Homologies in the primary structure of GTP-binding proteins: the nucleotide-binding site of EF-Tu and p21.

Leberman, Reuben; Egner, Ursula

doi:10.1002/j.1460-2075.1984.tb01808.x

Cited by 112 publications

(43 citation statements)

References 29 publications

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“…UV cross-linking studies performed with 8-azido GTP (Kim and Haley, personal communication) are consistent with residues 64-70 as the hydrophobic loop, and the analysis was consequently continued with this region. Residues 60-69 offl-tubulin were also independently implicated by Leberman and Egner [18] as a potential site for guanine base binding although their basis for selection differs from ours.…”

Section: Topological Considerationsmentioning

confidence: 42%

“…Since ligand-binding sites in ce/fl proteins are generally formed from two adjacent loops [20], the Mandelkow model appears to be incomplete and is incapable in its present form of explaining differences in GTP and GDP binding to fl-tubulin. (ii) In the Mandelkow model the guanine-binding site is formed from regions 60-69 and 240-244, and is a composite of sequences (region 60-69) observed in GTP-binding proteins such as EF-Tu [18] and sequences (region 240-244) observed in ATP/ADPand dinucleotide-binding proteins [25]. However, these regions lack a sequence thought to confer guaninebinding specificity on the G-proteins [21,23].…”

Section: Discussionmentioning

confidence: 99%

“…Despite the striking similarities between tubulin and the G-proteins, recent efforts to identify the nucleotide-binding site in tubulin on the basis of sequence homology with other G-proteins and thus confirm Hughes' hypothesis were largely unsuccessful [18,19]. In this paper we use a synthesis of sequence comparisons between tubulin, other Gproteins, and the ADP/ATP-binding proteins [3,7] and topological arguments [20] to generate a model for the GTP/GDP-binding site of ~-tubulin.…”

Section: Introductionmentioning

confidence: 99%

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A model of the nucleotide‐binding site in tubulin

1987

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Tubulin uses GTP to regulate microtubule assembly and is thought to be a member of a class of GDP/GTP‐binding proteins (G‐proteins) as defined by Hughes [(1983) Febs Lett. 164, 1–8]. How tubulin is structurally related to G‐proteins is not known. We use a synthesis of sequence comparisons between tubulin, other G‐proteins, and ADP/ATP‐binding proteins and topological arguments to identify potential regions involved in nucleotide binding. We propose that the nucleotide‐binding domain in the β‐subunit of tubulin is an α/β structure derived from amino acid residues ∼60–300. Five peptide sequences are identified which we suggest exist as ‘loops’ that extend from β‐strands and connect α‐helices in this structure. We argue that GDP binds to four of the five loops in an Mg2+‐independent manner while GTP binds in an Mg2+‐dependent manner to a different combination of four loops. We propose that this switch between loops upon GTP binding induces a conformational change essential for microtubule assembly.

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Section: Topological Considerationsmentioning

confidence: 42%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A model of the nucleotide‐binding site in tubulin

1987

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“…Recent data on the structure of other guanine nucleotide binding proteins provide further insight into the structure-function relationship in p21. For example, the bacterial elongation factor EF-Tu binds GTP and GDP with high specificity and has primary sequence homology with p21 and other guanine nucleotide binding proteins (20,22,23). The EF-Tu GDP binding site is composed of four polypeptide loops, each of which span a transition from a a-strand to an a-helical secondary structure (21,24).…”

Section: Resultsmentioning

confidence: 99%

Antibodies specific for amino acid 12 of the ras oncogene product inhibit GTP binding.

Clark¹,

Wong²,

Arnheim³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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An antibody (anti-p2lser) was raised against a ras p21-related synthetic peptide and was able to recognize specifically the substitution of serine for glycine at amino acid 12 of p21. This substitution causes oncogenic activation of p21. Anti-p2lser was found to immunoprecipitate v-Ki-ras p21 and to strongly inhibit its ability to autophosphorylate and to bind GTP in an immunoabsorption assay. Furthermore, binding of the antibody to p21 was specifically inhibited by GTP or GDP, suggesting that amino acids around position 12 are part of the GTP/GDP binding site. These results, taken together with the observation that the microinjection of anti-p2lser into cells transformed by v-Ki-ras p21 causes a transient reversion of the cells to a normal phenotype [Feramisco, J. R., Clark, R., Wong, G., Arnheim, N., Milley, R. & McCormick, F. (1985) Nature (London) 314; 639-642], support the idea that interaction of p21 with guanine nucleotides is crucial to the transforming function of this protein.The p21 proteins, encoded by members of the ras protooncogene family, cause the neoplastic transformation of certain cells in culture when activated through somatic mutation and have been implicated in the generation of a variety of human cancers. The most frequently observed mutation leading to the oncogenic activation of p21 results in the alteration of the glycine residue at position 12 of the protein to any of several other residues (1). p21 is known to specifically bind to GTP and hydrolyze it to GDP and inorganic phosphate (2-5). Substitution of valine for glycine at position 12 reduces the rate of GTP hydrolysis by a factor of about 10 (3-5). In forms of p21 that contain a threonine residue at position 59, phosphate is transferred to this residue by an autophosphorylation reaction (2, 6). The precise role of these activities in the transforming potential of activated p21 is not known. To learn more about the mechanism of p21 activation, we have used synthetic peptides (7-9) to produce an antibody (anti-p2lser) that specifically recognizes the substitution of serine for glycine at position 12 in an activated p21 (v-Ki-ras) and have used this antibody as a specific probe to study the structure and biochemical functions of p21. MATERIALS AND METHODSSynthetic Peptides. The peptide shown in Fig. 1 was synthesized with an automated SAM Peptide Synthesizer (Biosearch, San Rafael, CA) using standard solid-phase chemistry (11). Homogeneity of the peptide was confirmed by reversed-phase HPLC and amino acid analysis. Conjugation of the peptide to the carrier was achieved by a crosslinking reagent, a 6-(N-maleimido)aminocaproic acid ester of the sodium salt of 4-hydroxy-3-nitro-benzenesulfonic acid. This reagent couples via its active ester to amino groups of carrier proteins; the coupling can be quantitated by monitoring the release of 4-hydroxy-3-nitrobenzenesulfonate. Coupling of peptide to carrier results from the reaction of the maleimido group with the sulfhydryl group of the cysteine on the peptide. A complete descri...

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“…These plasma-membrane-associated proteins bind guanine nucleotides with high affinity (9,26,32) and display a low GTPase activity (11,19,22,34). Amino acid sequence comparisons have identified homologies between the ras proteins and other proteins which exhibit a high specific affinity for guanine nucleotides, including bacterial elongation and initiation factors, tubulin, and members of the G protein family (16,18). Of these regions of shared sequence homology, the region represented by ras amino acids 110 through 120 is the most striking.…”

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confidence: 99%

rasH mutants deficient in GTP binding.

Der¹,

Pan²,

Cooper³

1986

Mol. Cell. Biol.

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Single amino acid substitutions were introduced into a region of the rasH protein (residues 116, 117, and 119) homologous to a variety of diverse GTP-binding proteins. Each of the mutant p21 proteins displayed a significant reduction (10-to 5,000-fold) in GTP binding affinity. Activated rasH proteins deficient in GTP binding were unaltered in their ability to morphologically transform NIH 3T3 cells.The human ras family consists of three known genes (rasH, rasK, and rasN) which have been identified as active transforming genes in a variety of human neoplasms (5). The cellular ras genes encode closely related proteins designated p2is. These plasma-membrane-associated proteins bind guanine nucleotides with high affinity (9,26,32) and display a low GTPase activity (11,19,22,34). Amino acid sequence comparisons have identified homologies between the ras proteins and other proteins which exhibit a high specific affinity for guanine nucleotides, including bacterial elongation and initiation factors, tubulin, and members of the G protein family (16,18). Of these regions of shared sequence homology, the region represented by ras amino acids 110 through 120 is the most striking. We therefore used sitedirected mutagenesis to evaluate the possible role of these ras sequences in guanine nucleotide binding.A comparison of amino acid sequences representing a variety of guanine-nucleotide-binding proteins is shown in Table 1. A consensus sequence of Asn-Lys-X-Asp (human ras residues 116 through 119) was found in all GTP-binding proteins examined. To directly evaluate the involvement of these amino acids in the binding of GTP, oligonucleotidedirected mutagenesis (37, 38) was used to introduce single amino acid substitutions into a cDNA clone of a human rasH gene whose transforming potential was activated by substitution of leucine for glutamine at position 61 (rasH ; 8). Seventeen-base synthetic oligonucleotides, synthesized by the modified triester method, were used to introduce single nucleotide substitutions into a M13mp8 clone containing the rasH cDNA sequence. The following three mutations were isolated: rasH(61-Leu, 116-His), in which codon 116 was changed from AAC (Asn) to CAC (His); rasH(61-Leu, 117-Glu), in which codon 117 was changed from AAG (Lys) to GAG (Glu); and rasH(61-Leu, 119-His), in which codon 119 was changed from GAC (Asp) to CAC (His). The nucleotide sequences of the mutant clones were verified by dideoxy sequencing (31).To evaluate the guanine-nucleotide-binding properties of the mutated rasH proteins, each rasH mutation was constructed into a bacterial expression vector pXVR (8a), which directs synthesis of authentic mammalian p21, and then introduced into Escherichia coli PR13-Q. Upon induction with isopropylthio-3-D-galactoside, approximately 30% of the total bacterial protein was represented by p21. Bacterium-expressed p2is were purified as previously described

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Homologies in the primary structure of GTP-binding proteins: the nucleotide-binding site of EF-Tu and p21.

Cited by 112 publications

References 29 publications

A model of the nucleotide‐binding site in tubulin

A model of the nucleotide‐binding site in tubulin

Antibodies specific for amino acid 12 of the ras oncogene product inhibit GTP binding.

rasH mutants deficient in GTP binding.

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