso- ciation rates (k,,, and I&) are both very rapid even for high-affinity peptide ligands (41). Fast turnover rates could allow the rapid sampling of different binding sites and are observed for many protein-protein interactions involved in signal transduction. IO Lee, Cowburn, and Kurtyan 2.3. Recognition of Phosphotyrosine The recognition of phosphotyrosme 1s the defmmg feature of the SH2- pepttde interface. Although the details vary slightly from one SH2 complex to another, the overall features of the mteractron are strikmgly conserved. Rest- dues from aA, PB, PD, and the BC loop form the phosphotyrosine-bmdmg pocket and provide hydrophobtc mteractions with the phenoltc ring of phosphotyrosme and hydrogen-bonding interactions with the phosphate group (Figs. 3A,B). The most critical interaction wtth the phosphotyrosine is provided by Arg PBS, which forms a bidentate-tome interaction with the phosphate group. This arginine is located at the bottom of the binding pocket and becomes completely maccessible to solvent upon binding. Arg PB5 is strictly conserved m all SH2 domams, and even the conservative mutation of this residue to lysine abolishes bmdmg (42). With the backbone of the phosphotyrosme residue held m post- non by the outer strand of the central P-sheet (PD), the ionic mteraction between the phosphate group and Arg PBS provtdes a stereochemical “ruler” that appears to be the key for discrimmatmg between phosphotyrosme and other restdues. The location of Arg PBS is such that, in a fully extended conformation, this side chain IS Just long enough to interact with the phosphate group of a fully extended phosphotyrosme side chain, thus excludmg phosphoserme or phosphothreomne. An interesting feature often observed in the SH2-peptide complexes is the presence of an ammo-aromatic interaction between an ammo nitrogen of Arg aA and the phosphotyrosine rmg (18). Ammo-aromatic interactions have been observed in a number of protein structures as well as m some small molecules structures (43). The ammo mtrogen of this argmme hydrogen bonds with the phosphate group and the backbone-carbonyl group of the peptide. These mter- actions mediated by Arg aA appears to be optimal for phosphotyrosme and were first identified m the Src (18) and Lck structures (26) and later m other SH2-peptide structures, including ZAP-70 (34). However, the SH2 domains of the tyrosine phosphatases do not have Arg aA (it is replaced by glycme). In the SH2 structure of the phosphatase SH-PTP2 (28), the phosphate group rotates by -180”, facmg toward the BC loop (Fig. 3B), and the number of hydrogen bond with the phosphate group is almost the same as m Src or Lck. The ammo-aromatic mteraction is also not seen m other SH2 structures (such as p85 ref. 44) even when Arg aA 1s present. 2.4. Peptide Recognition The structures of the closely-related Src and Lck SH2 domams (25,26) m complex with a high-affmtty pepttde containmg the Tyr-Glu-Glu-Ile (YEEI) motif provided the first piece of structural mformation on sequence-specific Peptide Recognition Mechanisms 11 Fig. 3. Stereoviews of the phosphotyrosine binding sites of (A) Src and (B) N-terminal SH2 domains of SH-PTP2. The polypeptide backbone of the peptide is shown as a tube and the phosphotyrosine side chain is shown in black. Hydrogen bonds are indicated by dashed lines. (Adapted with permission from ref. 28.) recognition by the SH2 domain (Fig. 3A). In these structures, the peptide binds to the SH2 domain in an extended conformation and the interaction resembles a two-pronged plug (the peptide) engaging a two-holed socket (the SH2 domain). The two prongs refer to the phosphotyrosine and the Ile +3 residue of the, peptide, which fit into the corresponding pockets on the SH2 surface. This type of interaction is also observed in several other SH2-peptide complexes. 72 Lee, Cowburn, and Kuriyan 2.4.1. Type 1: Src and Lck Previous studies using random-peptlde libraries indicated that the Src-family SH2 domains strongly select large hydrophobic residues at +3 and, to a lesser extent, prefer nonbasic polar residues at +l and +2, with the optimal motif being YEEI (two glutamates and one isoleucme followmg the phosphotyrosme) (24). A peptide containing this optimal motif, derived from the hamster polyomavlrus middle-T antigen, binds to Src-family SH2 domains with high affinity and has been used for the structural studies. The most important feature m the Src and Lck structures is that the Ile +3 residue of the YEEI peptlde engages a well-defined hydrophobic pocket of the SH2 domain. This interaction 1s responsible for the selection of large hydro- phobic residues at +3 posltlon. The residues lmmg this pocket (which arise from PD, DE, loops EF and BG) are rather divergent. In particular, the two variable loops, BG and EF, shape the surface topography of this pocket. Muta- tions as well as large msertlons/deletlons are often found in this region, and these have been shown to be important for binding specificity. The glutamate residues at +l and +2 do not form extensive interactions with the SH2 domain, but are m the vlcmlty of basic residues that may account for the moderate selectivity against basic residues at these positions in the peptides (Fig. 2A). The prototypical two-pronged Interaction 1s also observed in two X-ray structures of the N-terminal SH2 domain of the ~85 subunit of PI-3 kmase (p85N), complexed separately with two high-affinity peptldes containing the optimal Y-M/V-X-M motif (44). Although m p85N SH2 the position of Met +3 shifts shghtly toward the central P-sheet, the interaction between this residue and the hydrophobic pocket 1s similar to that seen m Src and Lck. This resem- blance is expected, because both SH2 domains favor large hydrophobic residues at this posltlon (although p85N shows a higher preference for Met). A unique feature of the p85N SH2 domain 1s that this hydrophobic pocket 1s blocked by the side chain of Tyr BG5 m the absence of hgand, and this side chain has to move by 8 8, to open up the pocket for hgand binding. The large movement of the Tyr side chain might account for the changes in circular dlchroism and fluorescence spectra that had been noted upon peptide bmdmg, because no other large-conformatlonal change induced by peptide binding was found. A notable difference in the binding speciflcltles of p85N and Src 1s that, at the +1 position, p85N SH2 prefers hydrophobtc residues, whereas Src SH2 favors nonbasic polar residues. Ile PDS (m p85N) appears to be the major determinant for this difference, since replacement of this residue by Tyr (found in Src) shifts the selectlvlty toward that of the Src-family SH2 domains (27). In the p85N structure, the less bulky side chain of Ile pD5 opens up a shallow- [...]... were transmembrane protein-tyrosme kinases It seemed obvious that the key to understanding signal transmission would be to find and identify the substrate proteins phosphorylated by the liganded receptors, which must surely be the effecters responsible for stimulating the cell to proliferate When lysates of growth-factor stimu- From Methods m Molecular Bfology, Edlted by D Bar-Sagt Vol 84 0 Humana 33 Transmembrane. .. specificity and regulation of binding mteractions, review some of the classes of well-known protein-protein interactions known to be mvolved m intracellular signaling, and discuss how the significance of a particular interaction can be assessed interactions in Signaling Cascades 35 2 Specificity Two of the defining parameters of protein-protem mteractions are specificity and whether that specificity can be... sequence mformatton improves and as three-dtmensronal structural information accumulates, it is likely that many other binding modules will emerge Interactions in Signaling Cascades 37 Table 1 Protein-Binding Module Modules Implicated in Signaling No Repeats/ Core Sizea protein binding site” Regulated? SH2 -100 l-2 Y(P)nnn PTB -160 1 NPxY(P) SH3 -60 l-3 nxQPx@P PH 2100 l-2 Ankyrm 33 4-24 or QPx0Pxn... discoveries: 1 Many proteins rmphcated m signaling contained SH2 domams; 2 These proteins could often be shown to bind tightly to hgand-activated growthfactor receptors; and 3 Bacterially expressed SH2 domams could be shown to bind to tyrosmephosphorylated proteins, including activated receptors (7-11) We now know that these domains serve a general role in signaling m complex eukaryotes, mediating... (1993) Crystal structure of the SH3 domam m human Fyn; compartson of the three-dimensronal structures of SH3 domains m tyrosine kmases and spectrm EMBO J 12,2617-2624 2 Protein-Protein Interactions in Signaling Cascades Bruce J Mayer 1 Introduction The past decade has seen an explosion m our understanding of the mechamsms underlying the transmission of signals from outside the cell, and the ways m which... the two domains is similar, with the SHC-PTB domain somewhat larger and more complex (Fig SA) In both cases, the core architecture and topology of the protein fold is similar to that of the PH domain, a signaling module with various functions (4) In this PH-domain superfamily, two medium-size P-sheets pack against each other with inter-strand angles of about 60”, and a C-terminal a-helix lies along one... responsible for stimulating the cell to proliferate When lysates of growth-factor stimu- From Methods m Molecular Bfology, Edlted by D Bar-Sagt Vol 84 0 Humana 33 Transmembrane Sgnalmg Press Inc , Totowa, NJ Protocols Mayer lated cells were analyzed with phosphotyrosme-specific antibodies, however, a problem arose By far, the most promment tyrosme-phosphorylated protem was found to be the receptor itself Clearly... initiate mitogemc signalmg From such studies, a new paradigm emerged m which an enzyme’s predominant function can be to alter its bmdmg activities in response to ligand When closely exammed even “classical” signaling pathways reveal the crltl- cal importance of stable and regulated protein-protein interactions Among the best understood signalmg cascades are those mediated by heterotrimeric G proteins (3)... adenosine monophosphate (CAMP) in turn activates many molecules of protein kinase A, which then phosphorylate many intracellular proteins on serine and threomne residues The details of this relatively simple signaling apparatus reveal at least five critical protein-protein Interactions: The heterotrimeric G protein binds to the hganded (but not the unliganded) receptor; conformational changes brought about... (reviewed in ref 67) The molecular mechanism by which Nef promotes disease progress is not known in detail, although several lines of evidence suggest that Nef might function by interacting with cellular -signaling proteins Nef contains an invariant PxxP motif which is critical for optimal viral replication and has been shown to mediate specific interaction with certain Src familySH3 domains (68) A P- . bindmg/phosphotyrosine interaction (PTB/PI) From Methods m Molecular Bfology, Vol 84 Transmembrane Slgnahng Protocols Edlted by D Bar-Sag1 0 Humana Press Inc , Totowa, NJ 3 4 Lee, Cowburn,. signal transduction mediated by tyrosme phosphorylation. These intermolecular mteracttons target signaling proteins to particular cellu- lar locations and modulate the enzymatic activities that. target recognition and catalytic activity are usually functions of separate domains within the signaling molecules that participate m these pathways. Each of the signalmg molecules contains