Microtubule-stabilizing agents (MSAs) are efficacious chemotherapeutic drugs widely used for the treatment of cancer. Despite the importance of MSAs for medical applications and basic research, their molecular mechanisms of action on tubulin and microtubules remain elusive. Here we determined high-resolution crystal structures of aß-tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxane-pocket of ß-tubulin and used their respective side chain to induce structuring of the M-loop into a short helix. Because the M-loop establishes lateral tubulin contacts in microtubules, these findings explain how taxane-site MSAs promote microtubule assembly and stability. They further offer fundamental structural insights into the control mechanisms of microtubule dynamics. Here we determined high-resolution crystal structures of -tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxanepocket of -tubulin and used their respective side chain to induce structuring of the M-loop into a short helix. Because the M-loop establishes lateral tubulin contacts in microtubules, these findings explain how taxane-site MSAs promote microtubule assembly and stability. They further offer fundamental structural insights into the control mechanisms of microtubule dynamics.One sentence summary:Microtubule-stabilizing agents use a common mechanism to stabilize a major loop in tubulin that controls microtubule assembly and stability. suggesting that binding of MSAs and TTL does not induce significant structural changes in the T 2 R complex. Both Zampa and EpoA were deeply buried in a pocket formed by predominantly hydrophobic residues of helix H7, -strand S7, and the loops H6-H7, S7-H9 (designated the Mloop (7)) and S9-S10 of -tubulin; this pocket is commonly known as the 'taxane-pocket' (8, 9)In the T 2 R-TTL-Zampa complex, the C9 atom of Zampa was covalently bound to the NE2 atom of His229 of -tubulin (Fig. S1B), which is consistent with mass spectrometry data (10). In addition, two hydrogen bonds were formed between the OH20 group and the O1' atom of Zampa, and the main chain carbonyl oxygen and the NH group of Thr276, respectively. In the T 2 R-TTL-EpoA complex, the O1, OH3, OH7 and N20 groups of EpoA were hydrogen bonded to atoms of residues Thr276 (main chain NH), Gln281 (side chain amide nitrogen), Asp226 (side chain oxygen) and Thr276 (side chain hydroxyl group) of -tubulin, respectively. The binding 4 mode of EpoA in the tubulin-EpoA structure is fundamentally different from the one proposed based on electron crystallography data of zinc-stabilized tubulin sheets (Fig. S2A); however, the orientation of the ligand in the taxane-pocket was ambiguous in the electron crystallography structure because the density of the ligand in experimental omit maps was discontinuous and limited in quality (9, 11). In contrast, the density of EpoA in our tubulin-EpoA X-ray crystal structure is very well defined and allowed the o...

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contributed equally to this work Reovirus attaches to cellular receptors with the s1 protein, a ®ber-like molecule protruding from the 12 vertices of the icosahedral virion. The crystal structure of a receptor-binding fragment of s1 reveals an elongated trimer with two domains: a compact head with a new b-barrel fold and a ®brous tail containing a triple b-spiral. Numerous structural and functional similarities between reovirus s1 and the adenovirus ®ber suggest an evolutionary link in the receptorbinding strategies of these two viruses. A prominent loop in the s1 head contains a cluster of residues that are conserved among reovirus serotypes and are likely to form a binding site for junction adhesion molecule, an integral tight junction protein that serves as a reovirus receptor. The ®brous tail is mainly responsible for s1 trimer formation, and it contains a highly¯ex-ible region that allows for signi®cant movement between the base of the tail and the head. The architecture of the trimer interface and the observed¯exi-bility indicate that s1 is a metastable structure poised to undergo conformational changes upon viral attachment and cell entry. Keywords: adenovirus/evolution/reovirus/s1/ virus±receptor interactions IntroductionReoviruses form non-enveloped, icosahedral particles (Dryden et al., 1993) that contain a segmented doublestranded (ds) RNA genome. The virions measure~850 A Ê in diameter and are composed of eight structural proteins. Five of these (l1, l2, l3, m2 and s2) form the`core' or inner capsid particle, the crystal structure of which has been determined (Reinisch et al., 2000). A second layer of proteins (m1, s1 and s3) forms the reovirus outer capsid, with m1 and s3 comprising the bulk of this capsid and s1 protruding from the 12 vertices of the icosahedron. The s3 protein, whose crystal structure is known (Olland et al., 2001), is thought to serve as a protective cap for m1, and cleavage of s3 by endosomal proteases during viral infection results in the loss of s3 and generation of infectious subvirion particles. The reovirus s1 protein serves as the viral attachment protein (Weiner et al., 1980;Lee et al., 1981). Rotary shadowing studies show that s1 is a long, ®ber-like molecule with head-and-tail morphology and several de®ned regions of¯exibility within its tail (Fraser et al., 1990). The s1 tail partially inserts into the virion via`turrets' formed by the pentameric l2 protein, whereas the s1 head projects away from the virion surface (Furlong et al., 1988;Dryden et al., 1993).Reoviruses have been isolated from many mammalian species, including humans (Tyler and Fields, 1996). Three major reovirus serotypes have been described, which are represented by the prototype strains type 1 Lang (T1L), type 2 Jones (T2J) and type 3 Dearing (T3D). Reoviruses infect most children and can cause mild respiratory or gastrointestinal illnesses (Tyler and Fields, 1996). They also serve as important models for studies of viral replication and pathogenesis and, in particular, for analysis of viral determina...

Summary Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase III clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib’s target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.

Structural analysis of a complex of tubulin and tubulin tyrosine ligase (TTL) reveals insights into TTL’s enzymatic mechanism, how it discriminates between α- and β-tubulin, and its possible evolutionary origin.

The recent success of antibody-drug conjugates (ADCs) in the treatment of cancer has led to a revived interest in microtubuledestabilizing agents. Here, we determined the high-resolution crystal structure of the complex between tubulin and maytansine, which is part of an ADC that is approved by the US Food and Drug Administration (FDA) for the treatment of advanced breast cancer. We found that the drug binds to a site on β-tubulin that is distinct from the vinca domain and that blocks the formation of longitudinal tubulin interactions in microtubules. We also solved crystal structures of tubulin in complex with both a variant of rhizoxin and the phase 1 drug PM060184. Consistent with biochemical and mutagenesis data, we found that the two compounds bound to the same site as maytansine and that the structures revealed a common pharmacophore for the three ligands. Our results delineate a distinct molecular mechanism of action for the inhibition of microtubule assembly by clinically relevant agents. They further provide a structural basis for the rational design of potent microtubuledestabilizing agents, thus opening opportunities for the development of next-generation ADCs for the treatment of cancer.drug mechanism | microtubule-targeting agents | X-ray crystallography

Laulimalide and peloruside A are microtubule-stabilizing agents (MSAs), the mechanism of action on microtubules of which is poorly defined. Here, using X-ray crystallography it is shown that laulimalide and peloruside A bind to a unique non-taxane site on β-tubulin and use their respective macrolide core structures to interact with a second tubulin dimer across protofilaments. At the same time, they allosterically stabilize the taxane-site M-loop that establishes lateral tubulin contacts in microtubules. Structures of ternary complexes of tubulin with laulimalide/peloruside A and epothilone A are also solved, and a crosstalk between the laulimalide/peloruside and taxane sites via the M-loop of β-tubulin is found. Together, the data define the mechanism of action of laulimalide and peloruside A on tubulin and microtubules. The data further provide a structural framework for understanding the synergy observed between two classes of MSAs in tubulin assembly and the inhibition of cancer cell growth.

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