Abstract
Proper folding and assembly of tubulin alphabeta-heterodimers involves a stepwise progression mediated by a group of protein cofactors A through E. Upon release of the tubulin monomers from the chaperonin CCT, they are acted upon by each cofactor in the folding pathway through a unique combination of protein interaction domains. Three-dimensional structures have previously been reported for cofactor A and the C-terminal CAP-Gly domain of cofactor B (CoB). Here we report the NMR structure of the N-terminal domain of Caenorhabditis elegans CoB and show that it closely resembles ubiquitin as was recently postulated on the basis of bioinformatic analysis (Grynberg, M., Jaroszewski, L., and Godzik, A. (2003) BMC Bioinformatics 4, 46). CoB binds partially folded alpha-tubulin monomers, and a putative tubulin-binding motif within the N-terminal domain is identified from sequence and structure comparisons. Based on modeling of the homologous cofactor E ubiquitin-like domain, we hypothesize that cofactors B and E may associate via their beta-grasp domains in a manner analogous to the PB1 and caspase-activated deoxyribonuclease superfamily of protein interaction domains.
Highlights
The cytoskeleton of eukaryotic cells is a network of microfilaments, intermediate filaments, and microtubules
We report the NMR structure of the N-terminal domain of Caenorhabditis elegans cofactor B (CoB) and show that it closely resembles ubiquitin as was recently postulated on the basis of bioinformatic analysis (Grynberg, M., Jaroszewski, L., and Godzik, A. (2003) BMC Bioinformatics 4, 46)
We report the structure of the Caenorhabditis elegans cofactor B ubiquitin-like (CoB1 Ubl) domain, which was solved as part of a structural genomics effort directed at novel protein domains from eukaryotic organisms
Summary
Cloning—We developed an Escherichia coli-based expression system to produce the N-terminal half of tubulin folding cofactor B from C. elegans, residues 1–120. CoB, cofactor B; Ubl, ubiquitin-like; NTA, nitrilotriacetic acid; NOE, nuclear Overhauser effect; HSQC, heteronuclear single quantum coherence; r.m.s.d., root mean square deviation; CAP-Gly, glycine-rich cytoskeleton-associated protein; CAD, caspase-activated deoxyribonuclease; CoE, cofactor E; PB1, Phox and Bem; DCX, doublecortin-like domain. Fractions containing pure target protein were pooled and dialyzed into 2 ϫ 4 liters of 20 mM sodium phosphate, pH 6.5, 50 mM NaCl. Dialyzed protein was concentrated, and purity and identity were verified by SDS-PAGE and mass spectrometry. Final refinement in explicit solvent with experimental constraints and nonbonded energy terms was performed in XPLOR-NIH [20] using a recently described protocol [21] that improves the quality of NMR structures in terms of validation criteria such as Ramachandran statistics and Z-scores
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