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The Journal of Immunology, 2000, 164: 4170-4177.
Copyright © 2000 by The American Association of Immunologists

Six X-Linked Agammaglobulinemia-Causing Missense Mutations in the Src Homology 2 Domain of Bruton’s Tyrosine Kinase: Phosphotyrosine-Binding and Circular Dichroism Analysis1

Pekka T. Mattsson2,*,{dagger}, Ilkka Lappalainen{ddagger},§, Carl-Magnus Bäckesjö*, Eeva Brockmann{dagger}, Susanna Laurén{dagger}, Mauno Vihinen§ and C. I. Edvard Smith*

* Center for Biotechnology, Department of Biosciences, and Department of Immunology, Microbiology, Pathology and Infectious Diseases (IMPI), Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden; {dagger} Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland; {ddagger} Department of Biosciences, Division of Biochemistry, University of Helsinki, Helsinki, Finland; and § Institute of Medical Technology, University of Tampere, Tampere, Finland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Src homology 2 (SH2) domains recognize phosphotyrosine (pY)-containing sequences and thereby mediate their association to ligands. Bruton’s tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase, in which mutations cause a hereditary immunodeficiency disease, X-linked agammaglobulinemia (XLA). Mutations have been found in all Btk domains, including SH2. We have analyzed the structural and functional effects of six disease-related amino acid substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q. Also, we present a novel Btk SH2 missense mutation, H362R, leading to classical XLA. Based on circular dichroism analysis, the conformation of five of the XLA mutants studied differs from the native Btk SH2 domain, while mutant R307G is structurally identical. The binding of XLA mutation-containing SH2 domains to pY-Sepharose was reduced, varying between 1 and 13% of that for the native SH2 domain. The solubility of all the mutated proteins was remarkably reduced. SH2 domain mutations were divided into three categories: 1) Functional mutations, which affect residues presumably participating directly in pY binding (R307G); 2) structural mutations that, via conformational change, not only impair pY binding, but severely derange the structure of the SH2 domain and possibly interfere with the overall conformation of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural-functional mutations, which contain features from both categories above (Y361C).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bruton’s tyrosine kinase (Btk)3 is a cytoplasmic protein tyrosine kinase (PTK) expressed in all hemopoietic cell lineages except for T lymphocytes and plasma cells (1, 2). Btk is expressed at all stages of B lineage development, before plasma cells, starting from CD34+ pro-B to mature B cells (3). Expression of Btk seems to be important at different stages of differentiation. In the mouse, this effect is exerted at two stages, first during the transition from small pre-B cells to immature B cells in the bone marrow, and later during the maturation from IgDlow IgMhigh to IgDhigh IgMlow stages in the periphery (4). Btk belongs to the Tec family of PTKs, which all contain five domains: pleckstrin homology, Tec homology, SH3 and SH2, and kinase (SH1) domains (1, 5). Btk is the defective gene in XLA, a hereditary immunodeficiency disease (6, 7) caused by a block in early B cell development (8, 9, 10, 11). Bone marrow analysis shows a failure in pre-B cell clonal expansion leading to a total loss in the production of immature B cells (12, 13). XLA results in profound hypogammaglobulinemia and absence of mature B cells and plasma cells, causing susceptibility to bacterial and enteroviral infections (8, 9, 10, 11). XLA mutations are found in all of the five protein domains. The XLA mutation registry, BTKbase (14, 15, 16, 17, 18 ; http://www.uta.fi/imt/bioinfo/BTKbase.html) lists more than 600 mutation entries from more than 470 unrelated families, showing about 350 unique molecular events.

SH2 domains are nonenzymatic modules of about 100 aa residues, which were first identified by sequence similarities in Src-related PTKs (19). SH2 domains play a central role in a number of cellular processes, including growth, immune response, metabolism, mitogenesis, motility, and gene transcription (for reviews, see Refs. 20, 21, 22). SH2 domain-containing proteins contribute to the orchestration of signal transduction pathways and have binding affinities ranging from 200 to 800 nM (23, 24) to specific, pY-containing peptides (25, 26, 27). SH2 domains have been identified in about 150 proteins having either enzymatic activity (for example, kinases and phosphatases) or being adaptor molecules involved in protein-protein interactions, such as Shc, Grb2, Grb4, or Gads (28, 29, 30, 31).

Several high resolution three-dimensional structures of SH2 domains have been determined. The conserved fold contains a large central antiparallel ß-sheet and an associated smaller ß-sheet, flanked by {alpha}-helices on either side. The pY-containing ligand binds to the SH2 domain in an extended conformation. The most important conserved interaction between the SH2 domain and its ligand is the direct interaction between the pY phosphate group and the guanidinium group of arginine at position ßB5; numbering according to the established guidelines in which this arginine residue is amino acid number five in the second (B; in alphabetical order) ß-sheet of the Btk SH2 domain (32). The ßB5 amino acid corresponds to R307 in Btk. Usually, the hydrogen-bonding interactions between the pY side chain and the SH2 domain also include the side chains of another arginine residue and a serine residue at positions {alpha}A2 and ßB7, respectively (33). These amino acids are also present in Btk (R288 and S309). The ligand-binding specificity arises mainly from the nature of the ßD5 residue and pY + 1 and pY + 3 binding pockets (26, 34). The second-messenger phosphoinositides produced by phosphoinositide 3-OH kinase have also been shown to bind to SH2 domains (35), although the physiological significance of this interaction has been disputed recently (36).

To understand the structure-function relationships of the Tec family kinases and their connection to signal transduction, B cell development, and inherited diseases, we are studying the functional effects of XLA mutations. To date, 20 XLA-causing amino acid substitutions have been identified in the Btk SH2 domain. In this study, we have analyzed the structural and functional properties of six Btk SH2 missense mutations viz G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S (Fig. 1GoA). In addition, we have identified a new XLA-causing missense mutation, H362R, located in the Btk SH2 domain. CD and pY-binding assays indicated that all of the disease-causing missense mutations studied affect pY binding, but the structural alteration varies considerably among the mutant proteins.



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  		FIGURE 1. A, A stereo view of the modeled structure of Btk SH2 domain (53 ). The analyzed XLA-causing mutations are shown on the left. On the right are amino acids predicted to be involved in the binding of pY-containing ligands. The peptide bound to the domain has sequence pYEEI. Residues recognizing and binding phosphotyrosine are colored with orange, and those binding to pY + 1 are in green. The hydrophobic pY + 3 binding pocket is in yellow. Y334, participating in both pY + 1 and pY + 3 binding, is in white. B, Binding of the pYEEI peptide to the Btk SH2 domain. Residues involved are colored as in A. The SH2 domain is shown with the surface presentation, and the peptide with a stick model. The binding residues are clearly visible. The peptide-SH2 domain interaction indicates structural complementarity.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of SH2 domain expression plasmids and purification of recombinant proteins

The plasmid pSL301/hBtk (37) was used as the template for the amplification of native Btk SH2 (aa 281–377) and SH2 274–381(274–381) using standard PCR techniques (38). The PCR products for Btk SH2 281–377(281–377) and SH2 274–381(274–381) were digested with EcoRI-BamHI and NcoI-NotI and ligated to pGEX4T-3 (Amersham Pharmacia Biotech, Piscataway, NJ) and pBAT4 vectors (39), respectively. The overlap extension PCR technique (40) was used to create the mutants G302E, R307G, Y334S, C337S, L358F, and H362Q with SH2 274–381(274–381) or SH2 271–383(271–383) as the templates. The DNA fragments encoding native and the mutant SH2 274–381(274–381) regions were extended by amplification using Pfu polymerase (Stratagene, La Jolla, CA) with sense 5' primer 5'-GCG CCC ATG GAA GCA GAA GAC TCC ATA GAA and antisense 3' primer 5'-GAG CCT CGA GTT AAT TCT TGT TTT GTT GAG ACA CTG, and each of the products was cloned into T7 promoter-based GST-containing vector pGAT2 (J. Peränen and M. Hyvönen, unpublished data) from the NcoI and XhoI restriction sites. The native SH2 271–383(271–383) was also cloned into pBAT4. The SH2 variant Y361C was constructed using the QuickChange mutagenesis kit (Stratagene) and PCR using native SH2 271–383(271–383) as the template. The constructs were confirmed by dideoxy sequencing.

GST-SH2 proteins were overexpressed in Escherichia coli BL21 (DE3) using the phage T7 expression system, as described (41). For purification of the proteins, cells from 1.7l fermentation were lysed in 80 ml of 20 mM Tris-Cl, pH 7.5, 20 mM NaCl, 75 nM aprotinin, 1.2 µM leupeptin, 13 µM bestatin, and 1 mM EDTA. Triton X-100 was added to a final concentration of 1% (v/v), and the mixture was stirred at 4°C for 45 min. After centrifugation for 50 min at 30,000 x g at 4°C, the supernatant was recovered and the GST activity was measured using the GST-detection module kit (Amersham Pharmacia Biotech). The supernatant was incubated with glutathione-Sepharose bead at 4°C for 16 h after adding 0.02% NaN3 into the solution. The gel was washed extensively with 20 mM Tris-Cl (pH 7.5) and 20 mM NaCl (buffer A), and the protein was eluted out with 50 mM reduced glutathione in 100 mM Tris-Cl (pH 8) containing 150 mM NaCl. The native SH2 protein was purified in a two-step procedure. First, lysate prepared in a similar manner to that above was eluted through a pY-Sepharose affinity chromatography column equilibrated with 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, and eluted out with a NaCl gradient. The second step consisted of gel filtration with Superdex 75, with 10 mM HEPES, pH 7.5, 100 mM NaCl as the buffer.

For CD analysis, 0.78–1.3 µmol of the GST-SH2 fusion proteins dissolved in 50 mM reduced glutathione in 100 mM Tris-Cl, pH 8, containing 150 mM NaCl was incubated with thrombin (77 U/µmol of fusion protein) overnight at room temperature with gentle agitation. The final reaction mixtures were concentrated with Amicon stir-flow cell with a Nova 5K membrane (Filtron), and then underwent gel filtration on a Superdex 75 (Amersham Pharmacia Biotech) column equilibrated with 20 mM Na-K-phosphate buffer pH 7.

In vitro and in vivo solubility analysis of SH2 domains

The long-term in vitro solubility analysis was performed by storage of the purified GST-fused or thrombin-digested SH2 domains at 4°C on a time scale from several hours to weeks. After storage, the proteins were evaluated by SDS-PAGE. For in vivo studies, human cDNA of the native Btk and constructs carrying the mutations G302E, R307G, and Y334S at their SH2 domains were cloned into pSGT (42) and used to transfect COS-7 cells using standard calcium phosphate precipitation (43, 44). After expression for 2 days, the cells were solubilized in buffer containing 1% (v/v) Triton X-100, 50 mM HEPES, pH 7.4, 1 mM DTT, 20 µM leupeptin, 1.5 µM aprotinin, 1 mM PMSF, and 1 mM benzamide. After incubation for 10 min on ice, detergent-insoluble materials were removed by centrifugation. The cell lysates were aliquoted and stored for different time periods at 4°C, and the solubility of the Btk proteins was analyzed in a time course using ECL Western blotting (Amersham Pharmacia Biotech).

Screening of XLA mutations using SSCP analysis

Genomic DNA from EDTA-preserved blood samples was isolated using the Nucleon BACC2 kit (Amersham Pharmacia Biotech). The SSCP analysis was performed as described previously (45). DNA fragments amplified by PCR were purified and sequenced according to Amersham Pharmacia Biotech protocol.

Binding of SH2 domains to pY-Sepharose

The GST-fusion proteins of wild-type Btk SH2 and SH3 (Y223A) domains and mutation-containing SH2 variants were dialyzed against buffer A. After dialysis, the protein solutions were filtered and the amount of proteins was estimated both with determination of the protein concentration by using the method of Bradford (46) and by measuring the GST activity. The wild-type proteins (0.03–0.26 µmol) and variants (0.05 µmol) were run to a pY-Sepharose column equilibrated with buffer A. The pY-Sepharose was prepared by using the oxirane-coupling method (47) with L-phosphotyrosine (Sigma, St. Louis, MO) as the ligand. The bound proteins were eluted with a NaCl gradient (0.02–1 M), and the quantitative amounts of eluted proteins were calculated. The ratios of bound variants were compared with the binding of the native SH2 domain. Another GST-fusion construct, Btk SH3 (Y223A), was used as the control for unspecific adsorption.

CD analysis of SH2 proteins

The CD spectra of the purified SH2 proteins (~8 µM) dissolved in 20 mM phosphate buffer, pH 7, were recorded using a JASCO J-720 spectropolarimeter at 25°C using 1-mm path-length cuvette. The scanning speed was 20 nm/min, bandwidth 1 nm, response time 1 s, and sensitivity 20 mdeg. Ten consecutive scans were recorded from each sample, and the averaged spectra were smoothed after subtracting the baseline. The secondary structure estimations were performed using programs SELCON (48, 49, 50) and CONTIN (51), employing self-consistent method and ridge regression analysis, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A novel Btk SH2 domain missense mutation, H362R, leads to a classical XLA

To elucidate the structure-function relationships of the Btk, and its connection to signal transduction and XLA disease, we are studying the functional and conformational effects of XLA mutations. From 20 XLA-causing amino acid substitutions identified to date in the Btk SH2 domain (18), we have selected six missense mutations, i.e., G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S for further analysis (Fig. 1GoA). In parallel, we have screened patients by SSCP analysis to identify new mutations causing XLA. During preparation of the manuscript, we identified a novel SH2 missense mutation, H362R, causing a classical form of XLA. The SSCP analysis showed a single PCR fragment with an altered single strand conformation, and sequencing demonstrated a nucleotide change of A to G at position 1085 (data not shown). The mutation replaces the basic and nonionized histidine residue with basic and charged arginine residue at aa 362 in the Btk SH2 domain (BTKbase PIN H362R (1), accession number A0542). The history of the patient revealed a persistent enteroviral infection with severe neurological consequences, which is frequently observed in XLA. The patient has two male cousins with XLA, one of which died of pneumococcal meningitis.

The nature of Btk SH2 polypeptide termini is critical for domain solubility

The expression level of the various native and GST-SH2 fusion proteins, as estimated from both lysate and pellet, was similar, but their solubility differed considerably. The expression levels of soluble native SH2 271–381(271–381) and SH2 274–383(274–383) were 10–15 mg/L of fermented medium, whereas SH2 281–377(281–377) or SH2 274–381(274–381) was less soluble and had a tendency to aggregate. The most soluble protein was obtained with the construct extended from both termini, SH2 271–383(271–383). The yield of soluble protein, when purified, reached a value of 20–25 mg/L of fermented medium both for native and GST-fusion proteins. In addition, the extended proteins had a reduced tendency to aggregate, although they precipitated during prolonged storage. Storage of the proteins at 4°C, even in the presence of 2 mM DTT, resulted in the formation of a mixture of monomers and dimers, evident by nonreducing SDS-PAGE (data not shown). To prevent dimer formation and to further improve solubility, residue C337 was mutated to serine. This mutation has not been seen in any patient to date. The SH2 domain with mutation of C337 to serine was expressed under identical conditions and yields were as for native SH2. The protein showed the same or higher solubility than native SH2 protein, with no tendency to dimerize. Our results indicate that the hydrophilic nature of the protein chain termini is a critical parameter for obtaining large amounts of soluble Btk SH2 domains.

The expression level and solubility of mutant Btk SH2 domains are reduced

The six XLA mutants were introduced into the pGAT2/GST-SH2 271–383(271–383) construct. The conditions for the expression of different SH2 variants and the expression level of soluble fusion proteins, as measured using the GST detection module kit, were examined (Table IGo). The native GST-SH2 271–383(271–383) domain was expressed with high yields at 37°C within a few hours of induction. The expression of GST-SH2 mutants was feasible only at lower temperatures (16 and 22°C). The amount of soluble mutant SH2-GST fusion protein produced was considerably reduced compared with the level of soluble native protein at 37°C. The GST activity is in good agreement with the solubility of the mutant SH2 domain proteins after the removal of GST by thrombin digestion (Table IIGo). As an exception, H362Q fused with GST showed relatively high GST activity, but it had a remarkably strong tendency to aggregate during thrombin digestion. The solubility of transiently expressed native Btk and Btk proteins containing mutations at G302E, R307G, and Y334S was studied in COS-7 cells. The expression levels of native and mutation-containing domains were nearly identical, and the proteins had not aggregated during storage for 1 wk at 4°C, demonstrating that the properties of isolated mutated SH2 domains differ from those of full-length proteins.


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  		Table I. Fermentation conditions of different SH2 domain variants and levels of soluble GST fusion proteins in the lysates

 

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  		Table II. The solubility and binding assays of Btk SH2 constructs1

 
The conformation of Btk SH2 mutant R307G remained unchanged, while the conformation of the other mutants was altered

Proteins can be classified based on the content and organization of secondary structure elements. SH2 domains belong to {alpha}+ß proteins, and the three-dimensional structure of all known SH2 domains has similar folding, with a large antiparallel ß-sheet flanked by two {alpha}-helices on either side. The CD spectrum of {alpha}+ß proteins typically shows two minima between 208–210 and 222 nm and one maximum between 190 and 195 nm (52). The spectra of the native Btk and mutant R307G are identical (Fig. 2Go). They have a broad minimum in the range of 212–220 nm caused by two {alpha}-helices. The CD spectrum of C337S and Y361C (Fig. 2Go) is highly similar to the spectrum of the native SH2 domain. The minor changes observed in the conformation of Y361C might contribute to the ligand binding. The other mutants studied differed clearly from the native SH2 (Fig. 2Go). The spectra of G302E, Y334S, and H362Q have only one minimum at 207–208 nm. The spectrum of the L358F differs clearly from all the others by having two minima at 207 and 214 nm. The composition of the secondary structural elements of the different SH2 domain proteins was estimated using the CD spectra information (data not shown). The R307G mutation does not affect the secondary structure. C337S and Y361C do not change the amount of ß-strands, while the results of the estimation of {alpha}-helices using the different programs were contradictory. The G302E mutation decreases ß-strand content slightly, and the L358F mutation increases {alpha}-helical content. The {alpha}-helix content of G302E mutation and ß-strand content of L358F mutation could not be interpreted because of large variation in the results. The Y334S and H362Q mutations affect both the {alpha} and ß structures.



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  		FIGURE 2. Upper, The CD spectrum of the native Btk SH2 domain (black) is compared with the spectra of SH2 domains containing mutations R307G (green), C337S (blue), and Y361C (red). Lower, The CD spectra of the native SH2 (black), G302E (green), Y334S (magenta), L358F (blue), and H362Q (red) SH2 domains.

 
All Btk SH2 XLA mutants studied showed reduced pY binding

The native GST-SH2 fusion proteins showed both GST activity and pY-binding capacity, indicating that they retain their native conformation. The pY-Sepharose binding of GST-SH2 proteins carrying missense mutations was reduced by one to two orders of magnitude (Table IIGo). The R307G mutation reduces binding to the pY-Sepharose to 4.5% (Table IIGo), although its secondary structure remains unaltered according to the CD analysis (Fig. 2Go). The mutations in adjacent residues, Y361C and H362Q, had the lowest pY-resin-binding values, 1% and 1.5%, respectively, but differed considerably in secondary structure. The level of pY binding of mutant L358F was about 5%, and that of the mutants G302E and Y334S was about 10% compared with the native SH2. The binding of a domain with missense mutation C337S, not known to cause XLA, had an increased affinity (137%) and a solubility that was at least as high as that of the native SH2 domain.

Only one mutant, R307G, has decreased pY binding and unchanged SH2 domain conformation. Thus, R307G may be described as a functional mutation, resulting in loss of a critical pY-binding residue and destroying the important interaction between guanidinium and phosphate groups (Fig. 1GoB). The tyrosines 334 and 362 are likely to be involved in pY + 1 and pY + 3 site recognition according to the model of the Btk SH2 domain (53). The altered CD spectra (Fig. 2Go), changes in secondary structure elements, and diminished pY-binding abilities (Table IIGo) indicate that mutations Y334S and H362Q have both structural and functional contributions. Moreover, in the G302E, L358F, and Y361C mutant forms, the loss of pY-binding ability is accompanied by changes in protein conformation. Five of the six mutations causing XLA affected the SH2 domain conformation, while all six mutants showed reduced pY-binding properties and decreased solubility.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The XLA mutations are scattered all along the BTK gene, and the distribution of the mutations in the five domains is approximately according to the length of the domain, except for the TH domain (18). Moreover, missense mutations are underrepresented in the SH3 domain (18). The missense mutations are the most common mutational event in Btk. In the SH2 domain, 20 different missense mutations have been identified (18). By using SSCP analysis, we demonstrated a new, severe XLA-causing missense mutation, H362R, in the Btk SH2 domain. A missense mutation affecting the same residue, H362Q, has been reported previously in the Btk SH2 domain, leading also to a classical form of XLA (54).

A variety of SH2 domain constructs was created to enhance the solubility of the expressed proteins. The side chains of lysine and glutamate are highly hydrophilic (55), and the protein solubility can be increased by inclusion of these residues. Contrary to the isolated mutation-containing SH2 domains, the corresponding full-length Btk proteins or GST-fusion proteins carrying the same mutations did not aggregate during storage. This indicates that the mutated SH2 domains are by themselves more prone to aggregation. Mutations G302E and L358F decrease the Btk level in monocytes in vivo (56). In addition, the t1/2 of Y361C was decreased 3-fold in B cells (57). Whether the decreased Btk steady state level is secondary to the degradation of malformed Btk in the cells or is because Btk is turned over more rapidly when incorrectly localized (due to malfunction of SH2 domain) remains to be elucidated. The Btk SH2 domain contains a single cysteine residue, C337, which according to the model of SH2 domain structure (53) is situated on the surface of the domain. The storage of purified native SH2 domains resulted in the formation of a mixture of monomers and dimers and finally to aggregation. It is not known whether this aggregation is a direct consequence of dimer formation. Noteworthily, oxidation and subsequent dimer formation have been shown to impair protein crystallization of, for example, Grb2 (58). By replacing C337 with serine in Btk, we succeeded in preventing dimer formation.

Sequence alignments of SH2 domains have been presented in several studies (59, 60, 61). The side chains of ligand residues at positions from pY, pY + 1, and pY + 3 (Fig. 1GoB) are tightly anchored on the protein surface (62). The six XLA mutations were selected on the basis of their spatial locations and anticipated different roles in the SH2 domain function (Fig. 1GoA). With the exception of H362, all of the mutated residues are conserved in the Src SH2 domain. R307 is a crucial pY-contacting residue at position ßB5, which is directly involved in ligand binding. Therefore, the highly reduced pY-binding capacity of R307G is easily understood. Two different mutations have been identified from XLA patients, R307G and R307T. Northern blot of the R307G mutation showed a Btk transcript of normal size and abundance (63). This mutation almost completely abolished Btk function, leading to less than 1% B cells and undetectable Ig levels (64). According to our results, R307G does not alter protein structure, but it almost completely abolishes the pY binding. The substitution removes the essential pY interaction, and R307G is therefore classified as a functional mutation. Substitution of the corresponding residue by lysine in GAP and Abl also abolishes ligand binding (65, 66). Moreover, mutation R175L, at the corresponding position of c-Src, impairs the regulation of c-Src kinase activity most obviously by preventing the association of the phosphorylated tail with the SH2 domain (67, 68).

G302 precedes the ßB-strand in the interior of the molecule opposite to the pY-binding area (Fig. 1GoA). Glycine is almost invariant at this position in SH2 domains. The mutation G302E reduces the expression level of Btk in monocytes to 23% (56). Several missense mutations have been reported to destabilize Btk protein (57, 69, 70). According to our results, the G302E mutant folds in a structurally altered manner, which leads to decreased pY-binding ability and to reduced solubility. The substitution G302E introduces an acidic residue at a conserved site in the loop connecting the {alpha}A-helix and the ßB-strand. The change of charge in this area conceivably leads to alteration in conformation and might be the cause for the decrease in the amount of ß-strands. Also, glycine is the ultimate amino acid in the loop that is most likely important for a bend formation and orientation of the ß-strand following it, which is involved in pY binding. Without a conformational change in the G302E SH2 domain, the side chain of the bulky glutamate can be predicted to cause a clash with the K374 side chain. Evidently, this XLA mutation leads to a conformational modification and to loss of SH2 domain function in protein-protein interactions.

Y334 at position ßD5 forms part of the region recognizing residues pY + 1 and pY + 3 of the ligand. Studies using affinity selection of phosphopeptides from degenerate libraries have shown the pivotal role of the ßD5 residue in defining the sequence specificity of SH2 domains (26). The replacement of Btk Y334 with serine severely alters the protein conformation and function. This might be a consequence of slightly different relative orientation of the ß-sheets toward each other, leading to a change in the protein tertiary structure.

L358 is located in the {alpha}B-helix at the hydrophobic protein core. Leucine is highly conserved at this position among SH2 domains. In monocytes, the expression level of Btk protein in XLA patients carrying this mutation is 32% of wild type (56). According to the SH2 domain model (53), the side chain of phenylalanine is too large to fit into the structure and it will clash with L346 of the hydrophobic pY + 3 binding site (Fig. 1GoB).

Y361 is located in the {alpha}B-helix adjacent to the hydrophobic pY + 3 binding site (Fig. 1GoB). A mutation at this position causes an atypical form of XLA (57). The replacement of Y361 with cysteine shortens the t1/2 of Btk protein in vivo to one-third as compared with native Btk (57). However, whether this is due to an abnormal intracellular location of Btk or whether it is caused by increased susceptibility to degradation is not known. Our results show that the missense mutation Y361C leads to only minor alterations in the CD spectrum in spite of an essentially complete inability to bind to pY-Sepharose. H362, located in the {alpha}B-helix, participates in the phosphopeptide binding at position pY + 3. The H362Q mutation leads to a conformational change in the SH2 domain structure, and it markedly disturbs the pY-peptide binding. Based on these results, we regard G302E, Y334S, L358F, and H362Q as structural mutations and Y361C as structural-functional mutation.

Mutation R307K in the Btk SH2 domain has been shown to abrogate both the regulatory effect of Btk to the sustained increases in intracellular Ca2+ and the phosphorylation of PLC{gamma} isoforms (71). Altered PLC{gamma} activation has also been suggested to be responsible for the abnormal Ca2+ signaling in Xid B cells and in Btk-deficient chicken B lymphoma cells (72, 73). The requirement of the Btk SH2 domain for Ca2+ signaling suggests an interaction between the Btk SH2 domain and a tyrosine-phosphorylated ligand, allowing colocalization of phosphorylated PLC{gamma} and activated Btk (71, 74). Recently, B cell linker protein has been identified as a major Btk SH2 domain-binding protein in B cells (75). The interaction of B cell linker protein and SH2 domain of Btk leads to additional transphosphorylation of PLC{gamma}, and thereby to the sustained low-level accumulation of inositol-1,4,5-trisphosphate, a second messenger for the Ca2+ signaling (71, 75, 76). Noteworthily, by using a Btk gene carrying a R307A mutation, it has been shown that a functional SH2 domain is also important for the ability of Btk to act as a negative regulator of Fas-mediated apoptosis (77).

These data indicate that the Btk SH2 domain interactions with specific tyrosine-phosphorylated substrates during B cell differentiation are disrupted by the SH2 domain mutations leading to XLA phenotype. We found no evidence for mutations with intact pY-binding suggestive of a defect confined to the impairment of protein-protein interactions. Within Src family kinases, the SH2 domain associates with the phosphorylated tail segment and places the SH2 linker correctly for the interaction with SH3 domain (78, 79, 80). Btk does not possess the corresponding regulatory C-terminal phosphorylation site as the Src family members. However, we believe that structurally and functionally solid SH2 domain is central for both the biological function and the regulation of Btk and other Tec family members. However, in view of the recent structures of Src-family kinases (81, 82, 83) and the finding that the observed domain interactions are conserved among other cytoplasmic PTKs (68), it could be anticipated that the Btk SH2 domain has functions other than binding to pY-containing proteins. Mutants resulting in conformational changes but retaining some pY-binding capacity could potentially be involved in such functions.


   Acknowledgments
 
We are indebted to Stefan Knapp for critical reading of the manuscript. We also thank Lotta Asplund for the SSCP analysis.


   Footnotes
 
1 This work was supported by the Juselius Foundation, the Tampere University Hospital Medical Research Fund, the Jenny and Antti Wihuri Foundation, the Oskar Öflund Foundation, the Swedish Cancer Foundation, European Union Grant BIO4-CT98-0142, and European Union BIOMED2 Grant PL 963007. Back

2 Address correspondence and reprint requests to Dr. Pekka Mattsson, Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland. Back

3 Abbreviations used in this paper: Btk, Bruton’s tyrosine kinase; CD, circular dichroism; PLC, phospholipase C; PTK, protein tyrosine kinase; pY, phosphotyrosine; SH, Src homology; SSCP, single strand conformation polymorphism; XLA, X-linked agammaglobulinemia. Back

Received for publication November 22, 1999. Accepted for publication February 7, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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