The Local Electrostatic Environment Determines Cysteine Reactivity of Tubulin

Abstract

Of the 20 cysteines of rat brain tubulin, some react rapidly with sulfhydryl reagents, and some react slowly. The fast reacting cysteines cannot be distinguished with [ 14 C]iodoacetamide, N-[ 2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid]), since modification to mole ratios < <1 cysteine/dimer always leads to labeling of 6 -7 cysteine residues. These have been identified as Cys-305␣, Cys-315␣, Cys-316␣, Cys-347␣, Cys-376␣, Cys-241␤, and Cys-356␤ by mass spectroscopy and sequencing. This lack of specificity can be ascribed to reagents that are too reactive; only with the relatively inactive chloroacetamide could we identify Cys-347␣ as the most reactive cysteine of tubulin. Using the 3.5-Å electron diffraction structure, it could be shown that the reactive cysteines were within 6.5 Å of positively charged arginines and lysines or the positive edges of aromatic rings, presumably promoting dissociation of the thiol to the thiolate anion. By the same reasoning the inactivity of a number of less reactive cysteines could be ascribed to inhibition of modification by negatively charged local environments, even with some surface-exposed cysteines. We conclude that the local electrostatic environment of cysteine is an important, although not necessarily the only, determinant of its reactivity. C]ethylmaleimide, or IAE-DANS ([5-((((The 20 SH groups of the tubulin dimer have long led to speculation as to their function. Requirements for a few of the SH groups have been identified. Thus, Cys-12␤ is near the binding site of the exchangeable GTP of ␤-tubulin (1), and a C12S␤ mutation is lethal in haploid yeast, although a C12A␤ mutation is survivable (2). Cys-241␤ 1 and Cys-356␤ are near or are part of the binding site for colchicine and other agents (3)(4)(5). What the precise role of the involvement of these cysteines may be is for the most part not clear. Some of the SH groups of tubulin form thioesters with palmitic acid both in vivo and in vitro; these may be responsible for membrane localization of tubulin (6 -9). One of these has been located as Cys-376␣ (10). Except for this palmitoylation site, no specific functions have been identified for the 12 SH groups of ␣-tubulin, and the order of reactivities of the SH groups has not been definitively established. It has been repeatedly demonstrated (11) that reaction of an equivalent of 1 or 2 SH groups with the usual alkylating agents abolished polymerization competence, but their location in the sequence has not been unambiguously determined. Loss of colchicine binding requires modification of additional SH groups by these nonspecific SH reagents. For this reason we have approached the reactivity of tubulin SH groups in a more general sense, comparing the effects of thioether, disulfide, and thioester formation as well as their location in ␣-tubulin and ␤-tubulin.Protein sulfhydryl groups can be involved in numerous reactions such as oxidation, disulfide interchange, thioether, and thioester formation. For the purpose of this discussion, we shall exclude oxid...

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