Selected research achievements
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.
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.
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.
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.
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.
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.
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.
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.
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.