The specificity of the N-terminal SH2 domain of SHP-2 is modified by a single point mutation

SH2 domains are small protein domains of approximately 100 amino acids that bind to phosphotyrosine (pY) in the context of a specific sequence surrounding the target pY. In general, the residues C-terminal to the pY of the binding target are considered most important for defining the binding specificity, and in particular the pY + 1 and pY + 3 residues (i.e., the first and third amino acids C-terminal to the pY). However, our previous studies with the SH2 domains of the protein tyrosine phosphatase SHP-2 [Huyer, G., Li, Z. M., Adam, M., Huckle, W. R., and Ramachandran, C. (1995) Biochemistry 34, 1040-1049] indicated important interactions with the pY - 2 residue as well. In the SH2 domains of SHP-2, the highly conserved alphaA2 Arg is replaced by Gly. A comparison of the published crystal structures of the Src SH2 domain and the N-terminal SH2 domain of SHP-2 complexed with high-affinity peptides suggested that the alphaA2 Gly of SHP-2 creates a gap which is filled by the side chain of the pY - 2 residue of the bound peptide. It was predicted that replacing this Gly with Arg would alter or eliminate the involvement of the pY - 2 residue in binding. The alphaA2 Gly --> Arg mutant was constructed, and indeed, this mutant no longer required residues N-terminal to the target pY for high-affinity binding, making its specificity more like that of other SH2 domains. The alphaA2 Gly is clearly involved in directing the unusual requirement for the pY - 2 residue in the binding sequence of this SH2 domain, which has important implications for its in vivo targeting and specificity.     

Thermodynamic studies of tyrosyl-phosphopeptide binding to the SH2 domain of p56lck

The interaction between SH2 domains and tyrosine-phosphorylated proteins is essential in several cytosolic signal transduction pathways. Here we report thermodynamic studies of the interaction of the p56lck (lck) SH2 domain with several phosphopeptides, using the technique of isothermal titration calorimetry (ITC). This is the first report of the use of ITC to study SH2 domain binding reactions. The free energy of binding of the SH2 domain of lck to a phosphopeptide corresponding to the autoregulatory C-terminus of the protein (pY505) was found to be similar to that measured for a phosphopeptide modeled on the C-terminus of the epidermal growth-factor receptor (delta G degrees approximately -7.0 kcal mol-1 at pH 6.8), although significant differences in the enthalpy and entropy were observed. Binding of a phosphopeptide modeled on the C-terminus of p185neu was weaker (delta G degrees approximately -5.4 kcal mol-1 at pH 6.8). Lowering the pH to 5.5 reduced the binding affinity of pY505 by approximately 1 order of magnitude. We ascribe this to the protonation of a histidine side chain in the SH2 domain (H180), which is involved in a hydrogen-bonding network that optimizes the binding site geometry. No difference in affinity was observed between portions of lck corresponding to the SH3-SH2 (residues 63-228) and SH2 (residues 123-228) domains for the pY505 peptide. We also studied the effect upon pY505 peptide binding of mutations at two highly conserved arginine residues in the lck SH2 domain (R134 and R154).(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrochemical oxidation of N-acyldopamines and regioselective reactions of their quinones with N-acetylcysteine and thiourea

Affinity selection from peptide libraries is a powerful tool that has been used for determining the sequence specificities of a number of enzymes and protein binding domains, including protein kinases, src homology 2 domains, and PDZ domains. We have extended this approach to protein tyrosine phosphatases using peptide libraries containing a nonhydrolyzable phosphotyrosine analog, difluorophosphonomethylphenylalanine. A size-exclusion method is used to separate enzyme-peptide complexes from free peptide, providing several advantages over the traditional immobilized protein affinity column approach. In addition, the feasibility of using mass spectrometric detection to quantitate peptides rapidly and reproducibly is demonstrated as an alternative to quantitation by peptide sequencing. The validity of this analysis is demonstrated by synthesizing individual peptides and comparing their affinity for enzyme with the predictions from the affinity selection process. As a model for these studies the protein tyrosine phosphatase PTP1B is used, providing additional insights into the sequence specificity of this enzyme. In particular, a selection for aromatic amino acids at the pY - 1 position (immediately N-terminal to the phosphotyrosine), as well as a broad pY + 1 selectivity, is observed in addition to the general preference for acidic residues N-terminal to the phosphotyrosine. The approach described here should prove applicable to protein tyrosine phosphatases in general as well as for the study of nonpeptidyl combinatorial libraries.     

Determination of affinities for lck SH2 binding peptides using a sensitive fluorescence assay: comparison between the pYEEIP and pYQPQP consensus sequences reveals context-dependent binding specificity

The development of a sensitive fluorescence binding assay for evaluating the binding of phosphotyrosyl (pY) peptides to the recombinant SH2 domain of lck in solution is described. Several fluorescent peptides containing the consensus sequence of the viral hamster polyoma middle T antigen (pYEEI) were characterized. The peptides contained either the acetamido-anilino-naphthyl sulfonic acid (AANS), acrylodan, or dansyl groups as fluorophores. The spectral features of these probes were characterized in the presence and absence of the lck SH2 domain. The binding affinities (Kd) for the fluorescent peptides studied ranged from 40 to 500 nM. The fluorescent peptide containing the sequence FTATEC(AANS)QpYEEIP exhibited the highest binding affinity (Kd = 3.98 x 10(-8) M) and largest change in emission intensity (approximately 8.7-fold) upon binding the SH2 domain. This probe was subsequently used in competitive binding assays to study the interaction of the lck SH2 domain with a series of phosphopeptides related to the pYEEIP and pYQPQP (the pY505 C-terminal) consensus sequences. The effects of peptide length and substitutions of residues within the pYEEIP sequence are discussed in terms of binding affinities. Comparison between the two peptide series revealed that the contributions of individual substitutions to binding affinity are context-dependent. The data also led to the conclusion that the presence of P at +2 results in a functional "truncation" of the binding sequence; i.e., residues at positions higher than +2 do not participate significantly in binding. This implicit truncation may actually be a desired property for the autoregulatory nature of the pYQPQP sequence, since it retains specificity for the SH2 domain while adjusting the Kd to a value appropriate for maintaining the delicate balance of receptor-ligand interactions that are involved in signal transduction events.     

Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors

The transmembrane PTPase HPTP beta differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354-amino acid, 41-kDa human PTP beta catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTP beta shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9-11 residues from lck, src, and PLC gamma with Km values of 2, 4, and 1 microM, some 40-200-fold lower than the other two PTPases. With kcat values of 30-205 s-1, the catalytic efficiency, kcat/Km, of the HPTP beta 41-kDa catalytic domain is very high, up to 5.7 x 10(7) M-1 s-1. The peptides corresponding to PLC gamma (766-776) and EGFR (1,167-1,177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTP beta catalytic domain. While exchange of the alanine residue at the +2 position of the PLC gamma (Km of 1 microM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35-fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 microM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the -2 position of the EGFR peptide decreased the Km value 34-fold to 3 microM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.     

Flash-induced changes in buffering capacity of reaction centers from photosynthetic bacteria reveal complex interaction between quinone pockets

The Abl-SH3 domain is implicated in negative regulation of the Abl kinase by mediating protein-protein interactions. High-affinity SH3 ligands could compete for these interactions and specifically activate the Abl kinase, providing control and a better understanding of the molecular interactions that underlie diseases where SH3 domains are involved. The p41 peptide (APSYSPPPPP) is a member of a group of peptide ligands designed to bind specifically the Abl-SH3 domain. It binds to Abl-SH3 with a Kd of 1.5 microM, whereas its affinity for the Fyn-SH3 domain is 273 microM. We have determined the crystal structure of the Abl-SH3 domain in complex with the high-affinity peptide ligand p41 at 1.6 A resolution. In the crystal structure, this peptide adopts a polyproline type II helix conformation through residue 5 to 10, and it binds in type I orientation to the Abl-SH3 domain. The tyrosine side-chain in position 4 of the peptide is hydrogen bonded to two residues in the RT-loop of the Abl-SH3 domain. The tight fit of this side-chain into the RT-loop pocket is enhanced by conformational adjustment of the main chain at position 5. The SH3 ligand peptides can be divided into two distinct parts. The N-terminal part binds to the SH3 domain in the region formed by the valley between the nSrc and RT-loops. It determines the specificity for different SH3 domains. The C-terminal part adopts a polyproline type II helix conformation. This binds to a well-conserved hydrophobic surface of the SH3 domain. Analysis of two "half"-peptides, corresponding to these ligand parts, shows that both are essential components for strong binding to the SH3 domains. The crystal structure of the Abl-SH3:p41 complex explains the high affinity and specificity of the p41 peptide towards the Abl-SH3 domain, and reveals principles that will be exploited for future design of small, high-affinity ligands to interfere efficiently with the in vivo regulation of Abl kinase activity.     

Modified phage peptide libraries as a tool to study specificity of phosphorylation and recognition of tyrosine containing peptides

Tyrosine phosphorylation and protein recognition, mediated by phosphotyrosine containing peptides, play an important role in determining the specific response of a cell, when stimulated by external signals. We have used peptide repertoires displayed by filamentous phage as a tool to study the substrate specificity of the protein tyrosine kinase (PTK) p55(fyn) (Fyn). Peptide libraries were incubated for a short time in the presence of Fyn and phages displaying efficiently phosphorylated peptides were selected by panning over anti-phosphotyrosine antibodies. The characterization of the peptides enriched after three phosphorylation/selection rounds allowed us to define a canonical substrate sequence for the kinase Fyn, E-(phi/T)YGx phi, where phi represents any hydrophobic residue. A peptide conforming to this sequence is a better substrate than a second peptide designed to be in accord with the consensus sequence recognised by the Fyn SH2 domain. When the library phosphorylation reaction is carried out in saturation conditions, practically all the tyrosine containing peptides are phosphorylated, irrespective of their context. These "fully modified" peptide libraries are a valuable tool to study the specificity of phosphotyrosine mediated protein recognition. We have used this new tool to identify a family of peptides that bind the PTB domain of the adapter protein Shc. Comparison of the peptide sequences permits us to confirm the essential role of N at position -3, while P often found at position -2 in natural targets is not absolutely required. Furthermore, our approach permits us to reveal an "extended" consensus indicating that residues that do not seem to influence binding in natural peptides can make productive contacts, at least in linear peptides.     

Solution structure of the C-terminal SH2 domain of the human tyrosine kinase Syk complexed with a phosphotyrosine pentapeptide

BACKGROUND: Recruitment of the intracellular tyrosine kinase Syk to activated immune-response receptors is a critical early step in intracellular signaling. In mast cells, Syk specifically associates with doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) that are found within the IgE receptor. The mechanism by which Syk recognizes these motifs is not fully understood. Both Syk SH2 (Src homology 2) domains are required for high-affinity binding to these motifs, but the C-terminal SH2 domain (Syk-C) can function independently and can bind, in isolation, to the tyrosine-phosphorylated IgE receptor in vitro. In order to improve understanding of the cellular function of Syk, we have determined the solution structure of Syk-C complexed with a phosphotyrosine peptide derived from the gamma subunit of the IgE receptor. RESULTS: The Syk-C:peptide structure is compared with liganded structures of both the SH2 domain of Src and the C-terminal SH2 domain of ZAP-70 (the 70 kDa zeta-associated protein). The topologies of these domains are similar, although significant differences occur in the loop regions. In the Syk-C structure, the phosphotyrosine and leucine residues of the peptide ligand interact with pockets on the protein, and the intervening residues are extended. CONCLUSIONS: Syk-C resembles other SH2 domains in its peptide-binding interactions and overall topology, a result that is consistent with its ability to function as an independent SH2 domain in vitro. This result suggests that Syk-C plays a unique role in the intact Syk protein. The determinants of the binding affinity and selectivity of Syk-C may reside in the least-conserved structural elements that comprise the phosphotyrosine- and leucine-binding sites. These structural features can be exploited for the design of Syk-selective SH2 antagonists for the treatment of allergic disorders and asthma.     

SH2 domain protein interaction and possibilities for pharmacological intervention

SH2 domain containing proteins play a key role in the process of intracellular transmission of signalling events initiated at the cell surface. As a pre-requisite in the fulfillment of this function, these proteins bind to a variety of phospho-tyrosine (pY) containing target molecules. Delineation of these binding sites as essentially short linear peptides (both structurally and functionally) has led to the suggestion that the activity of these signalling complexes may be manipulated by the development of relatively simple peptide reagents. This review examines the range of possibilities open on this approach and the extent to which positive results have already been realised.     

Exploiting the basis of proline recognition by SH3 and WW domains: design of N-substituted inhibitors

Src homology 3 (SH3) and WW protein interaction domains bind specific proline-rich sequences. However, instead of recognizing critical prolines on the basis of side chain shape or rigidity, these domains broadly accepted amide N-substituted residues. Proline is apparently specifically selected in vivo, despite low complementarity, because it is the only endogenous N-substituted amino acid. This discriminatory mechanism explains how these domains achieve specific but low-affinity recognition, a property that is necessary for transient signaling interactions. The mechanism can be exploited: screening a series of ligands in which key prolines were replaced by nonnatural N-substituted residues yielded a ligand that selectively bound the Grb2 SH3 domain with 100 times greater affinity.     

CBL-GRB2 interaction in myeloid immunoreceptor tyrosine activation motif signaling

In this study, we provide the first evidence for role of the CBL adapter protein interaction in Fc gammaRI receptor signal transduction. We study the Fc gammaRI receptor, an immunoreceptor tyrosine activation motif (ITAM)-linked signaling pathway, using IFN-gamma-differentiated U937 myeloid cells, termed U937IF cells. CBL is constitutively associated with both GRB2 and the ITAM-containing receptor subunit, Fc gammaRIgamma of Fc gammaRI, providing direct evidence that CBL functions in myeloid ITAM signaling. Fc gammaRI cross-linking of U937IF cells induces the tyrosine phosphorylation of CBL that is associated with an altered CBL-GRB2 interaction. Both GRB2-SH3 and SH2 domains bind CBL in resting cell lysates; upon Fc gammaRI stimulation, phosphorylated CBL binds exclusively to the GRB2-SH2 domain. Glutathione-S-transferase fusion protein data demonstrate that the constitutive interaction of CBL with GRB2 and CRKL is mediated via two discrete regions of the CBL C terminus. The proximal C terminus (residues 461-670) binds to GRB2 constitutively, and under conditions of receptor activation binds to the tyrosine-phosphorylated SHC adapter molecule. The distal C terminus of CBL (residues 671-906) binds the CRKL adapter protein. The data demonstrate that the CBL-GRB2 and GRB2-SOS protein complexes are distinct and mutually exclusive in U937IF cells, supporting a model by which the CBL-GRB2 and GRB2-SOS complexes function in separate pathways for myeloid Fc gammaRI signaling.     

Calmodulin binding to myosin light chain kinase begins at substoichiometric Ca2+ concentrations: a small-angle scattering study of binding and conformational transitions

We have used small-angle scattering to study the calcium dependence of the interactions between calmodulin (CaM) and skeletal muscle myosin light chain kinase (MLCK), as well as the conformations of the complexes that form. Scattering data were measured from equimolar mixtures of a functional MLCK and CaM or a mutated CaM (B12QCaM) incompetent to bind Ca2+ in its N-terminal domain, with increasing Ca2+ concentrations. To evaluate differences between CaM-enzyme versus CaM-peptide interactions, similar Ca2+ titration experiments were performed using synthetic peptides based on the CaM-binding sequence from MLCK (MLCK-I). Our data show there are different determinants for CaM binding the isolated peptide sequence compared to CaM binding to the same sequences within the enzyme. For example, binding of either CaM or B12QCaM to the MLCK-I peptide is observed even in the presence of EGTA, whereas binding of CaM to the enzyme requires Ca2+. The peptide studies also show that the conformational collapse of CaM requires both the N and C domains of CaM to be competent for Ca2+ binding as well as interactions with each end of MLCK-I, and it occurs at approximately 2 mol of Ca2+/mol of CaM. We show that CaM binding to the MLCK enzyme begins at substoichiometric concentrations of Ca2+ (< or = 2 mol of Ca2+/mol of CaM), but that the final compact structure of CaM with the enzyme requires saturating Ca2+. In addition, MLCK enzyme does bind to 2Ca2+ x B12QCaM, although this complex is more extended than the complex with native CaM. Our results support the hypothesis that CaM regulation of MLCK involves an initial binding step at less than saturating Ca2+ concentrations and a subsequent activation step at higher Ca2+ concentrations.     

Tandem SH2 domains confer high specificity in tyrosine kinase signaling

SH2 domain proteins transmit intracellular signals initiated by activated tyrosine kinase-linked receptors. Recent three-dimensional structures suggest mechanisms by which tandem SH2 domains might confer higher specificity than individual SH2 domains. To test this, binding studies were conducted with tandem domains from the five signaling enzymes: phosphatidylinositol 3-kinase p85, ZAP-70, Syk, SHP-2, and phospholipase C-gamma1. Bisphosphorylated TAMs (tyrosine-based activation motifs) were derived from biologically relevant sites in platelet-derived growth factor, T cell, B cell, and high affinity IgE receptors and the receptor substrates IRS-1 (insulin receptor substrate-1) and SHPS-1/SIRP. Each tandem SH2 domain binds a distinct TAM corresponding to its appropriate biological partner with highest affinity (0.5-3.0 nM). Alternative TAMs bind the tandem SH2 domains with 1,000- to >10,000-fold lower affinity than biologically relevant TAMs. This level of specificity is significantly greater than the approximately 20-50-fold typically seen for individual SH2 domains. We conclude that high biological specificity is conferred by the simultaneous interaction of two SH2 domains in a signaling enzyme with bisphosphorylated TAMs in activated receptors and substrates.     

An NMR study on the DNA-binding SPKK motif and a model for its interaction with DNA

The solution structure of one and two repeats of the 'SPKK' DNA-binding motif is reported on the basis of NMR measurements. In dimethylsulphoxide (DMSO) the major population (approximately 90%) of peptides, SPRKSPRK(S2) and GSPKKSPRK(S2b), adopts a conformation, which has two trans prolines. The two 'SP(R/K)K' units in these peptides are equivalent and each adopts a turn structure exchanging with an extended structure. This is suggested by an NOE connectivity of the beta-turn type, between the backbone amide protons of residues (i+2) and (i+3) and NOE connectivities of the Asx(sigma)-turn type, between protons of the ith Ser and the backbone amide proton on residue (i+2). This suggests that each SP(R/K)K unit has a structural intermediate between (or a combination of) a beta-turn and an Asx(sigma)-turn. In 90-10% DMSO/H2O at 4 degrees C the two units of S2 are connected more tightly by folding into a short 3(10) helix, broken at the second proline. For another peptide, Thr-Pro-Arg-Lys(T1), the major population (75%) in 100% DMSO comprises a beta-turn in rapid exchange with an extended structure. We did not observe an NOE connectivity of the Asx(sigma) type with the T1 peptide. A possible structure of the SPKK motif in the complex with DNA is discussed.     

Immunogenicity and conformational properties of an N-linked glycosylated peptide epitope of human T-lymphotropic virus type 1 (HTLV-I)

The identification and characterization of epitopes of human T-lymphotropic virus type 1 (HTLV-I), which elicit an effective humoral or cell-mediated immune response, remains a central obstacle to the development of a peptide-based vaccine against the virus infection. The objective of the studies presented here was to examine the influence of N-linked glycosylation on peptide structure and immunogenicity. We engineered the 233-253 sequence of gp46 of HTLV-I to contain an N-acetylglucosamine (GlcNAc) residue at Asn244. Secondary structure prediction using computer algorithms indicated that this peptide may contain a beta-turn at residues 242-246. Recent work with model glycopeptides suggests that beta-turn conformation in peptides may be induced, and probably is stabilized, by the presence of even a single sugar residue. In the present study, the structures of the 233-253 peptide, SC1, and the 233-253(Asn244-GlcNAc) glycopeptide, SC2, were determined. Similar conformation was exhibited by both the glycosylated and nonglycosylated peptide displaying a beta-turn at residues 243-246 and extended-chain structure at the peptide/glycopeptide termini. Both peptides were engineered into chimeric constructs with a promiscuous T-cell epitope from measles virus and were used as immunogens in rabbits. Both chimeric peptides were highly immunogenic in rabbits, producing high-titered antibodies as early as primary + three weeks. The antibodies generated against either construct were able to bind to whole virus (ELISA) and to gp46 (radioimmunoprecipitation assay). Additionally, human sera of individuals known to be positive for HTLV-I recognized both the glycosylated and nonglycosylated constructs. It appears that the 233-253 peptide is able to adopt a conformation that mimics the structure in native gp46, and addition of a GlcNAc residue at Asn244 does not affect the conformational preference or stability of this construct; nor does glycosylation alter immunogenicity but instead appears to enhance immune recognition.     

Solution structure of the C-terminal SH2 domain of the p85 alpha regulatory subunit of phosphoinositide 3-kinase

Heterodimeric class IA phosphoinositide 3-kinase (PI 3-kinase) plays a crucial role in a variety of cellular signalling events downstream of a number of cell-surface receptor tyrosine kinases. Activation of the enzyme is effected in part by the binding of two Src homology-2 domains (SH2) of the 85 kDa regulatory subunit to specific phosphotyrosine-containing peptide motifs within activated cytoplasmic receptor domains. The solution structure of the uncomplexed C-terminal SH2 (C-SH2) domain of the p85 alpha subunit of PI 3-kinase has been determined by means of multinuclear, double and triple-resonance NMR experiments and restrained molecular-dynamics simulated-annealing calculations. The solution structure clearly indicates that the uncomplexed C-SH2 domain conforms to the consensus polypeptide fold exhibited by other SH2 domains, with an additional short helical element at the N terminus. In particular, the C-SH2 structure is very similar to both the p85 alpha N-terminal SH2 domain (N-SH2) and the Src SH2 domain with a root mean square difference (rmsd) for 44 C alpha atoms of 1.09 and 0.89 A, respectively. The canonical BC, EF and BG loops are less well-defined by the experimental restraints and show greater variability in the ensemble of C-SH2 conformers. The lower level of definition in these regions may reflect the presence of conformational disorder, an interpretation supported by the absence or broadening of backbone and side-chain NMR resonances for some of these residues. NMR experiments were performed, where C-SH2 was titrated with phosphotyrosine-containing peptides corresponding to p85 alpha recognition sites in the cytoplasmic domain of the platelet-derived growth-factor receptor. The ligand-induced chemical-shift perturbations indicate the amino-acid residues in C-SH2 involved in peptide recognition follow the pattern predicted from homologous complexes. A series of C-SH2 mutants was generated and tested for phosphotyrosine peptide binding by surface plasmon resonance. Mutation of the invariant Arg36 (beta B5) to Met completely abolishes phosphopeptide binding. Mutation of each of Ser38, Ser39 or Lys40 in the BC loop to Ala reduces the affinity of C-SH2 for a cognate phosphopeptide, as does mutation of His93 (BG5) to Asn. These effects are consistent with the involvement of the BC loop and BG loops regions in ligation of phosphopeptide ligands. Mutation of Cys57 (beta D5) in C-SH2 to Ile, the corresponding residue type in the p85 alpha N-SH2 domain, results in a change in peptide binding selectivity of C-SH2 towards that demonstrated by p85 alpha N-SH2. This pattern of p85 alpha phosphopeptide binding specificity is interpreted in terms of a model of the p85 alpha/PDGF-receptor interaction.     

A bacterial cytokine

T cell receptor (TCR) triggering induces association of the protein tyrosine kinase ZAP-70, via its two src-homology 2 (SH2) domains, to di-phosphorylated Immunoreceptor Tyrosine-based Activation Motifs (2pY-ITAMs) present in the intracellular tail of the TCR-zeta chain. The crystal structure of the SH2 domains complexed with a 2pY-ITAM peptide suggests that the 60-amino acid-long inter-SH2 spacer helps the SH2 domains to interact with each other to create the binding site for the 2pY-ITAM. To investigate whether the inter-SH2 spacer has additional roles in the whole ZAP-70, we raised antibodies against two peptides of this region and probed ZAP-70 structure under various conditions. We show that the reactivity of antibodies directed at both sequences was dramatically augmented toward the tandem SH2 domains alone compared with that of the entire ZAP-70. This indicates that the conformation of the inter-SH2 spacer is not maintained autonomously but is controlled by sequences C-terminal to the SH2 domains, namely, the linker region and/or the kinase domain. Moreover, antibody binding to the same two determinants was also inhibited when ZAP-70 or the SH2 domains bound to the zeta chain or to a 2pY-ITAM. Together, these two observations suggest a model in which intramolecular contacts keep ZAP-70 in a closed configuration with the two SH2 domains near to each other.     

Recognition specificity of individual EH domains of mammals and yeast

The Eps homology (EH) domain is a recently described protein binding module that is found, in multiple or single copies, in several proteins in species as diverse as human and yeast. In this work, we have investigated the molecular details of recognition specificity mediated by this domain family by characterizing the peptide-binding preference of 11 different EH domains from mammal and yeast proteins. Ten of the eleven EH domains could bind at least some peptides containing an Asn-Pro-Phe (NPF) motif. By contrast, the first EH domain of End3p preferentially binds peptides containing an His-Thr/Ser-Phe (HT/SF) motif. Domains that have a low affinity for the majority of NPF peptides reveal some affinity for a third class of peptides that contains two consecutive amino acids with aromatic side chains (FW or WW). This is the case for the third EH domain of Eps15 and for the two N-terminal domains of YBL47c. The consensus sequences derived from the peptides selected from phage-displayed peptide libraries allows for grouping of EH domains into families that are characterized by different NPF-context preference. Finally, comparison of the primary sequence of EH domains with similar or divergent specificity identifies a residue at position +3 following a conserved tryptophan, whose chemical characteristics modulate binding preference.     

A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of alpha chain, beta chain, and antigenic peptide, with immunogenicity in vitro and reduced affinity for bacterial superantigens

Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogeneous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 beta chain. Co-transfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated beta chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR alpha chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, beta chain, and alpha chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated alpha chain moved in electrophoresis as compact class II MHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding for the bacterial superantigens staphylococcal enterotoxin A and B and almost no binding for toxic shock syndrome toxin-1. The unique properties of these engineered HLA-DR1 molecules may facilitate our understanding of the complex nature of antigen recognition and aid in the development of novel vaccines with reduced superantigen binding.