The architecture of Trypanosoma brucei tubulin-binding cofactor B and implications for function

Abstract

Tubulin-binding cofactor (TBC)-B is implicated in the presentation of α-tubulin ready to polymerize, and at the correct levels to form microtubules. Bioinformatics analyses, including secondary structure prediction, CD, and crystallography, were combined to characterize the molecular architecture of Trypanosoma brucei TBC-B. An efficient recombinant expression system was prepared, material-purified, and characterized by CD. Extensive crystallization screening, allied with the use of limited proteolysis, led to structures of the N-terminal ubiquitin-like and C-terminal cytoskeleton-associated protein with glycine-rich segment domains at 2.35-Å and 1.6-Å resolution, respectively. These are compact globular domains that appear to be linked by a flexible segment. The ubiquitin-like domain contains two lysines that are spatially conserved with residues known to participate in ubiquitinylation, and so may represent a module that, through covalent attachment, regulates the signalling and/or protein degradation associated with the control of microtubule assembly, catastrophe, or function. The TBC-B C-terminal cytoskeleton-associated protein with glycine-rich segment domain, a known tubulin-binding structure, is the only such domain encoded by the T. brucei genome. Interestingly, in the crystal structure, the peptide-binding groove of this domain forms intermolecular contacts with the C-terminus of a symmetry-related molecule, an association that may mimic interactions with the C-terminus of α-tubulin or other physiologically relevant partners. The interaction of TBC-B with the α-tubulin C-terminus may, in particular, protect from post-translational modifications, or simply assist in the shepherding of the protein into polymerization.

Keywords: CAP-Gly domain; CD; crystallography; tubulin-binding; ubiquitin-like.

Fig 1

Schematic of TbTBC-B. Domains and termini are labelled. Residues 3–87 and 157–222 represent the assigned Ubl (blue box) and CAP-Gly (yellow box) domains, respectively. Black lines and residue numbers 2–87 and 153–232 represent the structures determined.

Fig 2

Ribbon diagram of the two domains of TbTBC-B. The Ubl domain β-strands are in light blue, α-helices are in pink, and the CAP-Gly domain β-strands are in yellow. The N-termini and C-termini and elements of secondary structure are labelled. The orientation of the two domains with respect to each other is arbitrary. Two lysines of note are depicted as pink spheres.

Fig 3

The primary and secondary structure of TbTBC-B. Residues strictly conserved in at least 75% of TBC-B sequences are encased in black. Red stars mark Lys32 and Lys60, which are conserved in ubiquitin.

Fig 4

Structural overlay of TbTBC-B Ubl (blue) and ubiquitin (PDB code 1UBQ; pink). Spatially conserved lysines are depicted as sticks.

Fig 5

(A) The TbTBC-B Ubl domain superimposed with other TBC-B Ubl structures. Structures are shown as tubes coloured by structure: yellow, TbTBC-B; cyan, C. elegans; magenta, D. melanogaster; slate, M. musculus. Affinity tags, where present, have been omitted from the models. The N-terminus is at the top, and the C-terminus points to the bottom. (B) The TbTBC-B CAP-Gly domain superimposed on other TBC-B CAP-Gly domains, in a similar way as in (A): yellow, TbTBC-B; cyan, C. elegans; pink, M. musculus. The N-terminus is on the left side, and the C-terminus is on the right.

Fig 6

The Asp191 and Arg159 salt bridge in the CAP-Gly domain. The main α trace is shown as grey sticks. Hydrogen bonds are shown as black dashed lines. Conserved residues are shown as sticks coloured by atom type: C, grey; N, blue; O, red, S, yellow.

Fig 7

(A) Chain A C-terminus interacting with the peptide-binding groove of a symmetry-related molecule, chain B. The chain B main chain is shown in grey ribbon style, and residues that surround the C-terminal tail are shown as sticks coloured according to atom type: C, grey; N, blue; O, red. Chain A is shown as sticks coloured according to atom type: C, slate blue; O, red. Hydrogen bonds are shown as black dashed lines. Residue numbers for chain A are shown in cyan. L217 and Q221 (chain B) and V231 (chain A) are shown with two side chain rotamer conformations. G199 and F216 are not labelled. (B) Relative positions of the C-terminus peptides in M. musculus CAP-Gly domain II of CLIP-170 structure (pale pink cartoon; peptide carbon atoms are in magenta) and TbTBC-B (chain B as a white cartoon; chain A carbon atoms are in slate). For both peptides: O, red; N, dark blue. Amino acids, for clarity, are shown as backbones, except for proline and the C-terminus residues. Peptides are labelled 1–3 from the C-terminus: A for TbTBC-B chain A; P for M. musculus polypeptide.