SSNA1 stabilizes dynamic microtubules and detects microtubule damage

Sjögren's Syndrome Nuclear Autoantigen 1 (SSNA1/NA14) is a microtubule-associated protein with important functions in cilia, dividing cells and developing neurons. However, the direct effects of SSNA1 on microtubules are not known. We employed in vitro reconstitution with purified proteins and TIRF microscopy to investigate the activity of human SSNA1 on dynamic microtubule ends and lattices. Our results show that SSNA1 modulates all parameters of microtubule dynamic instability - slowing down the rates of growth, shrinkage and catastrophe, and promoting rescue. We find that SSNA1 forms stretches along growing microtubule ends and binds cooperatively to the microtubule lattice. Furthermore, SSNA1 is enriched on microtubule damage sites, occurring both naturally, as well as induced by the microtubule severing enzyme spastin. Finally, SSNA1 binding protects microtubules against spastin's severing activity. Taken together, our results demonstrate that SSNA1 is both a potent microtubule stabilizing protein and a novel sensor of microtubule damage; activities that likely underlie SSNA1's functions on microtubule structures in cells.

Chemoinformatics Analyses of Tau Ligands Reveal Key Molecular Requirements for the Identification of Potential Drug Candidates against Tauopathies

Tau is a highly soluble protein mainly localized at a cytoplasmic level in the neuronal cells, which plays a crucial role in the regulation of microtubule dynamic stability. Recent studies have demonstrated that several factors, such as hyperphosphorylation or alterations of Tau metabolism, may contribute to the pathological accumulation of protein aggregates, which can result in neuronal death and the onset of a number of neurological disorders called Tauopathies. At present, there are no available therapeutic remedies able to reduce Tau aggregation, nor are there any structural clues or guidelines for the rational identification of compounds preventing the accumulation of protein aggregates. To help identify the structural properties required for anti-Tau aggregation activity, we performed extensive chemoinformatics analyses on a dataset of Tau ligands reported in ChEMBL. The performed analyses allowed us to identify a set of molecular properties that are in common between known active ligands. Moreover, extensive analyses of the fragment composition of reported ligands led to the identification of chemical moieties and fragment combinations prevalent in the more active compounds. Interestingly, many of these fragments were arranged in recurring frameworks, some of which were clearly present in compounds currently under clinical investigation. This work represents the first in-depth chemoinformatics study of the molecular properties, constituting fragments and similarity profiles, of known Tau aggregation inhibitors. The datasets of compounds employed for the analyses, the identified molecular fragments and their combinations are made publicly available as supplementary material.

Abstract P-33: Structural Basis for the Growing Microtubule End Recognition by End Binding Proteins

Background: Eukaryotic end binding proteins (EBs) can follow the growing microtubule end. EBs play a crucial role in microtubule dynamic instability and promote simultaneously growth rate and catastrophe frequency. It makes EB-like proteins perspective drag targets for a wide number of diseases. But the molecular mechanism of tip tracking by EB-like proteins remains unknown. Studies of mutants have revealed that the conservative amino acid Q102 (numbering relative to the human EB1 protein) plays a key role in the recognition of the growing microtubule end. However, the 3D structure studies revealed that this amino acid has no bonds with tubulin. In this work, we performed structural and phylogenetic analysis of EBs proteins to identify a possible molecular mechanism behind the plus end tracking. Methods: UCSF Chimera10 was used for structural analysis. Phylogenetic analysis was performed with MEGA X software. 3D structures of EBs and microtubules with different states of GTP hydrolysis were used (pdb 3JAK, 3JAS, 3JAT, 3JAW, 3JAL, 3JAR, 6DPU, 6DPV, 6DPW). Results: We have shown that two conservative amino acids (K100, E106) should play an important role in the recognition of the microtubule plus end in addition to Q102. It was concluded that these amino acids together form the plus-end «navigation site» of EBs. Analysis of possible interaction of the «navigation site» amino acids with microtubules in different conformational states suggested that the main mechanism of growing microtubule end recognition is not due to an affinity increase for a certain state of tubulin in microtubules at their end, but it due to a significant affinity decrease in other parts of the microtubule as a result of steric clashes. Conclusion: Thus, the results of the analysis suggested the possible molecular mechanism that provides the tip tracking by EB-like proteins and allowed us to identify the key amino acids of this mechanism.

SSNA1 stabilizes dynamic microtubules and detects microtubule damage

ABSTRACTSjögren’s Syndrome Nuclear Autoantigen 1 (SSNA1/NA14) is a microtubule-associated protein with important functions in cilia, dividing cells and developing neurons. However, the direct effects of SSNA1 on microtubules are not known. We employed in vitro reconstitution with purified proteins and TIRF microscopy to investigate the activity of human SSNA1 on dynamic microtubule ends and lattices. We find that SSNA1 modulates all parameters of microtubule dynamic instability – slowing down the rates of growth, shrinkage and catastrophe, and promoting rescue. SSNA1 accumulation on dynamic microtubule ends correlates with the growth rate slow-down. Furthermore, SSNA1 prevents catastrophe when soluble tubulin is removed or sequestered by Op18/Stathmin. Finally, SSNA1 detects spastin-induced damage and inhibits spastin’s severing activity. Therefore, SSNA1 is both a potent microtubule stabilizing protein and a sensor of microtubule damage; activities that likely underlie SSNA1’s cellular functions.

Atomistic Basis of Microtubule Dynamic Instability Assessed Via Multiscale Modeling

Microtubule “dynamic instability,” the abrupt switching from assembly to disassembly caused by the hydrolysis of GTP to GDP within the β subunit of the αβ-tubulin heterodimer, is necessary for vital cellular processes such as mitosis and migration. Despite existing high-resolution structural data, the key mechanochemical differences between the GTP and GDP states that mediate dynamic instability behavior remain unclear. Starting with a published atomic-level structure as an input, we used multiscale modeling to find that GTP hydrolysis results in both longitudinal bond weakening (~ 4 kBT) and an outward bending preference (~ 1.5 kBT) to both drive dynamic instability and give rise to the microtubule tip structures previously observed by light and electron microscopy. More generally, our study provides an example where atomic level structural information is used as the sole input to predict cellular level dynamics without parameter adjustment.

Cevipabulin-tubulin complex reveals a novel agent binding site on α-tubulin and provides insights into microtubule dynamic instability

Microtubule, composed of αβ-tubulin heterodimers, remains as one of the most popular anticancer targets for decades. To date, anti-microtubule drugs mainly target β-tubulin to inhibit microtubule dynamic instability (MDI) while agents binding to α-tubulin are less well characterized and also the molecular mechanism of MDI is far from being articulated. Cevipabulin, an oral microtubule-active antitumor clinical candidate, is widely accepted as a microtubule stabilizing agent (MSA) but binds to the microtubule-destabilization vinblastine site on β-tubulin and this unusual phenomenon has so far failed to be explained. Our X-ray crystallography study reveals that, in addition binding to the vinblastine site, cevipabulin also binds to a novel site on α-tubulin (named the seventh site) which located at the region spatially corresponding to the vinblastine site on β-tubulin. Interestingly, cevipabulin exhibits two unique site-dependent functions. Cevipabulin binding to the seventh site promotes tubulin degradation through interaction of the non-exchengeable GTP to reduce tubulin stability. Cevipabulin binding to the vinblastine site enhances longitudinal interactions but inhibits lateral interactions of tubulins, thus inducing tubulin protofilament polymerization (but not microtubule polymerization like MSAs), and then tangling into irregular tubulin aggregates. Importantly, the tubulin-cevipabulin structure is an intermediate between “bent” and “straight” tubulins and the involved bent-to-straight conformation change will be helpful to fully understand the molecular mechanism of tubulin assembly. Our findings confirm cevipabulin is not an MSA and shed light on the development of a new generation of anti-microtubule drugs targeting the novel site on α-tubulin and also provide new insights into MDI.

Atomistic basis of microtubule dynamic instability assessed via multiscale modeling

Microtubule “dynamic instability,” the abrupt switching from assembly to disassembly caused by the hydrolysis of GTP to GDP within the β subunit of the αβ-tubulin heterodimer, is necessary for vital cellular processes such as mitosis and migration. Despite existing high-resolution structural data, the key mechanochemical differences between the GTP and GDP states that mediate dynamic instability behavior remain unclear. Starting with a published atomic-level structure as an input, we used multiscale modeling to find that GTP hydrolysis results in both longitudinal bond weakening (~4 kBT) and an outward bending preference (~1.5 kBT) to both drive dynamic instability and give rise to the microtubule tip structures previously observed by light and electron microscopy. More generally, our study provides an example where atomic level structural information is used as the sole input to predict cellular level dynamics without parameter adjustment.