The Interaction of Proline-Rich Motifs in Signaling Proteins with Their Cognate Domains

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The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains

BRIAN K. KAY,*,1 MICHAEL P. WILLIAMSON,† AND MARIUS SUDOL‡ *Department of Pharmacology, University of Wisconsin-Madison, Madison, Wisconsin 53706-1532, USA; †Department of Molecular Biology and Biotechnology, University of Shefﬁeld, Western Bank, Shefﬁeld S10 2TN, United Kingdom; ‡Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029-6574, USA

ABSTRACT A common focus among molecular SH3 DOMAINS and cellular biologists is the identification of proteins that interact with each other. Yeast two- SH3 domains are 50–70 amino acids long and often hybrid, cDNA expression library screening, and present in eukaryotic signal transduction and cy- coimmunoprecipitation experiments are powerful toskeletal proteins such as Abl, actin-binding pro- methods for identifying novel proteins that bind to tein, Bem1, cdc25, cortactin, calcium channel ␤1B2 one’s favorite protein for the purpose of learning subunit, Grb2, myosin, Nck, PI3K regulatory subunit, more regarding its cellular function. These same PLC␥, ras GTPase activator, spectrin, and tight junc- techniques, coupled with truncation and mutagen- tion protein ZO-1. Computer-aided analysis of pro- esis experiments, have been used to define the tein sequences has even suggested that SH3 domains region of interaction between pairs of proteins. may also exist in bacteria (7a). For a long time, the One conclusion from this work is that many inter- function of the SH3 domain in eukaryotes was enig- actions occur over short regions, often less than 10 matic; however, since the domain was present in so amino acids in length within one protein. For many cytoskeletal proteins, it was assumed to play a example, mapping studies and 3-dimensional anal- role in mediating protein–protein interactions (8) or yses of antigen–antibody interactions have re- directing cell compartmentalization (9). The SH3 vealed that epitopes are typically 4–7 residues long domain clearly had an important function for some (1). Other examples include protein-interaction proteins, as mutations in the SH3 domains of cellular modules, such as Src homology (SH) 2 and 3 Src (10) and Abl (11, 12) are activating. domains, phosphotyrosine binding domains The ligand specificity of SH3 domains was first (PTB), postsynaptic density/disc-large/ZO1 revealed when a ␭-cDNA expression library was (PDZ) domains, WW domains, Eps15 homology screened with a glutathione S-transferase (GST)-Abl (EH) domains, and 14–3-3 proteins that typically SH3 fusion protein (13). Two different cDNA clones recognize linear regions of 3–9 amino acids. Each were isolated and the regions responsible for their of these domains has been the subject of recent binding to the Abl SH3 domain were shown to be reviews published elsewhere (2–7). Among the proline-rich segments (14). Examination of other primary structures of many ligands for protein– SH3 ligands identified in the same manner (15–17) protein interactions, the amino acid proline is has suggested that SH3 domains recognize proline- critical. In particular, SH3, WW, and several new rich sequences containing the core PxxP, where x protein-interaction domains prefer ligand se- denotes any amino acid (18, 19). quences that are proline-rich. In addition, even With the advent of combinatorial peptide libraries, though ligands for EH domains and 14–3-3 do- ligand specificity has been determined rapidly for a mains are not proline-rich, they do include a single large number of different SH3 domains. The SH3 proline residue. This review highlights the analysis domains of Src and the phosphatidyl inositol 3-ki- of those protein–protein interactions that involve nase (PI3K) regulatory subunit select proline-rich proline residues, the biochemistry of proline, and sequences from peptides displayed on beads (20) current drug discovery efforts based on proline peptidomimetics.—Kay, B. K., Williamson, M. P., 1 Sudol, M. The importance of being proline: the Correspondence: Department of Pharmacology, Univer- sity of Wisconsin, 1300 University Ave., Madison, WI 53706- interaction of proline-rich motifs in signaling pro- 1532, USA. E-mail: [email protected] teins with their cognate domains. FASEB J. 14, 2 The FASEB Journal cannot guarantee the availability of 231–241 (2000) references to the Web.

0892-6638/00/0014-0231/$02.25 © FASEB 231 TABLE 1. Protein-rich sequences that bind to speciﬁc protein complexes have shown that peptide ligands can bind a interaction modules in two orientations with respect to the SH3 domain (28, 29). The peptides, which bind in either an N to Proteins with SH3 domains Peptide ligand motif Reference C or C to N terminal orientation relative to the SH3 Src RPLPPLP (21–23, 28) domain, have been classiﬁed as either class I or class PPVPPR Yes RxLPxLP (26) Lyn RxxRPLPPLPxP (22) Abl PPP⌿PPPP␺P (22) PI3K RxxRPLPPLPP (22) PLC␥ PPVPPRP (26) Cortactin ϩPP␺P␺KP (26) p53BP2 RPx␺P␺R (26) Grb2 A PLPxLP (26) Crk A PxLPx(K/R) (134) Amphiphysin I PxRPx (R/H) (R/H) (27) Nck, SH3 B PxPPxRxxSL (25) CAP, SH3 C PxPPxRxSSL (135)

Proteins with WW domains Class I WW’s YAP65 PPxY (53) Nedd-4 PPxY (66) Dystrophin PPxY (69) Class II WW’s FBPs PPPPPPL/R (56) FE65 PPPPPPL/R (65) Class III WW’s FBPs (PxxGMxPP)2* (58) Class IV WW’s Essl/Pinl (pS/pT)P (59, 62, 110) Nedd-4 (pS/pT)P (62)

Other proteins Mena (EVH1) (D/E)FLPPPP (84) Homer (EVH1) PPxxFr (88) CD2-BP (BHB) (PPPGHR)2* (91) Proﬁlin G/LPPPPPP(PP) (92)

a Peptide sequences were deduced from screens of combinatorial peptide libraries, analysis of various interacting proteins, or amino acid replacements of individual sequences. In some cases, only the core of the deduced motifs is shown. Additional ligand sequences Figure 1. A) Three-dimensional model of how class I and II can be found on the Internet for SH3 (http://www.pharmacia.mod- ligands bind to SH3 domains, based on the structure of the ules/SH3/ligands) and WW domains (http://www.bork.embl-heidelberg.de/Modules.ww-gif.html). Residues labeled ⌿, ϩ, and x corre- Src SH3 domain (28). The protein’s topological surface is spond to aliphatic (A, I, L, V, and P), positively charged (R, K, or H), shown in gray, with acidic residues colored in blue. The and any residues, respectively. Lowercase letters refer to positions ligands are RALPPLPRY, a class I ligand (below) and AFAP- that are often (but not always) a certain residue. pS and pT refer to PLPRR, a class II ligand (above). All residues in the ligands phosphoserine and phosphothreonine, respectively. Residues in pa- are colored gray, except for the recognition prolines (yellow), rentheses have been shown to be equivalent. Proteins with multiple flanking prolines (cyan), leucine (brown), and arginine SH3 domains, such as Grb2, Nck, and CAP (Cbl-Associated Protein) (red). The side-chain of the first arginine in the class II ligand have the particular SH3 domains denoted with letters, starting at the has been omitted for clarity. The peptides interact with the amino terminus with A. 2* indicates a tandem repeat of the bracketed two LP dipeptide pockets (left and center) and the acidic sequence. specificity pocket (right), which form grooves in the surface

separated by ridges. The NH2- and carboxyl-termini of the and phage (21–23). Phage libraries have also been peptides are denoted with N and C, respectively. B) Three- dimensional model of how peptide ligands bind to the YAP65 used to identify the specificity of SH3 domains from WW domain. The protein’s topological surface is shown in Abl, amphiphysin I, cortactin, Crk, Fyn, Grb2, Lyn, white, with three of the key residues shown in green (from left Nck, p53BP2, PI3K, PLC␥, Tsk, and Yes (22, 24–27). to right, Trp39, Tyr28, and Leu30). The peptide ligand is The optimal ligand preference for each domain shown in gray, except for the core prolines (yellow), which sit varies around the PxxP core (Table 1), demonstrat- in a shallow pocket formed by Trp39 and Tyr28, the flanking prolines (cyan), and the tyrosine (red), which sits over the ing that a great deal of protein–protein interaction hydrophobic pocket containing Leu30. C) A space-filling specificity can be encoded by proline-rich sequences. model of a polyproline helix, colored as green, carbon; red, Investigations into the structure of peptide-SH3 oxygen; blue, nitrogen; white, hydrogen.

232 Vol. 14 February 2000 The FASEB Journal KAY ET AL. II ligands, respectively. The orientation of the pep- Recently, a novel ligand sequence has been ob- tide is dictated by the location of a positively charged served for the SH3 domain of the epidermal growth residue, relative to the PxxP core, which forms a salt factor receptor substrate, Eps8, which has been bridge with an acidic residue in the SH3 domain discovered to form intertwined dimers (37). Because (Fig. 1A). Thus, peptides with the motif ϩxxPxxP the site of dimerization includes the interface of the and PxxPxϩ (where ϩ refers to a positively charged SH3 domain that typically interacts with PxxP motifs, amino acid) correspond to class I and class II motifs, the specificity of the intertwined dimers is altered. respectively. Some SH3 domains, like that present in Analysis of the ligand preferences of this dimer has Src, have the capacity to bind peptides of either class revealed it to recognize not PxxP, but PxxDY instead (i.e., RPLPPLP and PPVPPR; where the scaffolding (37a). It will be interesting to learn how common prolines are underlined), although the biological this unusual arrangement is among other SH3 do- implications of this are unclear. (One frequently mains. raised possibility is that ligand binding in two orien- Analysis of several SH3 domain interactions has tations could access different partners in multicom- suggested that some form stable complexes and others ponent protein complexes.) In both orientations, may be regulated in the cell. For example, the interac- not only do proline and leucine side-chains fit into tion between Sos and Grb2, which is constitutively the same hydrophobic pockets on the SH3 domain complexed in cells, can be inhibited by phosphoryla- (Fig. 1A), but also very similar hydrogen bonds can tion of serine/threonine residues in the proline-rich be made from peptide carbonyls in the proline-rich carboxyl-terminal segment of Sos, after receptor activa- ligand (30). Typically, the Kd values of synthetic tion (38). Phosphorylation of a PxxP site in the Wis- peptides binding SH3 domains are 1–100 ␮M, al- kott-Aldrich syndrome protein (WASP) can inhibit its though amino acid analoging experiments have led interaction with the SH3 domain of the cytoskeletal- to the development of higher affinity peptide ligands associated protein, PSTPIP (39). Although the sites

(31). The Kdvalues of full-length proteins that inter- have not been mapped, phosphorylation of the Dros- act with SH3 domains are signiﬁcantly lower than for ophila-enabled (Ena) protein by Abl can inhibit the peptides; for example, the HIV Nef protein binds to interaction of Ena with certain SH3 domains (40). the Hck SH3 domain with a Kd of ϳ250 nm (32). Finally, binding of phosphopeptide ligands to the Crk There are three shallow pockets within the SH3 SH2 domain alters its conformation such that a pro- domain where the peptide ligands bind. Two of the line-rich insert in the Crk SH2 domain becomes an pockets are 25 Å long and 10 Å wide, large enough to SH3 domain-binding site (41). Very likely, the interac- accommodate each of the prolines in the PxxP motif, tion between other SH3 domains and ligand-contain- accompanied by a hydrophobic residue (i.e., A, I, L, V, ing proteins is regulated by allosteric changes (42). A and P). These two pockets are parallel to each other recent publication capitalizes on this conclusion by and have been termed the ‘LP dipeptide pockets’ (33). generating temperature-sensitive SH3 domains for the A third pocket, termed the ‘speciﬁcity pocket’ is bound purpose of dissecting SH3 domain function in vivo by two loops (i.e., RT, nSrc) of the SH3 domain (34). (43). Such an approach may be applicable to other

Within this pocket sit residues either NH2- or carboxyl- protein interaction modules. terminal of the positively charged residue in class I or Although most SH3 domain-mediated interactions class II ligands, respectively. are intermolecular, several intramolecular interactions Besides the two orientations described above, a have been described. In Src (44, 45), Hck (46, 47), and third SH3 ligand corresponding arrangement has Abl (48), the SH3 domains interact with a linker region been described. In this arrangement, residues between the SH2 and catalytic kinase domains of these spaced apart in a protein sequence come together, proteins, thereby adopting a catalytically inactive con- because of the tertiary structure of the protein, to formation (49). In some cases, this conformation is interact with the peptide-binding groove of the SH3 stabilized by the interaction of the SH2 domain with domain. One example of this arrangement occurs phosphorylated tyrosine residues present near the car- within the p53 binding protein, p53BP2. The SH3 boxyl termini of such proteins. The elucidation of domain of p53BP2 interacts with two segments of the these various intramolecular interactions explains the L3 loop of p53, in a manner analogous to the observations that mutations in the SH3 domains of Src contacts made in canonical SH3-PxxP complexes, (10) and Abl (50) are activating, because the mutant with the positions and orientations of the interacting forms fail to adopt an inactive conformation. Another residues determined by the overall 3-dimensional example occurs in the cellular oncogene, Itk, where a structure of p53, rather than as part of a PxxP core proline-rich sequence, KPLPPTP (residues 155–160) (35). It should be mentioned that p53 does contain binds to an SH3 domain (residues 170–232) within the two PxxP motifs that have been shown to be involved same protein (51). The intramolecular interaction is in growth suppression through interactions with quite stable, even without the involvement of an SH2 other SH3 domain-containing cellular proteins (36). domain, although the interaction of the Itk SH3 do-

PROLINE 233 main with a soluble form of the KPLPPTP peptide Immediately after its discovery, the WW domain sequence is undetectable. attracted attention because the signaling complexes it mediates have been implicated directly or indirectly in several human diseases including Liddle’s syndrome of hypertension, muscular dystrophy, and WW DOMAINS Alzheimer’s and Huntington’s diseases (64–69). Liddle’s syndrome results from genetic lesions that WW domains are small globular modules composed affect ␤ and ␥ subunits of the amiloride-sensitive of 38–40 amino acids. The name refers to two epithelial sodium channel (70). Most of the muta- conserved tryptophan (W) residues that are spaced tions that have been reported in patients with Lid- 20–22 amino acids apart and play an important role dle’s syndrome represent deletions that encompass a in the structure and function of the domain (52). minimum of 12 residues, which includes the PPxY The WW domain was initially identified by computer- motif. Three independent Liddle’s syndrome pa- aided analysis of imperfectly repeated sequences in tients were recently characterized who have single- the murine form of Yes-associated protein (YAP; point mutations in one of the three crucial positions http://www.bork.embl-heidelberg.de/Modules.ww- of the PPxY motif in the ␤ subunit of the sodium gif.html). Although the WW domain resembles the channel (70–72). These mutations (i.e., P615S, SH3 domain functionally by displaying affinity to- P616L, and Y618H) substantiate biologically the con- ward proline-rich ligands, their structures are dis- clusions first generated by in vitro alanine scanning tinct (53–55). WW domains have an antiparallel of a short region within YAP WW domain interacting three-stranded ␤-sheet structure that forms a shallow proteins (53, 63). Liddle’s mutations abolish binding binding pocket for ligands containing PPxY or PPLP of sodium channel subunits to the Nedd-4 protein, core motifs, usually flanked by additional prolines which in addition to containing three WW domains (53, 56, 57). Recently another motif, with a prelimi- includes an ubiquitin-ligase catalytic domain (66, 73, nary consensus PxxGMxPP (Table 1), was proposed 74). Nedd-4 normally participates in targeting the for ligands interacting with a subset of WW domains epithelial sodium channel subunits for ubiquitin- present in proteins participating in the pre-mRNA directed proteolysis, but in mutant epithelial cells splicing machinery (58). These WW domains have a the channel has a long half-life, leading to a sodium distinguishing feature of three consecutive tyrosine imbalance and subsequently high blood pressure. residues located centrally within them. Given that epithelial sodium channels are phosphor- The first structure of a WW domain was that of ylated on serine, threonine, and possibly tyrosine human YAP in complex with its cognate ligand, solved by Nuclear Magnetic Resonance spectroscopy (54). residues, it will be interesting in the future to learn Shortly thereafter, the structure of Pin1 WW domain whether or not these modifications modulate the was solved by X-ray (59). These two structures are interaction between the sodium channel and the almost completely superimposable. The WW domain is WW domains of Nedd-4 and thereby change the rate the most compact globular structure known to occur of channel degradation (75, 76). naturally, and it is the smallest ␤-sheet module that Recently, the PPxY motif has been observed in a folds as a monomer in solution without disulfide number of transcription factors (i.e., c-Jun, AP2, bridges or cofactors (60). The hallmarks of the binding NF-E2, C/EBP␣, PEBP2/CBF) where it may play a pocket of the WW domain of human YAP include three role in transcriptional activation. For example, the hydrophobic amino acids, leucine, tyrosine, the second hematopoietic transcription factor, NF-E2, contains conserved tryptophan and histidine, as confirmed by two PPxY motifs that can be recognized by the WW structural and mutational studies (54). Two prolines of domains contained within certain ubiquitin ligases the ligand (PPxY) form van der Waals contacts with the (61, 77). Interaction of WW domain-containing pro- second tryptophan, whereas the terminal tyrosine of teins with this motif is likely to be important for the ligand fits into a hydrophobic pocket and is coor- transcriptional activation, as deletion of one of the dinated by a hydrogen bond from the conserved histi- two PPxY motifs in NF- E2 (61) or the single copy in dine residue (Fig. 1B). The Kd of interaction for PEBP2 (78) inhibits their ability to transactivate WW-ligand complex formation is in the high nm to low genes. Thus, the presence of the PPxY motif within ␮M values for proline-rich ligands, and in the low ␮M transcription factors may function to recruit WW values for phosphoserine or phosphothreonine con- domain-containing proteins such as YAP (78) and taining ligands, depending upon the domain-ligand Npw38 (79), which have been discovered to act as pair, buffer conditions, and the binding assay (57, 61, transcriptional coactivators. Conversely, it is possible 62). Phosphorylation of the terminal tyrosine in the in some cases that WW domain-containing proteins ligand (PPxY) abolishes the binding in vitro, suggesting serve to negatively regulate transcription. Recently, that this modification could represent a negative regula- PQBP-1, a novel polyglutamine tract binding protein tion mechanism for a subset of WW domains in vivo (63). with a WW domain, has been shown to inhibit

234 Vol. 14 February 2000 The FASEB Journal KAY ET AL. transcription activation by Brn-2, although the role BIOPHYSICAL REASONS WHY PROLINE IS A of its WW domain is yet to be deﬁned (80). COMMON BINDING MOTIF

Proline is unique among the 20 common amino OTHER MODULES/PROTEINS THAT BIND acids in having the side-chain cyclized onto the PROLINE-RICH LIGANDS backbone nitrogen atom. This means that the conformation of proline itself is limited, with backbone The number of modules that bind proline-rich li- ␾ angles of ϳϪ65°. It also restricts the conformation gands is increasing (81). The EVH1 (Enabled, VASP, of the residue preceding the proline because of the Homology 1) domain is a protein interaction mod- bulk of the N-substituent and results in a strong ule present in Ena, vasodilator-stimulated phospho- preference for a ␤-sheet conformation (99). As a protein (VASP), and the WASP family of proteins consequence, polyproline sequences tend to adopt that regulate the dynamics of the actin cytoskeleton the PP II helix, which is an extended structure with (82–85). EVH1 domains bind the proline-rich con- three residues per turn. This implies that the two sensus sequence (D/E)FPPPP, which is present in prolines in the SH3 domain ligand core, PxxP, are ActA, a protein on the surface of the bacterial on the same face of the helix and are thus well pathogen Listeria monocytogenes as well as in vinculin placed to interact with the protein. The PP II helix is and zyxin, two protein components of focal adhe- an unusual structure: the prolines form a continuous sions (84). The EVH1 domain has also been detected hydrophobic strip round the surface of the helix, in Homer, a neuronal protein enriched in excitatory while the backbone carbonyls present ideal hydro- synapses (86), and which binds to proline-rich motifs gen bonding sites, being both conformationally re- (i.e., PPxxF) within glutamate and inositol trisphos- stricted (and therefore poorly hydrated) and elec- phate receptors (86–88) and Shank, a postsynaptic tron-rich (Fig. 1C). Therefore, PP II helices present density protein (89). Recently, the 3-dimensional an easily accessible hydrophobic surface, as well as a structure of the EVH1 domain has been solved for good hydrogen-bonding site. The accessibility of PP the mouse Ena protein (90). Much like SH3 and WW II helices is greatly enhanced by the fact that they are domains, aromatic residues within the EVH1 domain frequently found either at the NH2- or carboxyl create an interaction surface for proline-rich peptide termini of proteins where they form extended struc- ligands that adopt a polyproline II (PP II) conforma- tures that have been described as ‘sticky arms’ (100). tion (see below), even though the binding site is PP II helices are common in globular proteins with V-shaped instead of flat. solved 3-dimensional structures (101), where they Two other proteins that bind proline-rich peptides are generally solvent exposed and amphipathic should be mentioned as well. A cytosilic protein, (102). They are probably even more common in CD2 binding protein (CD2BP2), was recently de- proteins that have not been characterized structur- scribed in a pathway that regulates CD2-triggered T ally, because they tend to occur in extended regions lymphocyte (91). The CD2BP2 protein contains a that are hard to characterize using X-ray diffraction domain that was shown to interact with a tandemly or NMR spectroscopy. It does not require an unbro- repeated PPPGHR sequence (Table 1) and its struc- ken sequence of prolines to make a PP II helix; in ture was recently solved (91a). Profilin was originally fact, this type of left-handed helix can form in described as a regulator of actin polymerization (92). globular proteins without protein in the helix (101). Within its structure, profilin, like SH3, WW, and By contrast, a single nonproline residue may be EVH1 domains, exposes stacked aromatic and hydro- enough to interrupt a PP II helix in a completely phobic residues to form an interface that binds exposed chain (103), and it has commonly been proline-rich ligands (82, 93, 94). The binding of observed that the flanking prolines around or within polyproline has been shown to be essential for the core PxxP SH3 ligand sequence function to profilin function (95); profilin require a minimum maintain the required PP II structure (104). of 6–8 consecutive prolines for high affinity (92, 94, The relative rigidity of polyproline stretches means 96). Some of these cores are flanked by leucines, that they lose little conformational entropy on bind- providing a basis for speculation about degeneracy ing and thus bind more favorably than other ex- of certain proline-rich ligands that might interact posed (i.e., nonglobular) peptide sequences. Of with both profilin and SH3 and WW domains with course, proline-rich sequences cannot bind as tightly similar affinities (81, 94, 97). Indeed, it has been as globular domains can; however, weaker binding speculated that profilin may provide the link be- can be of great advantage, as it allows the binding of tween signaling pathways and remodeling of the proline-rich regions to be modulated rapidly. It also actin cytoskeleton (98), a good example of the permits large changes to be made in Kd by small versatile biological role made possible by the promis- changes in the sequence of the proline-rich se- cuity exhibited by proline-rich regions and their quence or of its binding domain, either by sequence ligands. changes or by covalent modification such as phos-

PROLINE 235 phorylation. The entropic stabilization has been intramolecular ligands, as described above for SH3 estimated to be ϳ1 kcal mol-1 per amino acid, both domains (42). Because an intramolecular interac- experimentally (105) and theoretically (100). The tion is generally more energetically favorable than interaction is largely hydrophobic and, therefore, the equivalent intermolecular interaction, the ma- does not require highly complementary surfaces. nipulation of intramolecular interactions, by confor- This accounts for the large sequence variability seen mational change or phosphorylation, is likely to be in polyproline binding and for the remarkable abil- common. ity, described above, of SH3 domains to bind Phosphorylation is a very common mechanism for polyproline sequences in both orientations, two fea- regulation of protein function and is used exten- tures that give proline-rich ligands great versatility in sively in the context of proline-rich regions (108). signaling pathways (30). Proline-rich regions often contain serine or threo- Because proline-rich regions are exposed, their nine, which frequently occur as the residue immedi- on-rates and off-rates for binding can be very fast. ately preceding the proline. Specific enzymes control However, the price to be paid for these fast rates is phosphorylation and dephosphorylation: for exam- that the complexes are not structurally very well ple, the microtubule-associated protein 2 (MAP2) is defined on a nanometer scale. Therefore, proline- developmentally regulated by phosphorylation, rich sequences are commonly found in situations which controls its binding to tubulin via a proline- requiring the rapid recruitment or interchange of cdc2 several proteins, such as during initiation of tran- rich region (109). Both MAP2 kinase and p34 scription, signaling cascades, and cytoskeletal rear- (108) have a preference for the sequence Px(S/T)P, rangements. Here, the role of proline-rich regions is in which the two prolines are separated by two not to provide a structurally defined complex but residues, suggesting that the proline-dependent ki- rather to bring proteins together in such a way that nase may well be recognizing a PP II conformation in subsequent interactions are more probable. For ex- the substrate. As noted above, some WW domain ample, the proline-rich region on Sos1 binds to the ligands represent target sites for proline-directed two SH3 domains of Grb2, thereby bringing Sos1 to serine-threonine kinases and have a phosphorylat- the cell membrane following receptor activation, able residue frequently in the vicinity of the prolines. where Sos1 in turn then activates the Ras pathway When decorated by phosphorylation, the serines and (100). It is significant that the proline-rich protein is threonines that flank selected PPxY cores of WW part of an ‘adaptor’ system bringing together other domain ligands have the ability to positively or proteins. Proline-rich regions also seem to partici- negatively regulate the binding (Korosi, T., Chang, pate in other such adaptor systems, for example, in A., and Sudol, M., unpublished results). Very re- synaptic vesicle endocytosis (106). cently, WW domains of two proteins, Ess1/Pin1 and Some proteins contain tandem proline-rich re- Nedd-4, were shown to recognize phosphoserine or peats. These generally seem to have a different phosphothreonine containing ligands (Table 1) in a function, in that they are involved in the formation phosphorylation-dependent manner (62). It is likely of networks of interactions that lead to precipitation that, in addition to the PPxY binding pocket, these or rigid meshes, as, for example, in salivary protein/ WW domains may have another crevice that accom- polyphenol interactions or insect eggshells (100). An modates the phosphoserine or phosphothreonine interesting role of multiple repeats appears to be in residues. It remains to be determined how phosphor- the actin-based movement of Listeria monocytogenes, ylation affects the ligand binding of the 160 WW where each repeat, within the EVH1 domain of ActA, domains known so far. contributes to the rate of actin-based movement It has also been shown that phosphorylation (107). This latter provides yet another example of changes the cis/trans interconversion rate of the the ‘adaptor’ role of proline-rich regions. The bind- peptide bonds between serine/threonine and pro- ing of ActA (as well as zyxin and vinculin) to VASP and of VASP to profilin are both mediated by line. A candidate enzyme is the peptidyl-prolyl cis/ proline-rich regions, thus using proline-rich ligands trans isomerase, Ess1/Pin1, which catalyzes the indirectly to bring together ActA and actin (82). isomerization of phosphorylated Ser/Thr-Pro (110). This activity is essential for progression through the cell cycle (111, 112) and restoration of the microtubule-binding activity of ␶, a protein component of THE REGULATION OF PROLINE-DEPENDENT neurofibrillar tangles of Alzheimer’s patients (113). INTERACTIONS Peptide bond isomerization could alter binding either directly, by changing the shape of the ligand, or We still have much to learn about the regulation of indirectly, by affecting enzyme-catalyzed hydrolysis the interactions mediated by proline-rich regions. It of the bond. Therefore, it will be of great interest is clear that an important mechanism is provided by when the 3-dimensional structures of the Ess1/Pin1

236 Vol. 14 February 2000 The FASEB Journal KAY ET AL. WW domain complexed with its phosphorylated ing specific protein–protein interactions in the cell ligands (59, 62) are determined. for the purposes of evaluating the functional conse- Proline can have several other specific functions in quences of the interaction and drug discovery. Stuart proteins unrelated to the theme of binding elaborated Schreiber and colleagues (Harvard University) have here. Proline can induce ␤-turns, particularly if pre- attempted to design inhibitors of the Src SH3 do- ceded by tyrosine (114) or followed by phenylalanine main by initially synthesizing a library of compounds or tryptophan residues (115). Proline can also intro- that contained nonpeptidic elements fused to the duce bends in transmembrane helices, and because of NH2 terminus of the core sequence PLPPLP. A its rigidity can act as a conformational ‘switch’, allowing combinatorial library of compounds, which was syn- parts of proteins to adopt alternative conformations thesized on beads, was incubated with a biotinylated such as domain-swapped dimers (116). Src SH3 domain, and positive beads were revealed when incubated with streptavidin-linked alkaline phosphatase and a chromogenic substrate. Only those beads with nonpeptidic elements that fit into POLYPROLINE PEPTIDOMIMETICS the specificity pocket of the Src SH3 domain yielded

positive beads, because the Kd of the PLPPLP pep- With the elucidation of peptide sequences that bind tide by itself is 1 mmol/l (128, 129). One ligand was to specific domains, it should be possible to use selected for further development. Through screen- peptide ligands to interfere with specific SH3 do- ing a second-generation ‘tuning library’, ligands with mains in the cells. Ligands for the SH3 domains of nonpeptidic elements were identified that fit the Src and Lyn have been injected into oocytes (23) or specificity (130) and LP pockets (33). electroporated into mast cells (117) and led to a Other research groups have attempted to create biological response (i.e., acceleration of progester- peptidomimetics of the scaffolding prolines of SH3 one stimulated oocyte maturation, inhibition of mast ligands. This could have major consequences for cell activation). Although most peptides fail to cross drug design, because it allows a search for novel the membrane on their own, in the future it should functionalities to replace the proline that can inter- be possible to link them to peptide segments of act with new regions of the SH3 and hence enhance Antennapedia (118–120), Kaposi fibroblast growth binding. Novel oligomers containing proline analogs factor (121), or HIV Tat (122), which do have the have been observed to adopt a PP II conformation capacity to cross the plasma membrane. Recently, (131, 132). It also appears to be possible to replace such an approach has been used to introduce a proline by other N-substituted amino acids. Recently, dimeric peptide segment of Sos into fibroblasts a paper from the laboratory of Wendell Lim (Uni- where it was observed to block the endogenous Ras versity of California, San Francisco) demonstrated signaling pathway (123). Work from the laboratory that the two prolines in the PxxP motif could tolerate of Stephan Feller (University of Wurtzburg) has substitution by N-substituted glycine, or ‘peptoid’, shown that a high-affinity peptide ligand for the residues (133). From a survey of six SH3 domain- NH2-terminal SH3 domain of Crk, when attached to peptide ligand pairs, N-substituted ligands were dis- a cell permeable peptide sequence, has biological covered to bind better than the original peptides. In activity when added to tissue culture cells (Kardinal, particular, one ligand was identified that selectively

C., and Feller, S., unpublished observation). bound the NH2-terminal SH3 domain of Grb2 with A number of proline-rich peptides, which are 100-fold greater affinity than the original 12-mer antimicrobial and adopt a PP II conformation, such peptide sequence. Thus, in conjunction with cell as indolicidin (124) and bactenecin (125), likely act permeable peptides, it may be possible in the future by crossing cell membranes on their own. When to generate peptidomimetics that are potent antag- neutrophils are incubated with the PR-39 peptide, a onists of specific SH3 and WW domain based pro- proline- and arginine-rich antimicrobial peptide, tein–protein interactions in cultured cells and whole NADPH oxidase production of superoxide anion O2- animals. is blocked. The peptide is presumed to act by binding to the SH3 domains of the NADPH oxidase subunits, thereby blocking assembly of a functional enzyme (126). Furthermore, when NIH 3T3 cells are FUTURE PROSPECTS incubated with the 15 residue NH2-terminal segment of PR-39, the peptide crosses the membrane and Availability of complete genomes and proteomes binds SH3 domain-containing proteins, such as makes it attractive to analyze a defined number of p130Cas (127). domains and to predict their cognate ligands and There has been great interest in designing pep- biochemical/genetic interactions. For example, tidomimetic antagonists of protein-protein interac- computer analysis of the Saccharomyces cerevisiae and tions. Such antagonists should have value in disrupt- Caenorhabditis elegans genomes has revealed that they

PROLINE 237 contain potentially 23 and 54 SH3 domain- and 6 14. Ren, R., Mayer, B. J., Cicchetti, P., and Baltimore, D. (1993) Identification of a ten-amino acid proline-rich SH3 binding and 10 WW domain-containing proteins, respec- site. Science 259, 1157–1161 tively. These domains are currently being analyzed in 15. Alexandropoulos, K., Cheng, G., and Baltimore, D. (1995) terms of their ligand predilections, using phage- Proline-rich sequences that bind to Src homology 3 domains with individual specificities. Proc. Natl. Acad. Sci. USA 92, displayed combinatorial peptide libraries, so that the 3110–3114 optimal ligand sequences can be used to identify 16. Knudsen, B. S., Feller, S. M., and Hanafusa, H. (1994) Four putative interacting proteins in either the yeast or proline-rich sequences of the guanine-nucleotide exchange factor C3G bind with unique specificity to the first Src homol- nematode proteomes. Once the rules of the ‘protein ogy 3 domain of Crk. J. Biol. Chem. 269, 32781–32787 recognition code’ (81) are more defined and the 17. Schumacher, C., Knudsen, B. S., Ohuchi, T., Di Fiore, P. P., structural determinants of the domains are illumi- Glassman, R. H., and Hanafusa, H. (1995) The SH3 domain of Crk binds specifically to a conserved proline-rich motif in nated, one should be able to make such predictions Eps15 and Eps15R. J. Biol. Chem. 270, 15341–15347 with the aid of computer modeling/simulation pack- 18. Feller, R., Ren, R., Hanafusa, H., and Baltimore, D. (1994) SH2 ages. Such analyses could provide useful paradigms and SH3 domains as molecular adhesives: the interactions of Crk and Abl. Trends Biochem. Sci. 19, 453–458 for analogous future approaches directed toward 19. Mayer, B. J., and Eck, M. J. (1995) SH3 domains: minding your characterizing the human proteome. p’s and q’s. Curr. Biol. 5, 364–367 20. Chen, J. K., Lane, W. S., Brauer, A. W., Tanaka, A., and Supported by grants from the Leukemia Society of Amer- Schreiber, S. L. (1993) Biased combinatorial libraries: novel ica, Muscular Dystrophy Association, and the NIH. We would ligands for the SH3 domain of phosphatidylinositol 3-kinase. J. Am. Chem. Soc. 115, 12591–12952 like to thank Trevor Creamer, Michael Eck, Xavier Espanel, 21. Cheadle, C., Ivashchenko, Y., South, V., Searfoss, G. H., Stephan Feller, Sam Gellman, Jeremy Kasanov, and James French, S., Howk, R., Ricca, G. A., and Jaye, M. (1994) Morken for helpful comments on the manuscript. B. K. K. Identification of a Src SH3 domain binding motif by screening dedicates this review in memory of his father. a random phage display library. J. Biol. Chem. 269, 24034– 24039 22. Rickles, R. J., Botfield, M. C., Weng, Z., Taylor, J. A., Green, O. M., Brugge, J. S., and Zoller, M. J. (1994) Identification of REFERENCES Src, Fyn, Lyn, PI3K, and Abl SH3 domain ligands using phage display libraries. EMBO J. 13, 5598–5604 1. Geysen, H. M., Barteling, S. J., and Meloen, R. H. (1985) Small 23. Sparks, A. 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