Highlights

2018

A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine-site of tubulin; Nat. Comm.(2018)

Microtubule-targeting agents (MTAs) like taxol and vinblastine are amongst the most successful chemotherapeutic drugs against cancer. In this study, we developed a fluorescence anisotropy-based assay that specifically probes for ligands targeting the maytansine-site of tubulin, which has been discovered by us recently. Using this assay, we have determined the dissociation constants of known maytansine-site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine-site on beta-tubulin. The high resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next generation MTAs for the treatment of cancer.

Structure-function relationship of the Bik1-Bim1 complex; Structure (2018)

The budding yeast microtubule plus-end tracking proteins Bik1 (CLIP-170) and Bim1 (EB1) form a complex to interact with partners involved in spindle alignment, including Stu2 (XMAP215/ch-TOG) and Kar9 (putative APC). In this study, we show that the CAP-Gly and coiled-coil domains of Bik1 interact with the C-terminal ETF peptide of Bim1 and the C-terminal tail region of Stu2, respectively, with low micromolar affinities. The crystal structures of the CAP-Gly domain of Bik1 (Bik1CG) alone and in complex with an ETF peptide revealed unique secondary structure CAP-Gly elements and establishes Bik1CG as the first specific C-terminal phenylalanine recognition domain. We further found that Bik1-Bim1 can form ternary complexes with EB1-binding SxIP or LxxPTPh motifs present in diverse proteins, including Kar9. Perturbation of the Bik1-Bim1 interaction in vivo affected localization of Bik1 and astral microtubule length. Together, this study provides insight into the role of the Bik1-Bim1 interaction for cell division.

2017

Deconvolution of Buparlisib's mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention; Nature Comm. (2017)

BKM120 (Buparlisib) is one of the most advanced phosphoinositide 3-kinase (PI3K) inhibitors for the treatment of cancer, but evidence exists that it interferes with microtubule polymerization. We developed two derivatives that differ from BKM120 by only one Dalton, and show that these minute changes separate the dual-activity of BKM120 into discrete PI3K and tubulin inhibitors. Analysis of cellular growth arrest phenotypes and microtubule dynamics suggest that the anti-proliferative activity of BKM120 is mainly due to a cytotoxic effect rather than through inhibition of PI3K. Crystal structures of BKM120 variants in complex with tubulin and PI3K provide insights into the selective mode of action of this class of drugs. Our results provide a unique basis to develop the next generation of PI3K inhibitors with improved safety profiles and flexibility for the use in combination therapies.

Microtubule minus-end regulation at spindle poles by an ASPM-katanin complex; Nature Cell Biol. (2017)

ASPM is a microcephaly-associated protein family that regulates spindle architecture, but the underlying mechanism is poorly understood. Here, we show that ASPM forms a complex with another protein linked to microcephaly, the microtubule-severing ATPase katanin. ASPM and katanin localize to spindle poles in a mutually dependent manner and regulate spindle flux. X-ray crystallography revealed that the heterodimer formed by the N- and C-terminal domains of the katanin subunits p60 and p80, respectively, binds conserved motifs in ASPM. Reconstitution experiments demonstrated that ASPM autonomously tracks growing microtubule minus ends and inhibits their growth, while katanin decorates and bends both ends of dynamic microtubules and potentiates the minus-end blocking activity of ASPM. ASPM also binds along microtubules, recruits katanin and promotes katanin-mediated severing of dynamic microtubules. We propose that the ASPM-katanin complex controls microtubule disassembly at spindle poles and that misregulation of this process can lead to microcephaly.

A novel sequence motif targets diverse proteins to growing microtubule ends; Structure (2017)

Microtubule plus-end tracking proteins (+TIPs) are involved in virtually all microtubule-based processes. End binding (EB) proteins are considered master regulators of +TIP interaction networks since they autonomously track growing microtubule ends and recruit a plethora of proteins to this location. We discovered and characterized two major EB-interacting elements in the past: CAP-Gly domains and linear SxIP sequence motifs. We now identified LxxPTPh as a novel EB-binding motif that enables major +TIPs to interact with EBs at microtubule ends. In contrast to EB-SxIP and EB-CAP-Gly, the EB-LxxPTPh binding mode does not depend on the C-terminal tail region of EB. Our study reveals that +TIPs developed additional strategies besides CAP-Gly and SxIP to target EBs at growing microtubule ends. They further provide a unique basis to discover novel +TIPs, and to dissect the role of key interaction nodes and their differential regulation for hierarchical +TIP network organization and function in diverse eukaryotic organisms.

2016

Centriolar CPAP/SAS-4 imparts slow processive microtubule growth; Dev. Cell (2016)

Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious. Here, we bring together crystallographic, biophysical and reconstitution assays to demonstrate that the human centriolar protein CPAP (SAS-4 in worms and flies) binds and “caps” microtubule plus-ends by associating with a site of β-tubulin engaged in longitudinal tubulin-tubulin interactions. Strikingly, we uncover that CPAP activity dampens microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues. We further establish that the capping function of CPAP is important to limit growth of centriolar microtubules in cells. Our results suggest that CPAP acts as a molecular lid that ensures slow assembly of centriolar microtubules and, thereby, contributes to organelle length control.

Termination of protofilament elongation by Eribulin induces lattice defects that promote microtubule catastrophes; Curr. Biol. (2016)

Microtubules are dynamic polymers built of tubulin dimers that attach in a head-to-tail fashion to form protofilaments, which further associate laterally to form a tube. Asynchronous elongation or curling of individual protofilaments can potentially lead to an altered microtubule-end structure that promotes sudden depolymerization, termed catastrophe. However, how the dynamics of individual protofilaments relates to overall growth persistence has remained unclear. Here we used the microtubule targeting anti-cancer drug Eribulin to explore the consequences of stalled protofilament elongation on microtubule growth. Using X-ray crystallography, we first revealed that Eribulin binds to a site on β-tubulin that is required for protofilament plus-end elongation. Based on the structural information, we engineered a fluorescent Eribulin molecule. We demonstrate that single Eribulin molecules specifically interact with microtubule plus-ends and are sufficient to either trigger a catastrophe or induce slow and erratic microtubule growth in the presence of EB3. Interestingly, we found that Eribulin increases the frequency of EB3 comet “splitting”, transient events where a slow and erratically progressing comet is followed by a faster comet. This observation possibly reflects the healing of a microtubule lattice. Because comet splitting was also observed in control microtubules in the absence of any drugs, we propose that Eribulin amplifies a natural pathway towards catastrophe by promoting the arrest of protofilament elongation.

SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture; Nat. Cell Biol. (2016)

Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a 9-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from 5- to 10-fold. Expression of a SAS-6 mutant that forms 6-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with 8- or 9-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, 6- to 8-fold symmetric cartwheels were observed, while the microtubule wall maintained 8- and 9-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in 9-fold symmetric centrioles that displayed impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.

2014

A new tubulin-binding site and pharmacophore for microtubule-destabilizing anticancer drugs; Proc. Natl. Acad. Sci. USA (2014)

The recent success of antibody-drug conjugates (ADCs) in the treatment of cancer has led to a revived interest in microtubule-destabilizing agents (MDAs). We determined the high resolution crystal structure of the complex between tubulin and maytansine, which is part of an ADC that is FDA-approved for the treatment of advanced breast cancer. We found that the drug binds to a site on -tubulin that is distinct from the vinca domain and 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 I drug PM060184. Consistent with biochemical and mutagenesis data, we found that the two compounds bound to the same site as maytansine and the structures revealed a common pharmacophore for the three ligands. Our results delineate a novel 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 MDAs, thus opening new opportunities for the development of next-generation ADCs for the treatment of cancer.

2013

Structural basis of tubulin tyrosination by tubulin tyrosine ligase; J. Cell Biol. (2013)

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 tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxane-pocket 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.

Molecular Mechanism of Action of Microtubule-Stabilizing Anticancer Agents; Science (2013)

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 tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxane-pocket 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.

2011

Structural basis of the nine-fold symmetry of centrioles; Cell (2011)

The centriole, and the related basal body, is an ancient organelle characterized by a universal 9-fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are not understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles.

2010

Molecular basis of coiled-coil oligomerization; Proc. Natl. Acad. Sci. USA (2010)

Coiled coils are extensively and successfully used nowadays to rationally design multi-stranded structures for applications, including basic research, biotechnology, nanotechnology, material science and medicine. The wide range of applications as well as the important functions these structures play in almost all biological processes highlight the need for a detailed understanding of the factors that control coiled-coil folding and oligomerization. Here, we address the important and unresolved question why the presence of particular oligomerization-state determinants within a coiled coil does frequently not correlate with its topology. We found an unexpected, general link between coiled-coil oligomerization-state specificity and trigger sequences, elements that are indispensable for coiled-coil formation. By using the archetype coiled-coil domain of the yeast transcriptional activator GCN4 as a model system, we show that well-established trimer-specific oligomerization-state determinants only switch the peptide’s topology from a dimer to a trimer when inserted into the trigger sequence. We successfully confirmed our results in two other, unrelated coiled-coil dimers, ATF1 and cortexillin-1. We furthermore show that multiple topology determinants can co-exist in the same trigger sequence, revealing a delicate balance of the resulting oligomerization state by position-dependent forces. Our experimental results should significantly improve the prediction of the oligomerization state of coiled coils, an issue that is still largely unresolved. They therefore should have major implications for the rational design of coiled coils and consequently all applications using these popular oligomerization domains.

2009

Discovery of a microtubule tip localization signal; Cell (2009)

Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, migration and morphogenesis. EB1 and its homologues are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general 'microtubule tip localization signal' (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process.

2007

Establishing the structure-function relationship of CAP-Gly domains; Nat. Struct. Mol. Biol. (2007)

In all eukaryotes, CAP-Gly proteins control important cellular processes. The molecular mechanisms underlying the functions of CAP-Gly domains, however, are still poorly understood. Here we use the complex formed between the CAP-Gly domain of dynactin/p150glued and the C-terminal zinc knuckle of CLIP170 as a model system to explore the structure-function relationship of CAP-Gly mediated protein interactions. We demonstrate that the conserved GKNDG motif of CAP-Gly domains is responsible for targeting to the C-terminal EEY/F sequence motifs of CLIP170, EB proteins, and microtubules. The CAP-Gly-EEY/F interaction is essential for the recruitment of dynactin/p150glued by CLIP170 and for activation of CLIP170. Our findings define the molecular basis of CAP-Gly domain function, including the tubulin detyrosination/tyrosination cycle. They further establish fundamental roles for the interaction between CAP-Gly proteins and C-terminal EEY/F sequence motifs in regulating complex and dynamic cellular processes.

Molecular basis of the specific inhibition of protein kinase G from Mycobacterium tuberculosis; Proc. Natl. Acad. Sci. USA (2007)

The pathogenicity of mycobacteria such as Mycobacterium tuberculosis is closely associated with their capacity to survive within host macrophages. A crucial virulence factor for intracellular mycobacterial survival is PknG, a eukaryotic-like serine/threonine protein kinase that blocks the intracellular degradation of mycobacteria in lysosomes. Inhibiting PknG with the highly selective low molecular weight inhibitor AX20017 results in mycobacterial transfer to lysosomes and killing of the mycobacteria. Here we report the 2.4 Å X-ray crystal structure of PknG in complex with AX20017. The unique multidomain topology of PknG reveals a central kinase domain that is flanked by N- and C-terminal rubredoxin and tetratrico-peptide repeat domains, respectively. Directed mutagenesis suggests that the rubredoxin domain functions as a regulator of PknG kinase activity. The structure of PknG-AX20017 further reveals that the inhibitor is buried deep within the adenosine binding site, targeting an active conformation of the kinase domain. Remarkably, while the topology of the kinase domain is reminiscent of eukaryotic kinases, the AX20017 binding pocket is shaped by a unique set of amino acid side chains which are not found in any human kinase. Directed mutagenesis of the unique set of residues resulted in a drastic loss of the compound's inhibitory potency. Our results explain the specific mode of action of AX20017 and demonstrate that virulence factors highly homologous to host molecules can be successfully targeted to block the proliferation of Mycobacterium tuberculosis.

Molecular basis of coiled-coil formation; Proc. Natl. Acad. Sci. USA (2007)

Coiled coils have attracted considerable interest as design templates in a wide range of applications. Successful coiled-coil design strategies therefore require a detailed understanding of coiled-coil folding. One common feature shared by coiled coils is the presence of a short autonomous helical folding unit, termed ‘trigger sequence’, that is indispensable for folding. Detailed knowledge of trigger sequences at the molecular level is thus key to a general understanding of coiled-coil formation. Using a multidisciplinary approach we identify and characterize here the molecular determinants that specify the helical conformation of the monomeric early folding intermediate of the GCN4 coiled coil. We demonstrate that a network of hydrogen bonding and electrostatic interactions stabilize the trigger-sequence helix. This network is rearranged in the final dimeric coiled-coil structure, and its destabilization significantly slows down GCN4 leucine zipper folding. Our findings provide a general explanation for the molecular mechanism of coiled-coil formation.

2006

Deciphering key interaction modes of dynamic +TIP networks; Mol. Cell (2006)

Dynamic microtubule plus-end tracking protein (+TIP) networks are implicated in all functions of microtubules, but their nature and molecular determinants of their interactions are largely unknown. Here we have used a multimodular system to explore key binding modes of +TIP interactions. X-ray crystallography and calorimetry combined with sequence information define the specificity determinants of CAP-Gly domains for binding EB proteins. Importantly, they establish that CAP-Gly domains are carboxy terminal EEY/F-COO- motif-recognition domains. EEY/F-COO- motifs were found to represent specific sequence signals of EB, CLIP-170, and a-tubulin, playing a key functional role in these major proteins. Our findings provide a basis for understanding interaction modes between a-tubulin, CLIP-170, EB proteins, and the dynactin/dynein motor complex and suggest that low-affinity multifactorial interactions control dynamic +TIP networks at microtubule ends. They further offer a basis for understanding genetic CAP-Gly domain defects found in fatal human disorders.

2005

Discovery of a trimerization motif that controls the topology of short coiled coils; Proc. Natl. Acad. Sci. USA (2005)

The potential of short coiled coils for protein engineering, biotechnological, biomaterial, basic research, and medical applications has recently been recognized. For many of these applications knowledge of the factors that control the topology of the engineered protein systems is essential. Here we demonstrate that trimerization of short coiled coils is determined by a distinct structural motif that encompasses specific networks of surface salt bridges and optimal hydrophobic packing interactions. The motif is conserved among intracellular, extracellular, viral, and synthetic proteins and defines a general molecular determinant for trimer formation of short coiled coils. In addition to being of particular interest for the biotechnological production of candidate therapeutic proteins, these findings may also be of interest for viral drug development strategies.

2004

Exploring amyloid formation by a de novo design; Proc. Natl. Acad. Sci. USA (2004)

Protein deposition as amyloid fibrils underlies many debilitating human disorders. The complexity and size of disease-related polypeptides, however, often hinders a detailed rational approach to study effects that contribute to the process of amyloid formation. We report here a simplified peptide sequence successfully designed de novo to fold into a coiled-coil conformation under ambient conditions but to transform into amyloid fibrils at elevated temperatures. We have determined the crystal structure of the coiled coil form and propose a detailed molecular model for the peptide in its fibrillar state. The relative stabilities of the two structural forms and the kinetics of their interconversion were found to be highly sensitive to small sequence changes. The results reveal the importance of specific packing interactions on the kinetics of amyloid formation and show the potential of this exceptionally favourable system for probing details of the molecular origins of amyloid disease.