Selection and Characterization of Artificial Proteins Targeting the Tubulin α Subunit

• Published in: Structure (London) 2019-03, Vol.27 (3), p.497-506.e4
• Main Authors: Campanacci, Valérie, Urvoas, Agathe, Consolati, Tanja, Cantos-Fernandes, Soraya, Aumont-Nicaise, Magali, Valerio-Lepiniec, Marie, Surrey, Thomas, Minard, Philippe, Gigant, Benoît
• Format: Article
• Language: English
• Subjects: Animals; artificial protein; Biochemistry; Biochemistry, Molecular Biology; Cellular Biology; Crystallography, X-Ray; Dose-Response Relationship, Drug; Humans; in vitro selection; Life Sciences; microtubule; Microtubules - drug effects; Microtubules - metabolism; Models, Molecular; Peptide Library; Protein Conformation; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - pharmacology; Repetitive Sequences, Amino Acid; Structural Biology; tubulin; Tubulin - chemistry; Tubulin - metabolism; αRep
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2018.12.001

Microtubules are cytoskeletal filaments of eukaryotic cells made of αβ-tubulin heterodimers. Structural studies of non-microtubular tubulin rely mainly on molecules that prevent its self-assembly and are used as crystallization chaperones. Here we identified artificial proteins from an αRep library that are specific to α-tubulin. Turbidity experiments indicate that these αReps impede microtubule assembly in a dose-dependent manner and total internal reflection fluorescence microscopy further shows that they specifically block growth at the microtubule (−) end. Structural data indicate that they do so by targeting the α-tubulin longitudinal surface. Interestingly, in one of the complexes studied, the α subunit is in a conformation that is intermediate between the ones most commonly observed in X-ray structures of tubulin and those seen in the microtubule, emphasizing the plasticity of tubulin. These α-tubulin-specific αReps broaden the range of tools available for the mechanistic study of microtubule dynamics and its regulation. [Display omitted] •Selection of α-tubulin-specific artificial αRep proteins•The αReps inhibit microtubule assembly and specifically block growth at the (−) end•The αReps target the longitudinal surface of α-tubulin•The αReps are useful tools for the mechanistic study of microtubule dynamics Campanacci et al. have selected artificial proteins, termed αReps, that bind to the tubulin alpha subunit. These αReps inhibit microtubule assembly with a specific blocking effect at the slower growing microtubule minus end. The structure of tubulin-αRep complexes gives a rationale for this mechanism.