title: Site-directed mutagenesis of the GTP-binding domain of beta-tubulin.
abstract: Tubulin binds guanine nucleotides with high affinity and specificity. GTP, an allosteric effector of microtubule assembly, requires Mg2+ for its interaction with beta-tubulin and binds as the MgGTP complex. In contrast, GDP binding does not require Mg2+. The structural basis for this difference is not understood but may be of fundamental importance for microtubule assembly. We investigated the interaction of beta-tubulin with guanine nucleotides using site-directed mutagenesis. Acidic amino acid residues have been shown to interact with nucleotide in numerous nucleotide-binding proteins. In this study, we mutated seven highly conserved aspartic acid residues and one highly conserved glutamic acid residue in the putative GTP-binding domain of beta-tubulin (N-terminal 300 amino acids) to asparagine and glutamine, respectively. The mutants were synthesized in vitro using rabbit reticulocyte lysates, and their affinities for nucleotide determined by an h.p.l.c.-based assay. Our results indicate that the mutations can be placed in six separate categories on the basis of their effects on nucleotide binding. These categories range from having no effect on nucleotide binding to a mutation that apparently abolishes nucleotide binding. One mutation at Asp224 reduced the affinity of beta-tubulin for GTP in the presence but not in the absence of Mg2+. The specific effect of this mutation on nucleotide binding is consistent with an interaction of this amino acid with the Mg2+ moiety of MgGTP. This residue is in a region sharing sequence homology with the putative Mg2+ site in myosin and other ATP-binding proteins. As a result, tubulin belongs to a distinct class of GTP-binding proteins which may be evolutionarily related to the ATP-binding proteins.
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106.
authors.forename: G W
authors.initials: GW
authors.lastname: Farr
authors.forename: H
authors.initials: H
authors.lastname: Sternlicht
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006160:Guanosine Triphosphate / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011108:Polymers; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011108:Polymers; D014404:Tubulin; D006160:Guanosine Triphosphate