Structural Changes, Biological Consequences, and Repurposing of Colchicine Site Ligands.

Microtubule-targeting agents (MTAs) bind to one of several distinct sites in the tubulin dimer, the subunit of microtubules. The binding affinities of MTAs may vary by several orders of magnitude, even for MTAs that specifically bind to a particular site. The first drug binding site discovered in tubulin was the colchicine binding site (CBS), which has been known since the discovery of the tubulin protein. Although highly conserved throughout eukaryotic evolution, tubulins show diversity in their sequences between tubulin orthologs (inter-species sequence differences) and paralogs (intraspecies differences, such as tubulin isotypes). The CBS is promiscuous and binds to a broad range of structurally distinct molecules that can vary in size, shape, and affinity. This site remains a popular target for the development of new drugs to treat human diseases (including cancer) and parasitic infections in plants and animals. Despite the rich knowledge about the diversity of tubulin sequences and the structurally distinct molecules that bind to the CBS, a pattern has yet to be found to predict the affinity of new molecules that bind to the CBS. In this commentary, we briefly discuss the literature evidencing the coexistence of the varying binding affinities for drugs that bind to the CBS of tubulins from different species and within species. We also comment on the structural data that aim to explain the experimental differences observed in colchicine binding to the CBS of ß-tubulin class VI (TUBB1) compared to other isotypes.

Interaction of Colchicine-Site Ligands With the Blood Cell-Specific Isotype of ß-Tubulin-Notable Affinity for Benzimidazoles.

Tubulin, the main component of microtubules, is an α-ß heterodimer that contains one of multiple isotypes of each monomer. Although the isotypes of each monomer are very similar, the beta tubulin isotype found in blood cells is significantly divergent in amino acid sequence compared to other beta tubulins. This isotype, beta class VI, coded by human gene TUBB1, is found in hematologic cells and is recognized as playing a role in platelet biogenesis and function. Tubulin from the erythrocytes of the chicken Gallus gallus contains almost exclusively ßVI tubulin. This form of tubulin has been reported to differ from brain tubulin in binding of colchicine-site ligands, previously thought to be a ubiquitous characteristic of tubulin from higher eukaryotes. In this study, we sought to gain a better understanding of the structure-activity relationship of the colchicine site of this divergent isotype, using chicken erythrocyte tubulin (CeTb) as the model. We developed a fluorescence-based assay to detect binding of drugs to the colchicine site and used it to study the interaction of 53 colchicine-site ligands with CeTb. Among the ligands known to bind at this site, most colchicine derivatives had lower affinity for CeTb compared to brain tubulin. Remarkably, many of the benzimidazole class of ligands shows increased affinity for CeTb compared to brain tubulin. Because the colchicine site of human ßVI tubulin is very similar to that of chicken ßVI tubulin, these results may have relevance to the effect of anti-cancer agents on hematologic tissues in humans.

4',6-Diamidino-2-phenylindole (DAPI) induces bundling of Escherichia coli FtsZ polymers inhibiting the GTPase activity.

FtsZ (Filamentous temperature sensitivity Z) cell division protein from Escherichia coli binds the fluorescence probe DAPI. Bundling of FtsZ was facilitated in the presence of DAPI, and the polymers in solution remained polymerized longer time than the protofilaments formed in the absence of DAPI. DAPI decreased both the maximal velocity of the GTPase activity and the Michaelis-Menten constant for GTP, indicating that behaves like an uncompetitive inhibitor of the GTPase activity favoring the GTP form of FtsZ in the polymers. The results presented in this work support a cooperative polymerization mechanism in which the binding of DAPI favors protofilament lateral interactions and the stability of the resulting polymers.

Domain folding and flexibility of Escherichia coli FtsZ determined by tryptophan site-directed mutagenesis.

FtsZ has two domains, the amino GTPase domain with a Rossmann fold, and the carboxyl domain that resembles the chorismate mutase fold. Bioinformatics analyses suggest that the interdomain interaction is stronger than the interaction of the protofilament longitudinal interfaces. Crystal B factor analysis of FtsZ and detected conformational changes suggest a connection between these domains. The unfolding/folding characteristics of each domain of FtsZ were tested by introducing tryptophans into the flexible region of the amino (F135W) and the carboxyl (F275W and I294W) domains. As a control, the mutation F40W was introduced in a more rigid part of the amino domain. These mutants showed a native-like structure with denaturation and renaturation curves similar to wild type. However, the I294W mutant showed a strong loss of functionality, both in vivo and in vitro when compared to the other mutants. The functionality was recovered with the double mutant I294W/F275A, which showed full in vivo complementation with a slight increment of in vitro GTPase activity with respect to the single mutant. The formation of a stabilizing aromatic interaction involving a stacking between the tryptophan introduced at position 294 and phenylalanine 275 could account for these results. Folding/unfolding of these mutants induced by guanidinium chloride was compatible with a mechanism in which both domains within the protein show the same stability during FtsZ denaturation and renaturation, probably because of strong interface interactions.