title: Microtubule assembly is dependent on a cluster of basic residues in alpha-tubulin.
abstract: Previous studies have shown that tubulin, a major protein component of the microtubule, is rendered assembly incompetent when a highly reactive lysine residue (HRL) in the alpha polypeptide of tubulin dimer is reductively methylated [cf. Sherman, G., Rosenberry, T. L., & Sternlicht, H. (1983) J. Biol. Chem. 258, 2148-2156]. In this study we demonstrate that the HRL in bovine brain tubulin is Lys-394, a residue proximal in the alpha-tubulin sequence to the highly negatively charged carboxy-terminus region (residues 412-450) previously implicated in assembly. pH studies were undertaken to probe the local environment of Lys-394. These studies indicated that Lys-394 reactivity toward HCHO is sensitive to the titration of a pKa 6.3 group presumed to be a histidine residue. This assignment is supported by our finding that histidine modification via diethyl pyrocarbonate strongly affects Lys-394 reactivity toward HCHO as well as microtubule assembly. We propose on the basis of secondary structure considerations and published sequence data for a variety of tubulins that Lys-394 is part of an evolutionarily conserved cluster of basic residues (effective charge: 2+ to 2.5+ at neutral pH) composed of Lys-394, His-393, and Arg-390, which is important for tubulin function and which renders Lys-394 reactive as a nucleophile.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D003488:Cyanogen Bromide; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine; D007700:Kinetics; D008745:Methylation; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D051381:Rats; D012568:Schizosaccharomyces / Q000378:metabolism; D013045:Species Specificity; D013552:Swine; D014404:Tubulin / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D014404:Tubulin; D006639:Histidine; D004047:Diethyl Pyrocarbonate; D003488:Cyanogen Bromide