Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk

Wu, Xiaodong; Knudsen, Beatrice S.; Feller, Stephan M.; Zheng, Jie; Šali, Andrej; Cowburn, David; Hanafusa, Hidesaburô; Kuriyan, John

doi:10.1016/s0969-2126(01)00151-4

Cited by 246 publications

(290 citation statements)

References 48 publications

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“…At the same time, binding of the (K?R)-mutant peptide to the Grb2SH3(N) domain was greatly increased. These results were con®rmed and explained by ultrastructural data, which showed that the lysine is tightly bound in a uniquely structured pocket largely formed by an SH3 region named the`RT-loop' (Wu et al, 1995).…”

Section: Introductionmentioning

confidence: 74%

“…An alanine scan through this C3G-CB-1 peptide demonstrated that the residues P 284 , L 286 , P 287 and K 289 are particularly important for the binding a nity towards the CrkSH3(1) domain. The lysine residue was shown to contribute strongly to the almost unique SH3 binding speci®city of CB-1 Wu et al, 1995). Replacement of K 289 by an arginine reduces the a nity of the CB-1 peptide approximately eightfold (to 17.2 mM) and signi®cantly a ects the interactions observed in the crystallised peptide-SH3 complex (Wu et al, 1995).…”

Section: Resultsmentioning

confidence: 99%

“…The lysine residue was shown to contribute strongly to the almost unique SH3 binding speci®city of CB-1 Wu et al, 1995). Replacement of K 289 by an arginine reduces the a nity of the CB-1 peptide approximately eightfold (to 17.2 mM) and signi®cantly a ects the interactions observed in the crystallised peptide-SH3 complex (Wu et al, 1995). The stronger binding of a SoS1-related peptide (PPPVPPRRRR; 5.2 mM) which does not contain a lysine but an arginine in the corresponding position was therefore surprising.…”

Section: Resultsmentioning

confidence: 99%

“…The determinants of CrkSH3(1) binding to peptides have been elucidated in some detail using C3G-and SoSderived sequences Wu et al, 1995). C3G, a guanine-nucleotide exchange factor for the GTPases Rap 1 A and B (Gotoh et al, 1995), contains four potential CrkSH3 binding motifs (CB1 to CB4) clustered in the central region of the protein (Knudsen et al, 1994;Tanaka et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

“…Since highly selective CrkSH3(1) binding peptides with very high a nity could be useful as speci®c inhibitors to study the signalling of Crk family proteins, we decided to perform a mutagenesis study based on the sequences of naturally occurring CrkSH3(1) binding motifs, as well as the available ultrastructural information of SH3 domain-peptide interactions (Wu et al, 1995). The resulting high a nity CrkSH3(1) binding peptides (HACBPs) have a K d of up to 100 nM to CrkSH3(1), while exhibiting an approximately 100-fold weaker interaction with the Grb2SH3(N) domain.…”

Section: Introductionmentioning

confidence: 99%

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Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G

Posern¹,

Zheng

Knudsen

et al. 1998

Oncogene

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Many Src Homology 3 (SH3) domains function as molecular adhesives in intracellular signal transduction. Based on previous ultrastructural studies, short motifs which bind to the ®rst SH3 domains of the adapters Crk and CRKL were selectively mutagenised to generate Crk/ CRKL SH3-binding peptides of very high a nity and selectivity. A nities were increased up to 20-fold compared to the best wildtype sequences, while the selectivity against a similar SH3 domain [Grb2SH3(N)] was not only retained, but sometimes increased. Blot techniques with GST-fusion peptides and in solution precipitation assays with biotinylated high a nity Crk binding peptides (HACBPs) were subsequently used to analyse the binding of these sequences to a large panel of SH3 domain-containing fusion proteins. Only those proteins which contained the CrkSH3(1) or CRKLSH3(1) domains bound e ciently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of 35 S-labelled and unlabelled cell lysates. Very little binding of other cellular proteins to HACBP was detectable, indicative of a great preference for Crk and CRKL when compared to the wide variety of other endogenous cellular proteins. Moreover, HACBP disrupted in vitro preexisting Crk-complexes with DOCK180 and the exchange factors SoS and C3G, which are known targets of Crk adapters, in a concentration dependent manner. HACBP-based molecules should therefore be useful as highly selective inhibitors of intracellular signalling processes involving Crk and CRKL.

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Section: Introductionmentioning

confidence: 74%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G

Posern¹,

Zheng

Knudsen

et al. 1998

Oncogene

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Stability and folding of the SH3 domain of bruton's tyrosine kinase

et al. 1996

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Bruton's tyrosine kinase (BTK) plays an important role in B cell development. Deletion of C-terminal 14 amino acids of the SH3 domain of BTK results in X-linked agammaglobulinemia (XLA), an inherited disease. We report here on the stability and folding of SH3 domain of BTK. Peptides corresponding to residues 216-273 (58 residues) and 216-259 (44 residues) of BTK SH3 domain were synthesized by solid phase methods; the first peptide constitutes the entire SH3 domain of BTK while the latter peptide lacks 14 amino acid residues of the C-terminal. The 58 amino acid peptide forms mainly a beta-barrel type folding unit. Although small and lacking disulfide bonds, this peptide is extremely stable to thermal denaturation. Based on circular dichroism measurements, its melting temperature was found to be high, 82 degrees C at pH 6.0. However, the Gibbs free energy (delta GH2O) of the intrinsic stability and thermodynamic spontaneity of unfolding were found to be low, 2.6 kcal/mol by Gdn.HCl denaturation experiments, as compared to 12 kcal/mol obtained for larger single domain proteins, indicating poor stability of SH3 domain. Addition of 500 mM of Na2SO4 increased the free energy change delta GH2O to 4.0 kcal/mol, suggesting an ionic strength effect. The truncated peptide fails to fold correctly and adopts random coil conformation in contrast to 58 amino acid beta-barrel peptide, which exhibits high thermal stability but normal or low stability at ambient temperature. These results, to our knowledge the first to delineate the importance of C-terminal in structural integrity of SH3 domains, indicate also that improper folding and/or poor stability of mutant SH3 domain in BTK likely causes XLA.

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SH3 domain of Bruton's tyrosine kinase can bind to proline-rich peptides of TH domain of the kinase and p120cbl

et al. 1997

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X-linked agammaglobulinemia (XLA), an inherited disease, is caused by mutations in the Bruton's tyrosine kinase (BTK). The absence of functional BTK leads to failure of B-cell differentiation; this incapacitates antibody production in XLA patients, who suffer from recurrent, sometimes lethal, bacterial infections. BTK plays an important role in B-cell development; it interacts with several proteins in the context of signal transduction. Point mutation in the BTK gene that leads to deletion of C-terminal 14 aa residues of BTK SH3 domain was found in a patient family. To understand the role of BTK, we studied binding of BTK SH3 domain (aa 216-273, 58 residues) and truncated SH3 domain (216-259, 44 residues) with proline-rich peptides; the first peptide constitutes the SH3 domain of BTK, while the latter peptide lacks 14 amino acid residues of the C terminal. Proline-rich peptides selected from TH domain of BTK and p120cbl were studied. It is known that BTK TH domain binds to SH3 domains of various proteins. We found that BTK SH3 domain binds to peptides of BTK TH domain. This suggests that BTK SH3 and TH domains may associate in inter- or intramolecular fashion, which raises the possibility that the kinase may be regulating its own activity by restricting the availability of both its ligand-binding modules. We also found that truncated SH3 domain binds to BTK TH domain peptide less avidly than does normal SH3 domain. Also, we show that the SH3 and truncated SH3 domains bind to peptide of p120cbl, but the latter domain binds weakly. It is likely that the truncated SH3 domain fails to present to the ligand the crucial residues in the correct context, hence the weaker binding. These results delineate the importance of C-terminal in binding of SH3 domains and indicate also that improper folding and the altered binding behavior of mutant BTK SH3 domain likely leads to XLA.

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Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk

Abstract: The c-Crk SH3 domain engages in an unusual lysine-specific interaction that is rarely seen in protein structures, and which appears to be a key determinant of its unique ability to bind the C3G peptides with high affinity.

Cited by 246 publications

References 48 publications

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G

Stability and folding of the SH3 domain of bruton's tyrosine kinase

SH3 domain of Bruton's tyrosine kinase can bind to proline-rich peptides of TH domain of the kinase and p120cbl

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