Crosstalk between the catalytic and regulatory domains allows bidirectional regulation of Src.

The catalytic activity of Src family tyrosine kinases is inhibited by intramolecular interactions between the regulatory SH3 and SH2 domains and the catalytic domain. In the inactive state, the critical alphaC-helix in the catalytic domain is positioned such that the formation of the Glu 310-Lys 295 salt bridge is precluded, Tyr 416 in the activation loop is unphosphorylated, and the SH2 and SH3 domains are unavailable for interactions with other proteins. We found that phosphorylation of the activation loop or mutation of the loop preceding the alphaC-helix activates Src and increases the accessibility of the SH3 domain for ligands. Interaction of the alphaC-helix with the activation loop is a central component of this regulatory system. Our data suggest a bidirectional regulation mechanism in which the regulatory domains inhibit Src activity, and Src activity controls the availability of the regulatory domains. By this mechanism, Src family kinases can be activated by proteins phosphorylating or changing the conformation of the catalytic domain. Once active, Src family kinases become less prone to regulation, implying a positive feedback loop on their activity.

Src regulated by C-terminal phosphorylation is monomeric.

The activity of the c-Src protein tyrosine kinase is regulated by phosphorylation of a tyrosine residue (Tyr-527) in the C-terminal tail of the molecule. Phosphorylation of Tyr-527 promotes association of the tail with the SH2 domain and a concomitant reduction of the enzymatic activity of Src. We asked the question whether regulation by C-terminal phosphorylation was accompanied by a change in the quaternary structure of the enzyme or if it occurred within a monomeric form of Src. For this purpose we purified to homogeneity a chicken Src form lacking the unique domain from Schizosaccharomyces pombe cells. The cells were engineered to express Src along with Csk, a protein kinase able to phosphorylate Tyr-527 efficiently. Mass spectrometric analysis showed that purified Src was homogeneously phosphorylated at Tyr-527. The enzyme was in the regulated form, because it could be activated by a phosphorylated peptide able to bind the SH2 domain with high affinity. Using gel filtration chromatography, dynamic light scattering, and ultracentrifugation, we found that the regulated form of Src was a monomer. We have obtained crystals diffracting to 2.4 A with space group P2(1)2(1)2(1) and one molecule per asymmetric unit, in agreement with the monomeric state. These results indicate that the structural rearrangements of regulated Src are of an intramolecular nature.

The role of the linker between the SH2 domain and catalytic domain in the regulation and function of Src.

The crystal structures of the regulated Src and Hck tyrosine kinases show intramolecular interactions between the phosphorylated tail and the SH2 domain as well as between the SH3 domain, the SH2-catalytic domain linker (SH2-CD linker) and the catalytic domain. The relative contribution of these interactions to regulation of activity is poorly understood. Mutational analysis of Src and Lck revealed that interaction of the SH2-CD linker with the SH3 domain is crucial for regulation. Moreover, three sites of interaction of the linker with the catalytic domain, one at the beginning (Trp260) and two at the back of the small lobe, opposite the catalytic cleft (beta2/beta3 loop; alphaC/beta4 loop), impinge on Src activity. Other activating mutations map to the front of the catalytic domain in the loop preceding the alphaC-helix (beta3/alphaC loop). SH2-CD linker mutants are deregulated in mammalian cells but transform fibroblasts weakly, suggesting that the linker may bind cellular components. Interpretation of our results on the basis of the crystal structure of Src favours a model in which the correctly positioned SH2-CD linker exerts an inhibitory function on catalysis of Src family members by facilitating displacement of the alphaC-helix. This study may provide a template for the generation of deregulated versions of other protein kinases.

The 2.35 A crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regulatory interactions.

The Src protein tyrosine kinase plays a critical role in a variety of signal transduction pathways. Strict regulation of its activity is necessary for proper signalling. We present here the crystal structure of chicken Src which is phosphorylated at Tyr527 and represents its least active form. Our structure, similar to the recently reported human Hck and Src structures, contains the SH3, SH2 and the kinase domains and the C-terminal regulatory tail but not the N-terminal unique domain. The SH3 domain uses its hydrophobic surface to coordinate the SH2-kinase linker such that residues Gln251 and Leu255 specifically interact with side chains in the beta2-beta3 and the alphaC-beta4 loops of the N-terminal lobe opposite of the kinase active site. This position of the SH3 domain and the coordination of the SH2-kinase linker also optimally places the SH2 domain such that the phosphorylated Tyr527 in the C-terminal tail interacts with the SH2 binding pocket. Analogous to Cdk2 kinase, the position of the Src alphaC-helix in the N-terminal lobe is swung out disrupting the position of the active site residues. Superposition of other protein kinases including human Hck and Src onto chicken Src indicate that the alphaC-helix position is affected by the relative position of the N-terminal lobe with respect to the C-terminal lobe of the kinase and that the presence of the SH3/SH2-kinase linker/N-terminal lobe interactions restricts the kinase lobes and alphaC-helix access to the active conformation. These superpositions also suggest that the highly conserved alphaC-beta4 loop restricts the conformational freedom of the N-terminal lobe by anchoring it to the C-terminal lobe. Finally, based on sequence alignments and conservation of hydrophobic residues in the Src SH2-kinase linker as well as in the alphaC-beta4 and beta2-beta3 loops, we propose that the Src-related kinases, Abl, Btk and Csk, share the same quaternary structure.

The purification and characterization of the catalytic domain of Src expressed in Schizosaccharomyces pombe. Comparison of unphosphorylated and tyrosine phosphorylated species.

The catalytic domain of chicken Src including the C-terminal tail (Src-CD), has been expressed in Schizosaccharomyces pombe and purified to homogeneity. The expressed protein is a mixture of unphosphorylated (80%) and mono-phosphorylated (20%) species, that can be separated from each other by Mono Q chromatography. By a novel mass spectrometric method that utilizes parent ion scans of unseparated peptide mixtures, we found that the mono-phosphorylated form is phosphorylated either at Tyr416 or at Tyr436. The stability of Src-CD is comparable to the wild-type protein. Src-CD auto-phosphorylates and efficiently phosphorylates substrate peptides and proteins. Auto-phosphorylation occurs by an intermolecular mechanism and is completely inhibited by an excess of substrate peptide. Kinetic measurements for two exogenous substrates, the Src substrate peptide (AEEEIYGEFEAKKKK) and denatured enolase, showed that the overall activity (kcat) of the Src-CD molecule is about 10 times higher than that of wild-type Src. The kcat values for phosphorylation of the Src substrate peptide are similar for the unphosphorylated and monophosphorylated Src-CD (50 min-1), but the apparent K(m) values differ significantly (approximately 3 microM and 10 microM, respectively). Therefore, at low substrate concentrations in vitro the mono-phosphorylated form is more active, in agreement with the importance of Tyr416 for in vivo activity. The apparent K(m) values of the mono-phosphorylated Src-CD and wild-type Src for the Src substrate peptide and enolase are similar, indicating that, under these conditions, the kinase domain is mainly responsible for substrate binding.

Elongation factor Tu D138N, a mutant with modified substrate specificity, as a tool to study energy consumption in protein biosynthesis.

Substitution Asp138-->Asn changes the substrate specificity of elongation factor (EF) Tu from GTP to XTP [Hwang & Miller (1987) J. Biol. Chem. 262, 13081-13085]. This mutated EF-Tu (EF-Tu D138N) was used to show that 2 XTP molecules are hydrolyzed for each elongation cycle [Weijland & Parmeggiani (1993) Science 259, 1311-1313]. Here we extend the study of the properties of this EF-Tu mutant and its function in the elongation process. In poly(U)-directed poly(phenylalanine) synthesis, the number of peptide chains synthesized using EF-Tu D138N.XTP was 30% higher than with EF-Tu wild type (wt).GTP. However, since in the former case the average peptide chain length was correspondingly reduced, the number of the residues incorporated turned out to be nearly the same in both systems. The K'd values of the XTP and XDP complexes of EF-Tu D138N were similar to those of the GTP and GDP complexes of EF-Tu wt. The extent of leucine misincorporation and the kirromycin effect were also comparable to those in the EF-Tu wt/GTP system. The hydrolysis of two XTP molecules, very likely as part of two EF-Tu D138N.XTP complexes, for each elongation cycle was found to be independent of (i) MgCl2 concentration, (ii) ribosome concentration, and (iii) temperature (5-40 degrees C). With rate-limiting amounts of XTP the K'm of its XTPase activity corresponded to the K'm for XTP of poly(phenylalanine) synthesis (0.3-0.6 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Why do two EF-Tu molecules act in the elongation cycle of protein biosynthesis?

In the elongation cycle of bacterial protein biosynthesis, the binding of aminoacyl-tRNA (aa-tRNA) to the A-site of mRNA-programmed ribosomes is mediated by elongation factor Tu (EF-Tu) and associated with the hydrolysis of GTP. Recently, in the case of cognate aa-tRNA, the participation of two GTP molecules has been implicated in this reaction. These are likely to be involved in preventing the indiscriminate binding of aa-tRNA to the ribosomal A-site. This article integrates this unexpected finding with our current knowledge of the structure-function relationships of the macro-molecules involved in the elongation cycle.

Asparagine-135 of elongation factor Tu is a crucial residue for the folding of the guanine nucleotide binding pocket.

This work studies the structure-function relationships of Asn135, a residue situated in the GTP binding pocket of elongation factor Tu (EF-Tu). For this purpose we constructed EF-TuN135D/D138N and assayed its reactivity towards various purine nucleotides. We found that EF-TuN135D/D138N had no functional effect with GTP, ATP, XTP and isoGTP. The lack of a productive interaction with isoGTP shows that the Asn135 side-chain does not recognize the exocyclic keto group of the guanine base. However, EF-TuN135D/D138N, whose native conformation is stabilized by either elongation factor Ts or kirromycin, was able to support the enzymatic binding of aa-tRNA to the ribosome in the absence of any nucleotide, when in complex with the antibiotic. Taken together, these results show that Asn135 is important for the correct folding of the nucleotide binding site and that EF-Tu.kirromycin can mediate the binding of aa-tRNA to the mRNA-programmed ribosomes independently of the native conformation of this site.

Toward a model for the interaction between elongation factor Tu and the ribosome.

In the elongation cycle of bacterial protein synthesis the interaction between elongation factor-Tu (EF-Tu).guanosine triphosphate (GTP), aminoacyl-transfer RNA (aa-tRNA), and messenger RNA-programmed ribosomes is associated with the hydrolysis of GTP. This interaction determines the selection of the proper aa-tRNA for incorporation into the polypeptide. In the canonical scheme, one molecule of GTP is hydrolyzed in the EF-Tu-dependent binding of aa-tRNA to the ribosome, and a second molecule is hydrolyzed in the elongation factor-G (EF-G)-mediated translocation of the polypeptide from the ribosomal A site to the P site. Substitution of Asp138 with Asn in EF-Tu changed the substrate specificity from GTP to xanthosine triphosphate and demonstrated that the EF-Tu-mediated reactions involved the hydrolysis of two nucleotide triphosphates for each Phe incorporated. This stoichiometry of two is associated with the binding of the correct aa-tRNA to the ribosome.

Elongation factor Tu: a molecular switch in protein biosynthesis.

Elongation factor Tu (EF-Tu), the most abundant protein in Escherichia coli, is a guanine nucleotide-binding protein that in the 'on' state acts as a carrier of amino acyl-tRNA to the ribosome. Our knowledge of this essential component of translation has brought substantial progress in the past decade thanks to the co-ordinated application of biochemical, physico-chemical and genetic methods. Crystallographic analysis at 2.6 A resolution and site-directed mutagenesis have revealed structural and functional similarities between the guanine nucleotide-binding domains of EF-Tu and human H-ras p21 protein. The regulation of the expression of the two EF-Tu-encoding genes in E. coli, particularly that of tufB, has been shown to involve diverse mechanisms. Several aspects of the functions of EF-Tu in the elongation cycle have been reinvestigated, leading to new insights. These studies have emphasized the manifold aspects of the mechanisms regulating the activity of EF-Tu in the bacterial cell.

Structure-function relationships of elongation factor Tu as studied by mutagenesis.

We have modified elongation factor Tu (EF-Tu) from Escherichia coli via mutagenesis of its encoding tufA gene to study its function-structure relationships. The isolation of the N-terminal half molecule of EF-Tu (G domain) has facilitated the analysis of the basic EF-Tu activities, since the G domain binds the substrate GTP/GDP, catalyzes the GTP hydrolysis and is not exposed to the allosteric constraints of the intact molecule. So far, the best studied region has been the guanine nucleotide-binding pocket defined by the consensus elements typical for the GTP-binding proteins. In this area most substitutions were carried out in the G domain and were found to influence GTP hydrolysis. In particular, the mutation VG20 (in both G domain and EF-Tu) decreases this activity and enhances the GDP to GTP exchange; PT82 induces autophosphorylation of Thr82 and HG84 strongly affects the GTPase without altering the interaction with the substrate. SD173, a residue interacting with (O)6 of the guanine, abolishes the GTP and GDP binding activity. Substitution of residues Gln114 and Glu117, located in the proximity of the GTP binding pocket, influences respectively the GTPase and the stability of the G domain, whereas the double replacement VD88/LK121, located on alpha-helices bordering the GTP-binding pocket, moderately reduces the stability of the G domain without greatly affecting GTPase and interaction with GTP(GDP). Concerning the effect of ligands, EF-TuVG20 supports a lower poly(Phe) synthesis but is more accurate than wild-type EF-Tu, probably due to a longer pausing on the ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenesis of the NH2-terminal domain of elongation factor Tu.

Mutagenesis was carried out in the N-terminal domain of elongation factor Tu (EF-Tu) to characterize the structure-function relationships of this model GTP binding protein with respect to stability, the interaction with GTP and GDP, and the catalytic activity. The substitutions were introduced in elements around the guanine nucleotide binding site or in the loops defining this site, in the intact molecule or in the isolated N-terminal domain (G domain). The double substitution Val88----Asp and Leu121----Lys, two residues situated on two vicinal alpha-helices, influences the basic activities of the truncated factor to a limited extent, probably via long-range interactions, and induces a destabilisation of the G domain structure. The functional alterations brought about by substitutions on the consensus sequences 18-24 and 80-83 highlight the importance of these residues for the interaction with GTP/GDP and the GTPase activity. Mutations concerning residues interacting with the guanine base lead to proteins in large part insoluble and inactive. In one case, the mutated protein (EF-TuAsn135----Asp) inhibited the growth of the host cell. This demonstrates the crucial role of the base specificity for the active conformation of EF-Tu. The obtained results are discussed in the light of the three-dimensional structure of EF-Tu.