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. Author manuscript; available in PMC: 2009 Sep 28.
Published in final edited form as: J Mol Biol. 2005 Sep 9;352(1):1–10. doi: 10.1016/j.jmb.2005.06.072

Structural Basis for Recognition of the T Cell Adaptor Protein SLP-76 by the SH3 Domain of Phospholipase Cγ1

Lu Deng 1,†, C Alejandro Velikovsky 1,†, Chittoor P Swaminathan 1, Sangwoo Cho 1, Roy A Mariuzza 1,*
Editor: R Huber1
PMCID: PMC2753203  NIHMSID: NIHMS136551  PMID: 16061254

Abstract

The enzyme phospholipase Cγ1 (PLCγ1) is essential for Tcell signaling and activation. Following T cell receptor ligation, PLCγ1 interacts through its SH2 and SH3 domains with the adaptors LAT and SLP-76, respectively, to form a multiprotein signaling complex that leads to activation of PLCγ1 by Syk tyrosine kinases. To identify the binding site for PLCγ1 in SLP-76, we used isothermal titration calorimetry to measure affinities for the interaction of PLCγ1-SH3 with a set of overlapping peptides spanning the central proline-rich region of SLP-76. PLCγ1-SH3 bound with high specificity to the SLP-76 motif 186PPVPPQRP193, which represents the minimal binding site. To understand the basis for selective recognition, we determined the crystal structures of PLCγ1-SH3 in free form, and bound to a 10-mer peptide containing this site, to resolutions of 1.60 Å and 1.81 Å, respectively. The structures reveal that several key contacting residues of the SH3 shift toward the SLP-76 peptide upon complex formation, optimizing the fit and strengthening hydrophobic interactions. Selectivity results mainly from strict shape complementarity between protein and peptide, rather than sequence-specific hydrogen bonding. In addition, Pro193 of SLP-76 assists in positioning Arg192 into the compass pocket of PLCγ1-SH3, which coordinates the compass residue through an unusual aspartate. The PLCγ1-SH3/SLP-76 structure provides insights into ligand binding by SH3 domains related to PLCγ1-SH3, as well as into recognition by PLCγ1 of signaling partners other than SLP-76.

Keywords: crystal structure, SH3, calorimetry, T cell signaling, phospholipase Cγ1

The enzyme phospholipase Cγ1 (PLCγ1) is an essential signal transducing element in T cell activation and development.1,2 This multidomain protein comprises two Src homology 2 (SH2), one Src homology 3 (SH3), one pleckstrin homology (PH), and two catalytic domains (lipase and C2).3 PLCγ1 interacts with tyrosine-phosphorylated sites on target proteins through its SH2 domains, and with proline-rich sequences through its SH3, resulting in formation of specific complexes that mediate T cell signaling.1,2,4In addition, PLCγ1 plays a pivotal role in cellular growth and proliferation through interactions with various growth factor receptors, such as epidermal growth factor receptor,3,5–7 and has been shown to function as a guanine nucleotide exchange factor for the nuclear GTPase PIKE.8

Following engagement of the T cell receptor (TCR) by MHC/peptide ligands, the trans-membrane adaptor protein, linker for activation of T cells (LAT), becomes phosphorylated at multiple tyrosine residues by Src or Syk family protein kinases.1,2 Phosphorylation of LAT creates binding sites for the SH2 domains of other signaling molecules, notably PLCγ1 and the soluble adaptor Gads. In addition, PLCγ1 and Gads interact through their SH3 domains with the adaptor SLP-76.4,9–11 The resulting multiprotein signaling complex, comprising LAT, Gads, PLCγ1, and SLP-76, leads to phosphorylation of PLCγ1 by Syk tyrosine kinases.1,2 Once activated in this manner, PLCγ1 translocates to the plasma membrane and catalyzes production of the second messengers, inositol 1,4,5-trisphosphate and diacylglycerol, which, respectively, trigger calcium flux and contribute to protein kinase C and Ras activation.3,5

SLP-76 is composed of three distinct domains: (1) an N-terminal domain bearing tyrosine residues that become phosphorylated following TCR engagement, enabling SLP-76 to bind the SH2-containing proteins Vav and Nck; (2) a C-terminal SH2 domain which binds phosphorylated ADAP; and (3) a central proline-rich region that contains binding sites for the SH3 domains of Gads and PLCγ1.1,2 The binding site for Gads-SH3 has been precisely mapped to residues 233−241 of the SLP-76 proline-rich domain.12 In addition, three-dimensional structures of Gads-SH3 in complex with peptides representing this site have been determined.13,14 By contrast, the binding site for PLCγ1-SH3 has only been localized to a 67 amino acid residue stretch between the N-terminal domain of SLP-76 and the Gads binding site.10 This segment, designated the P-I region, is required for TCR-induced activation of PLCγ1 and NFAT.10,15 Likewise, although the NMR structure of human PLCγ1-SH3 in the unliganded form has been reported,16 no structural information is available on the interaction of PLCγ1-SH3 with SLP-76. Indeed, no three-dimensional structure has been determined for PLCγ1-SH3 in complex with a target sequence from any of the multiple signaling partners that this SH3 is known to recognize, which include MIST,17 SOS1,18 CAIR-1/BAG-3,19 and dynamin.20

Here, we used isothermal titration calorimetry (ITC) to delineate the binding site for PLCγ1-SH3 in the proline-rich region of SLP-76. We then determined the crystal structure of rat PLCγ1-SH3, both in the free form and bound to a peptide representing this site.

Identification of the SLP-76 sequence motif recognized by PLCγ1-SH3

Our first goal was to identify the binding site for PLCγ1-SH3 in the central proline-rich domain of SLP-76 (residues 157−420). Immunoprecipitation and glutathione-S-transferase (GST) pull-down experiments previously localized this site to a 67 residue segment (the P-I region) encompassing amino acid residues 157−223.10 However, this region includes 20 proline residues and seven XPXXP core motifs, several of which overlap (Table 1).

Table 1.

Equilibrium dissociation constants for the binding of SLP-76 peptides to PLCγ1-SH3 at 3 °C

graphic file with name nihms-136551-f0001.jpg
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PLCγ1-SH3 has been classified as a class II SH3 domain, signifying that it preferentially binds peptides containing XPXXPXR sequences, where X is any amino acid.21,22 Phage-displayed peptide libraries have further shown that the PLCγ1-SH3 consensus motif is PPVPPRPXXTL.23 The same study identified other polyproline sequences, not conforming to the class II motif, that also bind PLCγ1-SH3. However, as phage display methods do not readily permit quantitative comparisons of affinity, we used ITC to determine dissociation constants (Kd) for the interaction of PLCγ1-SH3 with 11 overlapping peptides (P1 through P11) of 12−18 residues each that were designed to span the entire P-I region of SLP-76 (Table 1).

The highest affinities were measured for peptides P5 and P6, which bound PLCγ1-SH3 with Kd values of 11.9 μM and 11.8 μM, respectively (Table 1, Figure 1). These are typical affinities for known SH3–peptide interactions.21,22 Both peptides contain the core sequence XPXXPXR present in the library consensus motif XPPVPPRPXXTL.23 Moreover, PLCγ1-SH3 is highly specific for this site in SLP-76, since it binds weakly, or not at all, to other peptides bearing the XPXXP motif (P1–P3, P7–P11). The only exception is P4 (Kd 33.1 μM); however, this peptide overlaps with P5 and P6 at nine positions (184QQPPVPPQR192).

Figure 1.

Figure 1

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Calorimetric titration of PLCγ1-SH3 with peptide derived from SLP-76. ITC was carried out on a MicroCal VP-ITC titration microcalorimeter as described.40 SLP-76 peptides were synthesized and purified by Biosource. Purified PLCγ1-SH3 was exhaustively dialyzed against PBS (5 mM phosphate buffer (pH 7.2), 136 mM NaCl, 4 mM KCl); the final dialysate was used to prepare SLP-76 peptide solutions. A non-linear, least-squares fitting method was used to determine the thermodynamic parameters. Data acquisition and analysis were performed using the software package Origin. (a) Raw data obtained from 30 automatic injections of 3 μl aliquots of 9.2 M SLP-76 184QQPPVPPQRPMAALPP199 peptide solution into 0.206 mM PLCγ1-SH3 solution in PBS at 3 °C. (b) Non-linear, least-squares fit (continuous line) of the incremental heat per mole of added ligand (open squares) for the titration in (a). A stoichiometry (n) value of 1.02 and Kd of 11.8 μM with uncertainties of 0.3 and 2%, respectively, were obtained for this experiment.

To define the shortest SLP-76 sequence retaining full affinity for PLCγ1-SH3, as determined using the 15-mers P5 and P6, we tested truncated peptides P12 (12-mer) and P13 (8-mer) (Table 1). Both bound this SH3 with Kd values (12.5 μM for P12 and 12.9 μM for P13) essentially identical with those for P5 and P6. Thus, the minimal binding site for PLCγ1-SH3 in SLP-76 is 186PPVPPQRP193.

Structures of PLCγ1 in free form and bound to an SLP-76 motif peptide

The crystal structures of unbound PLCγ1-SH3, and of PLCγ1-SH3 in complex with the SLP-76-derived 10-mer 185QPPVPPQRPM194 (SLP-76 185−194), were determined by the molecular replacement method and refined to resolutions of 1.60 Å and 1.81 Å, respectively (Table 2). The overall fold of rat PLCγ1-SH3 can be described as a β-barrel typical of all known SH3 domains,21,22 formed by two antiparallel, three-stranded β-sheets (Figure 2(a)). The root-mean-square deviation (r.m.s.d.) of main chain atoms, excluding the five N-terminal residues, is 1.97 Å for unliganded rat and human (PDB code of 1HSQ)16 PLCγ1-SH3. The structure of the PLCγ1-SH3 domain bound to the SLP-76 peptide ligand retains the same compact scaffold as its free form (Figure 3(a)). The r.m.s.d. of main chain atoms of free and peptide-bound PLCγ1-SH3 is 0.55 Å, indicating that the structure of the SH3 domain does not substantially change upon binding its cognate ligand. The most obvious difference is a local rearrangement of the n-Src loop (nomenclature according to Musacchio22) adjacent to the βc strand (residues 823−828; main chain atom r.m.s.d. of 0.77 Å). Binding of the SLP-76 peptide causes a small displacement of Gly827 and Trp828, which in turn initiates a conformational rearrangement of residues 823−826, with the Cα atom of Asp825 shifted by 1.82 Å relative to its position in the unbound SH3 (Figure 3(b)).

Table 2.

X-ray crystallographic statistics

PLCγ1-SH3 PLCγ1-SH3/SLP-76
Data collection statistics
Resolution (Å)a 50.0−1.60 (1.66−1.60) 50.0−1.81 (1.87−1.81)
Space group P21 P43212
Cell dimensions (Å, deg.) a=29.0, b=31.3, c=29.8, β=97.1 a=b=46.7, c=58.1
Unique reflectionsa 6979 (669) 6149 (527)
Completeness (%)a 97.5 (94.5) 97.4 (85.8)
Rmerge (%)a,b 3.7 (16.8) 9.7 (35.3)
Redundancya 4.1 (4.2) 5.3 (2.7)
I/σ〈I〉a 42.2 (10.5) 21.7 (1.8)
Refinement statistics
Resolution range (Å) 29.49−1.60 36.42−1.81
Rwork (%)c 14.2 17.1
Rfree (%)c 18.8 23.1
Number of reflections used 6604 5831
Number of reflections in Rfree set 323 279
Number of non-H protein atoms 513 548
Number of water molecules 120 67
Mean B-factor (Å2) 17.4 23.9
    Main-chain atoms 11.9 20.4
    Side-chain atoms 15.2 21.9
    Water molecules 33.8 44.2
r.m.s.d from ideality
    Bond lengths (Å) 0.017 0.014
    Bond angles (deg.) 1.7 1.4
Ramachandran plot statistics
    Most favored (%) 94.3 92.0
    Additionally allowed (%) 5.7 8.0
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Unbound PLCγ1-SH3 was crystallized in 36% (w/v) PEG4000, 0.1 M sodium acetate, 0.1 M Tris–HCl (pH 9.0) at room temperature. Co-crystallization with the SLP-76-derived 10-mer 185QPPVPPQRPM194 was carried out using a 4:1 molar ratio of peptide to protein. Crystals of the complex were grown in 2.4 M sodium malonate (pH 7.0). Crystals of free PLCγ1-SH3 were cryoprotected by step-wise soaking in solutions of mother liquor containing 2−25% (v/v) glycerol before being flash-cooled. Sodium malonate (3.4 M) was used as a cryoprotectant for the complex crystals. Diffraction data were collected at 100 K on beamline X29 of the Brookhaven National Synchrotron Light Source. The data were processed separately using the program HKL2000.44 Phases for the free PLCγ1-SH3 domain were determined by the molecular replacement method using Phaser45 with the Gads SH3 domain (PDB code 1OEB)14 as the search model. The graphics program XtalView46 was used to examine the crystal packing and initial electron density map. Refinement was carried out using RefMac 5.0.47 The final model comprises residues 790−851, one N-terminal serine from the GST fusion partner, and 120 water molecules. The structure of the PLCγ1-SH3/SLP-76 complex was solved with Phaser46 using the free PLCγ1-SH3 structure as the search probe. The final model includes residues 793−848 of the SH3 domain, all ten residues of the SLP-76 peptide, and 67 water molecules. Stereochemical parameters were evaluated with PROCHECK48 and MolProbity.49

a

Values in parentheses are statistics of the highest resolution shell.

b

Rmerge = Σ|Ij – 〈I〉|/ΣIj, where Ij is the intensity of an individual reflection and 〈I〉 is the average intensity of that reflection.

c

Rwork = Σ||Fo| – |Fc||/Σ|Fo|, where Fc is the calculated structure factor. Rfree is as for Rwork but calculated for a randomly selected 5.0% of reflections not included in the refinement.

Figure 2.

Figure 2

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(a) Structure of the PLCγ1 SH3 domain with bound SLP-76 peptide. Ribbon diagram of PLCγ1-SH3 in complex with the SLP-76-derived 10-mer 185QPPVPPQRPM194. The peptide is shown in ball-and-stick representation. Carbon atoms are gray, nitrogen atoms are blue, oxygen atoms are red, and the sulfur atom is yellow. A bridging water molecule (WAT) is represented by a red ball. Residues of PLCγ1-SH3 forming salt-bridge or hydrogen bond interactions (broken lines) with the ligand are drawn. The view is looking down the β-barrel of the SH3 domain. Secondary structure elements are labeled following the nomenclature for Src-SH3,22 and are colored as follows: 310 helix, brown; β-strands, magenta; loop regions, cyan. This Figure, together with Figures 3 and 4, were produced with MOLSCRIPT.41 (b) Electrostatic potential surface representation of PLCγ1-SH3 in complex with SLP-76 peptide. Solvent-accessible surfaces are colored according to electrostatic potential, with positively charged regions in blue and negatively charged regions in red. Electrostatic surface potentials were calculated with GRASP.42 The peptide is shown in stick representation. Carbon atoms are white, nitrogen atoms are blue, oxygen atoms are red, and the sulfur atom is green.

Figure 3.

Figure 3

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Comparison of PLCγ1-SH3 in free and SLP-76-bound forms. (a) Superposition of the polypeptide chain of unbound PLCγ1-SH3 (gold) onto that of PLCγ1-SH3 in the PLCγ1-SH3/SLP-76 complex (cyan). The SLP-76 peptide is drawn in ball-and-stick representation. The arrow points to the region where the two structures deviate most. (b) Close-up view of the rearrangement of the n-Src loop of PLCγ1-SH3 following peptide binding. Glycine residues are represented by their carbonyl oxygen atoms. (c) Adjustments in the PLCγ1-SH3 binding site upon ligation of SLP-76 peptide. Free PLCγ1-SH3 was superposed on SLP-76-bound PLCγ1-SH3. The view is looking down on the binding groove. The SLP-76 peptide and the rest of the SH3 structure are omitted for clarity. Only residues contacting the ligand are shown. Glycine residues are represented by their carbonyl oxygen atoms. Dual conformations were observed for the side-chains Gln805 and Arg806 in the PLCγ1-SH3/SLP-76 complex.

The electron density map of the PLCγ1-SH3/SLP-76 complex allowed unambiguous interpretation of the entire SLP-76 peptide chain. The sequence 186PPVPP190 (XPXXP) forms the core of this SH3 ligand, with the peptide docked onto the relatively flat surface of the SH3 domain in a class II binding mode (Figure 2(a)).21,22 The positively charged arginine at position 192, highly conserved in class II ligands, establishes the orientation of the SLP-76 peptide through formation of a salt-bridge with Asp808 of PLCγ1-SH3. Because Arg192 determines peptide orientation, it is termed the “compass residue”.22 The polyproline type II (PPII) helix starts from the second residue (Pro186) and ends at the last residue (Met194) with three residues per turn. There are three shallow pockets, or slots, evenly distributed along the surface of PLCγ1-SH3 to accommodate the peptide, two XP sites for binding the XP dipeptide moieties, and the neighboring compass slot, formed by residues from the RT and n-Src loops, the βc strand and the 310 helix, for binding Arg192.21,22 The PLCγ1-SH3/SLP-76 complex buries a total solvent-accessible surface of 871 Å2, of which 394 Å2 is contributed by PLCγ1-SH3 and 477 Å2 by SLP-76. The surface of PLCγ1-SH3 buried in the interface is mostly hydrophobic, except for the compass slot, which is negatively charged (Figure 2(b)).

As with other known PPII helixes, a continuous hydrophobic strip is formed around the surface of the bound SLP-76 peptide, while the backbone carbonyl groups present three hydrogen-bonding groups towards PLCγ1-SH3 (Figure 2(a)). The first peptide residue, Gln185, is held in an extended conformation. The first two PII helix residues, Pro186 and Pro187 are inlaid into the first XP slot formed by the side-chains of Phe800, Asn844 and Tyr845. The second XP slot holds SLP-76 residues Pro189 and Pro190 from the second turn of the PPII helix. Asn844 and Tyr845 form a wall separating the two XP slots, while Gln805 and Trp828 partition the second XP slot from the compass slot (Figure 2(b)). The third residues of the first two PPII helix turns, Val188 and Gln191, are positioned directly above the two walls and are oriented with their side-chains facing the solvent. Three short hydrogen bonds are present between the main chain carbonyl oxygen atoms of Pro187, Val188, and Pro190, and the hydroxyl oxygen atom of Tyr845, the side-chain amido nitrogen atom of Asn844, and the indole nitrogen atom of Trp828, respectively (Figure 2(a)). The side-chain of Arg192, the first residue of the third turn of the PPII helix, is tightly intercalated into the negatively charged compass pocket via salt-bridge interactions with Asp808 from the RT loop of PLCγ1-SH3. The side-chain conformation of Arg192 is further stabilized by a hydrogen bond to the side-chain of Glu809. A water molecule was found to assist binding by bridging the main chain nitrogen atom of Arg192 of SLP-76 and the side-chain Nη2 and Nη1 atoms of Arg806 of PLCγ1-SH3 (Figure 2(a)). The other two residues of the third PPII helix turn (Pro193 and Met194) point away from the SH3, towards the N terminus of the peptide ligand from a symmetry-related molecule. Thus, the pyrrolidine ring of Pro193 is parallel with that of Pro187 from a symmetry-related peptide; in addition, the carbonyl oxygen atom of Pro193 makes hydrogen-bond contact with the main chain nitrogen atom of Val188 from the same symmetry-related peptide (not shown).

Binding specificity of PLCγ1-SH3

Despite sequence differences of up to 75%, all SH3 domains of known three-dimensional structure adopt a similar β-barrel fold, with their proline-rich ligands forming classical PPII helices. However, subtle geometrical differences among these domains impart a significant degree of selectivity towards specific proline-rich targets.21,22 As shown in Figure 3(c), residues of PLCγ1-SH3 involved in ligand binding were identified by calculating differences in solvent accessibility between the free and complexed molecules. No major changes in binding site topology are evident upon engagement of SLP-76, with most side-chains retaining the same conformation as in the unbound SH3. Thus, the r.m.s.d. of residues (all atoms) involved in ligand binding is only 0.83 Å, indicating that relatively small adjustments suffice to accommodate the SLP-76 peptide. It is, however, notable that the side-chains of several contacting residues, in particular those of Phe800, Trp828 and Trp840, shift towards the SLP-76 peptide, optimizing the fit of the binding groove to the ligand and strengthening hydrophobic interactions (Figure 3(c)).

The PLCγ1-SH3/SLP-76 structure suggests that the selectivity of the interaction is primarily determined by strict geometric, or shape, complementarity between protein and peptide, rather than by sequence-specific hydrogen bonding. Thus, the hydrogen bonds formed by Asn844, Tyr845 and Trp828 are directed to main chain atoms of the peptide, and are therefore independent of peptide sequence. Although it has been proposed that the specificity of SH3–ligand interactions may arise from recognition of non-proline residues by non-conserved protein side-chains,21,22 we are unable to identify such residues in the PLCγ1-SH3/SLP-76 complex: the two XP binding slots are exclusively occupied by proline residues, which mediate most of the interactions with the SH3 domain. However, the non-core proline at position 189, which is most often leucine or valine in the class II consensus motifs of other SH3 domains (e.g. Src, Grb2, Crk),21–23 may contribute to optimal binding via hydrophobic interactions with PLCγ1-SH3 residues Tyr802, Gln805, Trp828, Pro842, Asn844 and Tyr845, in which the pyrrolidine ring of Pro189 stacks against Tyr802 and Pro842. In this regard, replacement of Pro189 by leucine or valine would be expected to disrupt shape complementarity at this site, resulting in decreased affinity.

ITC measurements indicate that Pro193, which does not directly contact the SH3 domain, is nevertheless required for optimal binding, since peptide P4, which lacks this residue, exhibits a threefold increment in Kd compared to peptide P5 (Table 1). As proline restricts the conformational freedom of the preceding residue in a polypeptide chain,24 we hypothesize that Pro193 assists in positioning the side-chain of Arg192 into the compass pocket of PLCγ1-SH3, thereby indirectly increasing peptide affinity. Indeed, the phage library consensus motif, XPPVPPRPXXTL, includes a proline following the compass arginine.23 However, Pro193 is not absolutely critical for PLCγ1-SH3 recognition, as its absence does not totally abrogate binding. Additionally, PLCγ1-SH3 has been shown to recognize sequence motifs lacking a corresponding proline in several target molecules besides SLP-76 (see below).

For PLCγ1-SH3 residues in contact with SLP-76 185−194, sequence alignments with other SH3 domains (not shown) indicate that Gln805, Arg806, Asp808, Gly826 and Gly827, located within the RT and n-Src loops, are the most variable. In the PLCγ1-SH3/SLP-76 structure, the side-chains of Gln805 and Arg806 exhibit dual conformations, one directed towards the ligand and the other away, implying that these residues probably do not contribute greatly to complex stabilization. Likewise, Gly826 and Gly827 make only a few van der Waals contacts with peptide. On the other hand, Asp808, which appears unique to PLCγ1-SH3, could contribute to peptide selectivity. Instead of using the highly conserved Glu809 (Asp in some SH3 domains; Figure 4(c)), as do other SH3 domains,21,22 PLCγ1-SH3 employs Asp808 to coordinate the critical Arg192 compass residue of SLP-76.

Figure 4.

Figure 4

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(a) Structure-based sequence alignment of the SH3 domains of PLCγ1, PLCγ2 and Lck. Secondary structure elements are denoted by violet arrows (β-strands) and a pink cylinder (310 helix). These, and the loop regions (brown lines), are labeled according to the nomenclature for Src-SH3.22 Residues of PLCγ1-SH3 making van der Waals contacts with SLP-76 are shaded yellow; residues interacting with SLP-76 through hydrogen bonds are shaded cyan. Asp808, which forms a salt-bridge with the bound peptide, is shaded red. (b) Superposition of PLCγ1-SH3 in bound form (cyan) onto free Lck-SH3 (green; PDB accession code 1LCK).28 The view is the same as that in Figure 3(c). Residues of Lck-SH3 that potentially contact bound peptide, based on sequence alignment with PLCγ1-SH3 (a), are shown. (c) Interactions at the SH3 compass pocket. The PLCγ1/SLP-76 complex (cyan) was superposed onto the Src-SH3/App12 complex (PDB accession code 1QWE)30 and free Lck-SH3. Only interactions with the compass residue of the bound peptide (Arg192 of SLP-76 and Arg9 of App12) are shown. Salt-bridges are drawn as broken lines.

Structure comparison with other SH3 domains

There exist two PLCγ isozymes devoted to tyrosine-dependent signaling pathways: PLCγ1 and PLCγ2.3 Whereas PLCγ1 is mainly involved in T cell signaling, PLCγ2 is the predominant PLCγ isoform in B cells. Activation of PLCγ2 upon antigen binding to the B cell receptor closely parallels that of PLCγ1 following TCR engagement, and requires the B cell cytosolic adaptor BLNK, which is analogous to SLP-76 in T cells.25,26

PLCγ1 and PLCγ2 share the same overall domain organization, including SH3 modules displaying 60% sequence identity. The PLCγ1-SH3 residues contacting the SLP-76 peptide in the complex structure are highly conserved in PLCγ2-SH3 (Figure 4(a)), indicating that PLCγ2 probably interacts with BLNK at a site similar to the one on SLP-76 recognized by PLCγ1 (186PPVPPQRP193). Sequence analysis of mouse BLNK (Swiss-Prot ID O88504) showed that this adaptor does not contain the consensus motif XPXXPXRX. However, two sequences were detected wherein the basic arginine in SLP-76 is substituted by lysine in BLNK: 161KPQVPPKP168 and 252KPATPLKT259. Based on their locations in BLNK relative to that of the PLCγ1-SH3 binding site in SLP-76, 161KPQVPPKP168 is the sequence most likely recognized by PLCγ1-SH3. Significantly, this potential docking site also contains a proline following the putative Lys167 compass residue.

Stimulation of the TCR activates the protein tyrosine kinase Lck and leads to phosphorylation of PLCγ1.1,2 It has further been shown that the SH3 domain of Lck interacts with SLP-76 and that the best peptide candidate (186PPVPPQRP193) is, coincidentally, the same as we have defined here for PLCγ1-SH3.27 Although Lck-SH3 and PLCγ1-SH3 bear only ∼30% sequence identity, it is apparent from their three-dimensional structures that the two SH3 domains probably bind SLP-76 similarly. Thus, least-squares superposition of the main chain atoms of Lck-SH3 in free form28 onto those of PLCγ1-SH3 in the PLCγ1-SH3/SLP-76 complex shows that most side-chains in the ligand-binding sites of these SH3 modules display similar conformations (Figure 4(b)). These include those of SLP-76-contacting residues Tyr802, Trp828, Pro842, Asn844 and Tyr845 (Phe115 in Lck).

The most significant structural difference between PLCγ1-SH3 and Lck-SH3 is located in the RT loop, where Asp808, which establishes the peptide orientation in the PLCγ1-SH3/SLP-76 complex by making a salt-bridge with SLP-76 Arg192, is substituted by Gly78 in Lck-SH3 (Figure 4(a)). However, as both flanking residues (Asp77 and Asp79) are negatively charged, these represent good alternative candidates for recognition of Arg192 (Figure 4(c)). Indeed, Asp79 of Lck-SH3 has been proposed to interact with a corresponding arginine (Arg182) in a class II peptide from tyrosine kinase interacting protein (Tip),29 while Src-SH3 uses a corresponding aspartate (Asp99) for salt-bridge formation to arginine-oriented class II peptides (Figure 4(c)).30 The putative Lck binding motif in Tip also includes a proline following the Arg182 compass residue. Lck-SH3 differs from PLCγ1-SH3 at two other potential ligand-binding residues: Glu96 (Gly827 in PLCγ1) and Gly109 (Trp840 in PLCγ1) (Figure 4(a)). However, in the PLCγ1/SLP-76 structure, only the carbonyl oxygen atom of Gly827 contributes to the binding site, whereas the Lck Glu96 side-chain points away from this site in the free Lck-SH3 structure (Figure 4(b)). By contrast, replacement of the bulky Trp840 in PLCγ1 by Gly109 in Lck is expected to result in the loss of several van der Waals contacts to the bound peptide, possibly reducing affinity.

Interaction of PLCγ1-SH3 with other signaling proteins

In addition to interacting with SLP-76 in the T cell signaling pathway, PLCγ1-SH3 has been implicated in other functions, including targeting PLCγ1 to the cytoskeleton,31 agonist-induced Ca2+ entry,32 and mitogenesis.33,34 Sequence analysis of proteins known to interact with PLCγ1-SH3, in conjunction with the crystal structure of the PLCγ1-SH3/SLP-76 complex, enables us to identify potential binding sites for this SH3 in several of its natural targets (Table 3). In common with SLP-76, MIST,17 c-Cbl,35,36 SOS1,18 TNK137 and dynamin20 contain at least one XPXXPXRP motif (group A in Table 3). This motif should assure optimal (or near optimal) binding to PLCγ1-SH3, as it retains, in addition to the XPXXPXR sequence, a proline immediately following the arginine compass residue. Consistent with this analysis, direct binding of PLCγ1-SH3 to peptides from TNK1 and dynamin representing putative recognition sites has been demonstrated.20,37 Other XPXXPXR sequences in c-Cbl and SOS1 (group B), while lacking proline at the position following the compass arginine, may nevertheless exhibit significant affinity for PLCγ1-SH3, since elimination of the corresponding proline in SLP-76 did not abolish binding (Table 1). Although both CAIR-1/BAG-3 and SAM 68 interact with PLCγ1-SH3,19,38 neither contains a class II motif (group C), suggesting that PLCγ1-SH3 may also recognize other motifs, as recently demonstrated for Gads-SH3.39

Table 3.

Binding sites for PLCγ1-SH3 in selected signaling partners

Group Sequence Positions Protein Reference
A PPVPPQRPa 186−193 SLP-76 This work
XPXXPXRP IPLPPPRP 158−165 MIST 17
PPPPPDRP 544−551 c-Cbl 36
PPPVPLRP 1216−1223 SOS1 18
PPGLPPRPa 528−535 TNK1 37
APPVPSRP 812−819 Dynamin 20
PPQVPSRPa 829−836 Dynamin 20
B LPPVPPRL 493−500 c-Cbl 36
XPXXPXRX PPPPPPRD 1203−1210 SOS1 18
PPPLPPRK 1146−1153 SOS1 18
PPAIPPRQ 1172−1179 SOS1 18
APPVPPRQ 1288−1295 SOS1 18
C GPVGPELP 326−333 CAIR-1/BAG-3 19
XPXXPXXP VPCPPPSP 371−378 CAIR-1/BAG-3 19
APPPPPVP 294−301 SAM 68 38
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Sequences of proteins known to bind PLCγ1-SH3 were compared to the SLP-76 186−193 sequence to identify potential binding sites. Consensus motifs for each group are indicated. Possible critical residues are in bold.

a

Direct interaction of peptides bearing this sequence with PLCγ1-SH3 has been reported.

Data bank accession codes

Coordinates and structure factors for unbound PLCγ1 and the PLCγ1/SLP-76 complex have been deposited under accession codes 1YWP and 1YWO, respectively.

Acknowledgements

We thank Graham Carpenter for providing the rat PLCγ1 cDNA. Data for this study were measured at beamline X29 of the National Synchrotron Light Source. Financial support comes principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy, and from the National Center for Research Resources of the National Institutes of Health. This work was supported by the Sandler Program for Asthma Research and National Institutes of Health Grant GM052801 (to R.A.M.).

Abbreviations used

PLCγ1

phospholipase Cγ1

SH2

Src homology 2

SH3

Src homology 3

PH

pleckstrin homology

TCR

T cell receptor

LAT

linker for activation of T cells

ITC

isothermal titration calorimetry

GST

glutathione-S-transferase

r.m.s.d.

root-mean-square deviation

References

  • 1.Samelson LE. Signal transduction mediated by the T cell antigen receptor: role of adapter proteins. Annu. Rev. Immunol. 2002;20:371–394. doi: 10.1146/annurev.immunol.20.092601.111357. [DOI] [PubMed] [Google Scholar]
  • 2.Jordan MS, Singer AL, Koretzky GA. Adaptors as central mediators of signal transduction in immune cells. Nature Immunol. 2003;4:110–116. doi: 10.1038/ni0203-110. [DOI] [PubMed] [Google Scholar]
  • 3.Carpenter G, Ji QS. Phospholipase C-γ. as a signal-transducing element. Expt. Cell Res. 1999;253:15–24. doi: 10.1006/excr.1999.4671. [DOI] [PubMed] [Google Scholar]
  • 4.Zhang W, Trible RP, Zhu M, Liu SK, McGlade J, Samelson LE. Association of Grb2 Gads, and phospholipase C-γ1 with phosphorylated LAT tyrosine residues. J. Biol. Chem. 2000;275:23355–23361. doi: 10.1074/jbc.M000404200. [DOI] [PubMed] [Google Scholar]
  • 5.Rhee SG, Bae YS. Regulation of phosphoinositide-specific phospholipase C isozymes. J. Biol. Chem. 1997;272:15045–15048. doi: 10.1074/jbc.272.24.15045. [DOI] [PubMed] [Google Scholar]
  • 6.Chattopadhyay A, Vecchi M, Ji Q, Mernaugh R, Carpenter G. The role of individual SH2 domains in mediating association of phospholipase C-γ1 with the activated EGF receptor. J. Biol. Chem. 1999;274:26091–26097. doi: 10.1074/jbc.274.37.26091. [DOI] [PubMed] [Google Scholar]
  • 7.Ji QS, Chattopadhyay A, Vecchi M, Carpenter G. Physiological requirement for both SH2 domains for phospholipase C-γ1 function and interaction with platelet-derived growth factor receptors. Mol. Cell. Biol. 1999;19:4961–4970. doi: 10.1128/mcb.19.7.4961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Ye K, Aghdasi B, Luo HR, Moriarity JL, Wu FY, Hong JJ, et al. Phospholipase C-γ1 is a physiological guanine nucleotide exchange factor for the nuclear GTPase PIKE. Nature. 2002;415:541–544. doi: 10.1038/415541a. [DOI] [PubMed] [Google Scholar]
  • 9.Lin J, Weiss A. Identification of the minimal tyrosine residues required for linker for activation of T cell function. J. Biol. Chem. 2001;276:29588–29595. doi: 10.1074/jbc.M102221200. [DOI] [PubMed] [Google Scholar]
  • 10.Yablonski D, Kadlecek T, Weiss A. Identification of a phospholipase C-γ1 (PLC-γ1) SH3 domain-binding site in SLP-76 required for T-cell receptor-mediated activation of PLC-γ1 and NFAT. Mol. Cell. Biol. 2001;21:4208–4218. doi: 10.1128/MCB.21.13.4208-4218.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Zhu M, Janssen E, Zhang W. Minimal requirement of tyrosine residues of linker for activation of T cells in TCR signaling and thymocyte development. J. Immunol. 2003;170:325–333. doi: 10.4049/jimmunol.170.1.325. [DOI] [PubMed] [Google Scholar]
  • 12.Berry DM, Nash P, Liu SK, Pawson T, McGlade CJ. A high-affinity Arg-X-X-Lys SH3 binding motif confers specificity for the interaction between Gads and SLP-76 in T cell signaling. Curr. Biol. 2002;12:1336–1341. doi: 10.1016/s0960-9822(02)01038-2. [DOI] [PubMed] [Google Scholar]
  • 13.Liu Q, Berry D, Nash P, Pawson T, McGlade CJ, Li SS. Structural basis for specific binding of the Gads SH3 domain to an RxxK motif-containing SLP-76 peptide: a novel mode of peptide recognition. Mol. Cell. 2003;11:471–481. doi: 10.1016/s1097-2765(03)00046-7. [DOI] [PubMed] [Google Scholar]
  • 14.Harkiolaki M, Lewitzky M, Gilbert RJ, Jones EY, Bourette RP, Mouchiroud G, et al. Structural basis for SH3 domain-mediated high-affinity binding between Mona/Gads and SLP-76. EMBO J. 2003;22:2571–2582. doi: 10.1093/emboj/cdg258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Singer AL, Bunnell SC, Obstfeld AE, Jordan MS, Wu JN, Myung PS, et al. Roles of the proline-rich domain in SLP-76 subcellular localization and T cell function. J. Biol. Chem. 2004;279:15481–15490. doi: 10.1074/jbc.M313339200. [DOI] [PubMed] [Google Scholar]
  • 16.Kohda D, Hatanaka H, Pauptit, Odaka M, Mandiyan V, Ullrich A, et al. Solution structure of the SH3 domain of phospholipase C-γ. Cell. 1993;72:953–960. doi: 10.1016/0092-8674(93)90583-c. [DOI] [PubMed] [Google Scholar]
  • 17.Goitsuka R, Tatsuno A, Ishiai M, Kurosaki T, Kitamura D. MIST functions through distinct domains in immunoreceptor signaling in the presence and absence of LAT. J. Biol. Chem. 2001;276:36043–36050. doi: 10.1074/jbc.M106390200. [DOI] [PubMed] [Google Scholar]
  • 18.Kim MJ, Chang JS, Park SK, Hwang JI, Ryu SH, Suh PG. Direct interaction of SOS1 Ras exchange protein with the SH3 domain of phospholipase C-γ1. Biochemistry. 2000;39:8674–8682. doi: 10.1021/bi992558t. [DOI] [PubMed] [Google Scholar]
  • 19.Doong H, Price J, Kim YS, Gasbarre C, Probst J, Liotta LA, et al. CAIR-1/BAG-3 forms an EGF-regulated ternary complex with phospholipase C-g and Hsp70/Hsc70. Oncogene. 2000;19:4385–4395. doi: 10.1038/sj.onc.1203797. [DOI] [PubMed] [Google Scholar]
  • 20.Scaife R, Gout I, Waterfield MD, Margolis RL. Growth factor-induced binding of dynamin to signal transduction proteins involves sorting to distinct and separate proline-rich dynamin sequences. EMBO J. 1994;13:2574–2582. doi: 10.1002/j.1460-2075.1994.tb06547.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Kay BK, Williamson MP, Sudol M. The importance of being proline: the interaction of pro-line-rich motifs in signaling proteins with their cognate domains. FASEB J. 2000;14:231–241. [PubMed] [Google Scholar]
  • 22.Musacchio A. How SH3 domains recognize proline. Advan. Protein Chem. 2002;61:211–268. doi: 10.1016/s0065-3233(02)61006-x. [DOI] [PubMed] [Google Scholar]
  • 23.Sparks AB, Rider JE, Hoffman NG, Fowlkes DM, Quillam LA, Kay BK. Distinct ligand preferences of Src homology 3 domains from Src Yes, Abl, Cortactin, p53bp2, PLCγ, Crk, and Grb2. Proc. Natl Acad. Sci. USA. 1996;93:1540–1544. doi: 10.1073/pnas.93.4.1540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.MacArthur MW, Thornton JM. Influence of proline residues on protein conformation. J. Mol. Biol. 1991;218:397–412. doi: 10.1016/0022-2836(91)90721-h. [DOI] [PubMed] [Google Scholar]
  • 25.Fu C, Turck CW, Kurosaki T, Chan AC. BLNK: a central linker protein in B cell activation. Immunity. 1998;9:93–103. doi: 10.1016/s1074-7613(00)80591-9. [DOI] [PubMed] [Google Scholar]
  • 26.Ishiai M, Kurosaki M, Pappu R, Okawa K, Ronko I, Fu C, et al. BLNK required for coupling Syk to PLCγ2 and Rac1-JNK in B cells. Immunity. 1999;10:117–125. doi: 10.1016/s1074-7613(00)80012-6. [DOI] [PubMed] [Google Scholar]
  • 27.Sanzenbacher R, Kabelitz D, Janssen OJ. SLP-76 binding to p56lck: a role for SLP-76 in CD4-induced desensitization of the TCR/CD3 signaling complex. J. Immunol. 1999;163:3143–3152. [PubMed] [Google Scholar]
  • 28.Eck MJ, Atwell SK, Shoelson SE, Harrison SC. Structure of the regulatory domains of the Src-family tyrosine kinase Lck. Nature. 1994;368:764–769. doi: 10.1038/368764a0. [DOI] [PubMed] [Google Scholar]
  • 29.Bauer F, Hofinger E, Hoffmann S, Rosch P, Schweimer K, Sticht H. Characterization of Lck-binding elements in the herpesviral regulatory Tip protein. Biochemistry. 2004;43:14932–14939. doi: 10.1021/bi0485068. [DOI] [PubMed] [Google Scholar]
  • 30.Feng S, Chen JK, Yu H, Simon JA, Schreiber SL. Two binding orientations for peptides to the Src SH3 domain: development of a general model for SH3-ligand interactions. Science. 1994;266:1241–1247. doi: 10.1126/science.7526465. [DOI] [PubMed] [Google Scholar]
  • 31.Bar-Sagi D, Rotin D, Batzer A, Mandiyan V, Schlessinger J. SH3 domains direct cellular localization of signaling molecules. Cell. 1993;74:83–91. doi: 10.1016/0092-8674(93)90296-3. [DOI] [PubMed] [Google Scholar]
  • 32.Patterson RL, van Rossum DB, Ford DL, Hurt KJ, Bae SS, Suh PG, et al. Phospholipase C-γ is required for agonist-induced Ca2C entry. Cell. 2002;111:529–541. doi: 10.1016/s0092-8674(02)01045-0. [DOI] [PubMed] [Google Scholar]
  • 33.Smith MR, Liu Y, Matthews NT, Rhee SG, Sung WK, Kung H. Phospholipase C-γ1 can induce DNA synthesis by a mechanism independent of its lipase activity. Proc. Natl Acad. Sci. USA. 1994;91:6554–6558. doi: 10.1073/pnas.91.14.6554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Huang PS, Davis L, Huber H, Goodhart PJ, Wegrzyn RE, Oliff A, Heimbrook DC. An SH3 domain is required for the mitogenic activity of microinjected phospholipase C-γ1. FEBS Letters. 1995;358:287–292. doi: 10.1016/0014-5793(94)01453-8. [DOI] [PubMed] [Google Scholar]
  • 35.Tvorogov D, Carpenter G. EGF-dependent association of phospholipase C-γ1 with c-Cbl. Expt. Cell Res. 2002;277:86–94. doi: 10.1006/excr.2002.5545. [DOI] [PubMed] [Google Scholar]
  • 36.Rivero-Lezcano OM, Sameshima JH, Marcilla A, Robbins KC. Physical association between Src homology 3 elements and the protein product of the c-cbl proto-oncogene. J. Biol. Chem. 1994;269:17363–17366. [PubMed] [Google Scholar]
  • 37.Felschow DM, Civin CI, Hoehn GT. Characterization of the tyrosine kinase Tnk1 and its binding with phospholipase C-γ1. Biochem. Biophys. Res. Commun. 2000;273:294–301. doi: 10.1006/bbrc.2000.2887. [DOI] [PubMed] [Google Scholar]
  • 38.Shen Z, Batzer A, Koehler JA, Polakis P, Schlessinger J, Lydon NB, Moran MF. Evidence for SH3 domain directed binding and phosphorylation of Sam68 by Src. Oncogene. 1999;18:4647–4653. doi: 10.1038/sj.onc.1203079. [DOI] [PubMed] [Google Scholar]
  • 39.Lewitzky M, Harkiolaki M, Domart M-C, Jones EY, Feller SM. Mona/Gads SH3C binding to hematopoietic progenitor kinase 1 (HPK1) combines an atypical SH3 binding motif, R/KXXK, with a classical PXXP motif embedded in a polyproline type II (PPII) helix. J. Biol. Chem. 2004;279:28724–28732. doi: 10.1074/jbc.M402745200. [DOI] [PubMed] [Google Scholar]
  • 40.Cho S, Velikovsky CA, Swaminathan CP, Houtman JCD, Samelson LE, Mariuzza RA. Structural basis for differential recognition of tyrosine-phosphorylated sites in the linker for activation of Tcells (LAT) by the adaptor Gads. EMBO J. 2004;23:1441–1451. doi: 10.1038/sj.emboj.7600168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Kraulis PJ. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallog. 1991;24:946–950. [Google Scholar]
  • 42.Nicholls A, Sharp KA, Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins: Struct. Funct. Genet. 1999;11:281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  • 43.Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995;4:2411–2423. doi: 10.1002/pro.5560041120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997;276:307–326. doi: 10.1016/S0076-6879(97)76066-X. [DOI] [PubMed] [Google Scholar]
  • 45.Storoni LC, McCoy AJ, Read RJ. Likelihood-enhanced fast rotation functions. Acta Crystallog. sect. D. 2004;60:432–438. doi: 10.1107/S0907444903028956. [DOI] [PubMed] [Google Scholar]
  • 46.McRee DE. XtalView/Xfit–a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 1999;125:156–165. doi: 10.1006/jsbi.1999.4094. [DOI] [PubMed] [Google Scholar]
  • 47.Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallog. sect. D. 1997;53:240–255. doi: 10.1107/S0907444996012255. [DOI] [PubMed] [Google Scholar]
  • 48.Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: a program to check the stereo chemical quality of protein structures. J. Appl. Crystallog. 1993;26:283–291. [Google Scholar]
  • 49.Lovell SC, Davis IW, Arendall WB, III, de Bakker PIW, Word JM, Prisant MG, et al. Structure validation by Cα geometry: ϕ ψ and Cβ deviation. Proteins: Struct. Funct. Genet. 2003;50:437–450. doi: 10.1002/prot.10286. [DOI] [PubMed] [Google Scholar]

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