Characterization of human mucin (MUC15) and identification of ovine and caprine orthologs.

The glycoprotein MUC15 (mucin 15) was initially isolated from the bovine milk fat globule membrane. The present work demonstrates the existence of immunologically similar proteins ( approximately 130 kDa) in ovine, caprine, porcine, and buffalo milk samples. Purification and N-terminal amino acid sequencing confirmed the presence of ovine and caprine MUC15 orthologs in milk fat globule membranes. Expression of MUC15 in human milk was demonstrated by immunostaining ( approximately 150 kDa) as well as by mass spectrometry. Screening of a human multiple tissue expression array showed abundant MUC15 gene expression in placenta, salivary gland, thyroid gland, trachea, esophagus, kidney, testis, and the leukemia K-562 cell line. Furthermore, moderate expression was seen in the pancreas, adult and fetal lung, fetal kidney, lymph node, adult and fetal thymus, and parietal lobe. Structural motifs for interactions (epidermal growth factor receptor and Src homology 2 domains) are identified in the intracellular region. Implication of the mucin in signal transduction and the potential physiological function of MUC15 are discussed.

Crystallization of the yeast elongation factor complex eEF1A-eEF1B alpha.

Crystals of the Saccharomyces cerevisiae elongation factor eEF1A (formerly EF-1 alpha) in complex with a catalytic C-terminal fragment of the nucleotide-exchange factor eEF1B alpha (formerly EF-1 beta) were grown by the sitting-drop vapour-diffusion technique, using polyethylene glycol 2000 monomethyl ether as precipitant. Crystals diffract to better than 1.7 A and belong to the space group P2(1)2(1)2(1). The unit-cell parameters of the crystals are sensitive to the choice of cryoprotectant. The structure of the 61 kDa complex was determined with the multiple anomalous dispersion technique using three selenomethionine residues in a 11 kDa eEF1B alpha fragment generated by limited proteolysis of full-length eEF1B alpha expressed in Escherichia coli.

Mutational analysis of Glu272 in elongation factor 1A of E. coli.

In our previous work (Mansilla et al. (1997) Protein Eng. 10, 927-934) we showed that Arg7 of Escherichia coli elongation factor Tu (EF1A) plays an essential role in aminoacyl-tRNA (aa-tRNA) binding. Substitution of Arg7 by Ala or Glu lost this activity. We proposed that Arg7 forms a salt bridge with the charged conserved amino acid Glu272 (Asp284 in Thermus aquaticus) thereby binding the N-terminal region of the protein to domain 2 and thus completing the conformational rearrangement needed for binding aa-tRNA. In this work we have mutated Glu272 to arginine, either alone (Glu272Arg), or in combination with one of the above mentioned mutations (Arg7Glu/Glu272Arg) in order to test this hypothesis. Our results show that, in confirmation of our thesis based on structural knowledge, the substitution of Glu272 (Asp284) decreases the ability of EF1A:GTP to bind aa-tRNA.

The elongation factor 1 A-2 isoform from rabbit: cloning of the cDNA and characterization of the protein.

Eukaryotic elongation factor 1 A (eEF1A, formerly elongation factor-1 alpha) is an important component of the protein synthesis apparatus. Here we report the isolation and characterization of the cDNA sequence encoding rabbit eEF1A-2, an isoform of eEF1A, as well as a structural and functional comparison of the two rabbit isoforms. Northern analysis of the expression pattern of eEF1A-2 showed that this isoform is expressed in skeletal muscle, heart, brain and aorta, while transcripts are not detected in liver, kidney, spleen and lung. In contrast, the previously characterized eEF1A-1 isoform is expressed in all tissues examined except skeletal muscle. We have recently purified eEF1A-2 from rabbit skeletal muscle. By partial amino acid sequencing and determination of the post-translational modifications of eEF1A-2 we found that both of the glycerylphosphorylethanolamine modifications observed in eEF1A-1 appear to be present in eEF1A-2. However, two of the residues found dimethylated in eEF1A-1 appeared to be trimethylated in eEF1A-2. A comparison of the enzymatic activity showed that eEF1A-1 and eEF1A-2 have indistinguishable activity in an in vitro translation system. In contrast, the GDP dissociation rate constant is approximately 7 times higher for eEF1A-1 than for eEF1A-2. The nucleotide preference ratio (GDP/GTP) for eEF1A-1 was 0.82, while the preference ratio for eEF1A-2 was 1.50.

The role of Glu259 in Escherichia coli elongation factor Tu in ternary complex formation.

Determination of the crystal structure of the ternary complex formed between elongation factor Tu:GTP and aminoacylated tRNA revealed three regions of interaction between elongation factor Tu and tRNA. The structure indicates that the conserved glutamic acid at position 271 in Thermus aquaticus EF-Tu could be involved in the binding of the 3' CCA-Phe end of the aminoacylated tRNA. Therefore, the corresponding residue, Glu259, of Escherichia coli EF-Tu was mutated into alanine, aspartic acid, glutamine and tyrosine, in order to substantiate the crystallographic structural evidence and to obtain further knowledge of the importance of this residue. All of the mutated proteins showed nucleotide binding properties similar to the wild type. In addition the GTPase activities were similar to the wild type. The mutation of Glu259 to either alanine or aspartic acid resulted in a reduced strength of interaction with tRNA, while mutation to tyrosine abolished completely the interaction with tRNA. Finally, mutation to glutamine resulted in an elongation factor Tu variant behaving like the wild type. In conclusion, the environment around the site binding the CCA-Phe end of the tRNA is very restricted spatially and chemically so that only a residue with almost the same size and chemical properties as glutamic acid fulfils the requirements with regard to size, salt bridge-formation potential and maintenance of the backbone conformation at the 259 position.

Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach.

The function of the N-terminal region of elongation factor Tu is still unexplained. Until recently, it has not been visible in electron density maps from x-ray crystallography studies, but the presence of several well conserved basic residues suggest that this part of the molecule is of structural importance for the factor to function properly. In this study, two lysines at positions 4 and 9 were mutated separately to alanine or glutamate. The resulting four point mutants were expressed and purified using the pGEX system. The untagged products were characterized with regard to guanine-nucleotide interaction, intrinsic GTPase activity, and binding of aminoacyl-tRNA (aa-tRNA). The results show that Lys9 is especially strongly involved in the association with guanine nucleotides and the binding of aa-tRNA. Also Lys4 plays a role in the association of GDP and GTP and is also of some importance in aa-tRNA binding. Our results are discussed in structural terms with the conclusion that a complex network of interactions across the interface between domains 1 and 2 with Lys9 being a key residue seems to be important for the fine tuning of the dimensions of the cleft accommodating the acceptor end of aa-tRNA as well as delineating the structure of the effector region.

Contribution of Arg288 of Escherichia coli elongation factor Tu to translational functionality.

The recently solved structure of the ternary complex formed between GTP-bound elongation factor Tu and aminoacylated tRNA reveals that the elements of aminoacyl-tRNA that interact with elongation factor Tu can be divided into three groups: the T stem; the 3'-end CCA-Phe; and the 5' end. The conserved residues Arg288, Lys89 and Asn90 are involved in the binding of the 5' end. In the active, GTP-bound form of the elongation factor, Arg288 and Asn90 are involved in the formation of a network of hydrogen bonds connecting the switch regions I and II of domain 1 with the rest of the molecule. This network is disrupted upon formation of the ternary complex. Arg288 was replaced by alanine, isoleucine, lysine or glutamic acid, and the resulting mutants have been subjected to an in vitro characterisation with the aim of clarifying the function of Arg288. Unexpectedly, the mutants behaved like the wild-type factor with regard to the association and dissociation of guanine nucleotides, and the intrinsic GTPase activities are unchanged. Furthermore, the mutants were as efficient as the wild-type factor in carrying out protein synthesis in vitro in the presence of an excess of aminoacyl-tRNA. However, the mutants' abilities to bind aminoacyl-tRNA and protect the labile aminoacyl bond were impaired, especially where the charge had been reversed.

Mutational analysis of Escherichia coli elongation factor Tu in search of a role for the N-terminal region.

We have mutated lysine 2 and arginine 7 in elongation factor Tu from Escherichia coli separately either to alanine or glutamic acid. The aim of the work was to reveal the possible interactions between the conserved N-terminal part of the molecule, which is rich in basic residues and aminoacyl-tRNA. The enzymatic characterization, comprising GDP and GTP temperature stability assays and measurement of nucleotide dissociation and association rate constants, GTPase activity and aminoacyl-tRNA binding, shows that position 2 is not involved in aminoacyl-tRNA binding, while position 7 is necessary to accomplish this activity. Furthermore, arginine 7 seems to play a role in regulating the binding of GTP. The three-dimensional structure of the ternary complex, EF-Tu:GTP:Phe-tRNAPhe, involving Thermus aquaticus EF-Tu and yeast Phe-tRNA(Phe), shows that Arg7 is in a position which permits salt bridge formation with Asp284, thus binding the N-terminus tightly to domain 2. We propose that this interaction is needed for aminoacyl-tRNA binding, and also for completing the structural rearrangement, which takes place when the factor switches from its GDP to its GTP form.

Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA.

Two residues of Escherichia coli elongation factor Tu involved in binding of aminoacyl-tRNA were identified and subjected to mutational analysis. Lys-89 and Asn-90 were each replaced by either Ala or Glu. The four single mutants were denoted K89A, K89E, N90A, and N90E, respectively. The mutants were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl-tRNA, which suggested an important role of Lys-89 and Asn-90 in tRNA binding. Furthermore, our results indicate helix B to be an important target site for nucleotide exchange factor EF-Ts. Also the mutants His-66 to Ala and His-118 to either Ala or Glu were characterized in an in vitro translation assay. Their functional roles are discussed in relation to the structure of elongation factor Tu in complex with aminoacyl-tRNA.

Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding.

Elongation factor Tu from Escherichia coli was mutated separately at positions Asp86 and Arg58, in order to shed light both on the GTPase mechanism of elongation factor Tu and on the binding of aminoacyl-tRNA. In addition, the binding of guanine nucleotides was investigated by determination of the dissociation and association rate constants. The results imply that Arg58 is unimportant for the intrinsic GTPase mechanism and the binding of guanine nucleotides, whereas it is strongly involved in the binding of aminoacyl-tRNA and of the ribosome. Asp86 appears to be essential for the regulation of guanine-nucleotide affinities, and it may also play a role in the intrinsic GTPase mechanism.

Mutation of the conserved Gly94 and Gly126 in elongation factor Tu from Escherichia coli. Elucidation of their structural and functional roles.

All guanine-nucleotide-binding proteins cycle between an inactive, GDP-bound and an active, GTP-bound conformation whereby they function as molecular switches. Elongation factor Tu from Escherichia coli is used as a model for defining residues important in the switch mechanism. Gly94 and Gly126 were separately mutated to alanine residues to study their role in the switch mechanism. The mutant proteins are denoted [G94A]EF-Tu and [G126A]EF-Tu, respectively. Both mutations affect the affinities for guanine nucleotides considerably, resulting in a decrease in the characteristic preference for GDP over GTP. Furthermore the [G94A]EF-Tu mutant possesses an increased GTPase activity. The aminoacyl-tRNA affinity is much reduced for [G94A]EF-Tu, as reflected in an increase of the dissociation rate constant for the ternary complex by a factor of 40. Surprisingly, however, both mutants in their GDP forms have a low, but significant affinity for aminoacyl-tRNA, which is not seen for the wild-type elongation factor Tu. The mutants only exhibit minor changes compared to the wild type with respect to in vitro translation of a poly(U) messenger.

Mutation of the conserved Gly83 and Gly94 in Escherichia coli elongation factor Tu. Indication of structural pivots.

Elongation factor Tu from Escherichia coli cycles between an active conformation where GTP is bound, and an inactive conformation where GDP is bound. Between the two conformations, elongation factor Tu undergoes major structural changes. The aim of this work has been to reveal the role of two very well conserved glycine residues, Gly83 and Gly94, in the switch mechanism. Gly83 has been mutated alone or in combination with Gly94, both glycine residues being mutated to alanine. Enzymic characterisation of the two mutants have shown that they have an altered nucleotide affinity, a decrease in aminoacyl-tRNA affinity, an increase in intrinsic GTP hydrolysis, different behaviours in effector stimulation of the intrinsic GTPase activity, and that they are completely unable to sustain poly(Phe) synthesis in an in-vitro poly(U)-directed system. Our results indicates that particularly Gly83 is an important pivot point in elongation factor-Tu.

Towards an understanding of structure-function relationships of elongation factor Tu.

In light of the recently determined structure of elongation factor Tu, and taking into account chemical studies mapping functional sites, a number of residues have been selected for site-directed mutagenesis studies. Gly94, Gly126, His66, His118, Lys89 and Asp90 have each been point-mutated. Preliminary in vitro characterization data are presented.

One-step purification of E. coli elongation factor Tu.

The tuf A gene, encoding the E. coli elongation factor Tu, was cloned in the pGEX gene fusion system. Upon expression EF-Tu is fused to glutathione-S-transferase serving as a purification handle with affinity for glutathione immobilised on agarose. This allows purification of EF-Tu in a one-step procedure. The construct was designed in order to make possible the release of authentic EF-Tu by cleaving the fusion protein with the protease factor Xa.