Himan Sternlicht

Site-directed mutagenesis of the GTP-binding domain of β-tubulin

Abstract Tubulin binds guanine nucleotides with high affinity and specificity. GTP, an allosteric effector of microtubule assembly, requires Mg2+ for its interaction with β-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 β-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 β-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 β-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.

Theory for modeling the copolymerization of tubulin and tubulin-colchicine complex

Substoichiometric concentrations of tubulin-colchicine complex (TC) inhibits microtubule assembly through a copolymerization reaction between tubulin and TC. We have determined the rates and extent of TC incorporation into bovine brain microtubules and developed a theory that models copolymerization. Our analysis suggests that while the apparent association rate constants for tubulin and TC are similar, the apparent dissociation rate constants for TC are a factor of five or more larger than those of tubulin. Copolymer composition showed only slight changes during assembly despite changes in the solution phase and showed little dependence at high TC upon the initial tubulin concentration. The theory was based on coupled Oosawa-Kasai equations that allow for the co-assembly of two components, tubulin and TC. An expression was derived that relates copolymer composition to reaction mixture composition and to the affinity of microtubule ends for tubulin and TC. This expression predicts copolymer composition at TC concentrations less than 10 microM and correlates composition with assembly inhibition. We perceive copolymerization as a facilitated incorporation of TC requiring the presence of tubulin. TC incorporation was dependent on the ratio of total tubulin to the dissociation constant for TC bound to microtubule ends. The copolymerization reaction is thus characterized by an interplay of two effects (a) where tubulin facilitates the incorporation of TC into the microtubule, and (b) where TC inhibits the assembly of tubulin into microtubules.

Site-directed mutagenesis of alpha-tubulin. Reductive methylation studies of the Lys 394 region.

Previous studies have implicated at least two regions in alpha-tubulin that are important for the regulation of microtubule assembly. These regions include a cluster of basic residues consisting of Arg 390, His 393, and Lys 394 and the highly acidic carboxyl terminus. Lys 394 is highly reactive to HCHO and NaCNBH3. The reductive methylation of Lys 394 by these reagents is thought to be responsible for the profound inhibitory effects of low concentrations of HCHO on microtubule assembly (cf. Szasz J., M. B. Yaffe, M. Elzinga, G. S. Blank, and H. Sternlicht. 1986. Biochemistry. 25:4572-4582). In this study we reexamined the basis for this inhibition. Lys 394 in a human keratinocyte alpha-tubulin (k alpha 1) was replaced by a glutamic acid residue using site-directed mutagenesis. The mutant K394E was synthesized in vitro using rabbit reticulocyte lysates, and its ability to coassemble with bovine brain microtubule protein (MTP) before and after reaction with HCHO and NaCNBH3 was compared with that of wild-type. No differences in the coassemblies of the unmethylated proteins were detected suggesting that Lys 394 is not essential for microtubule assembly. However, methylated K394E prepared at low HCHO concentrations (< 1 mM) incorporated into microtubules to a greater extent (approximately 30-40%) than methylated wild-type. This result is consistent with the hypothesis that methylation of Lys 394 interferes with microtubule assembly. However, the extent of protection afforded by the replacement of Lys 394 with Glu 394 was less than half as large as that predicted from the earlier studies. We tentatively conclude that another residue(s) besides Lys 394 contributes significantly to the assembly-inhibition observed with low concentrations of HCHO. Since this residue(s) is less reactive than Lys 394, it would have to inhibit assembly substoichiometrically when methylated. Potential candidates for this residue include bulk lysyl residue(s), a lysyl residue(s) with intermediate reactivity toward HCHO, and the NH2-termini. The NH2-termini are especially attractive candidates since they appear to have a structural role in microtubule assembly.

A kinetic model for colchicine inhibition of microtubule assembly

Colchicine is a potent drug used to probe microtubule dependent processes. We have recently shown that substoichiometric concentrations of colchicine-tubulin complex (CD), a 1:1 tight binding complex of drug with tubulin, copolymerizes with tubulin to form microtubule copolymers. The affinity of the microtubule ends for tublin decreased as the CD mole fraction in the microtubule increased. Mole fraction ratios as small as 1 CD to approx. 50 to 100 tubulins in the copolymers were accompanied by a significant change in binding affinities and polymerization rates. We have further extended our investigation of the CD-tubulin copolymerization reaction. A kinetic model was derived which relates the composition of the microtubule copolymer to the composition of the reaction mixture. This model allowed a predictive correlation to be made between copolymer composition and the extent of assembly inhibition.