Sequence and structural analysis of BTB domain proteins

Abstract

Background: The BTB domain (also known as the POZ domain) is a versatile protein-protein interaction motif that participates in a wide range of cellular functions, including transcriptional regulation, cytoskeleton dynamics, ion channel assembly and gating, and targeting proteins for ubiquitination. Several BTB domain structures have been experimentally determined, revealing a highly conserved core structure.

Results: We surveyed the protein architecture, genomic distribution and sequence conservation of BTB domain proteins in 17 fully sequenced eukaryotes. The BTB domain is typically found as a single copy in proteins that contain only one or two other types of domain, and this defines the BTB-zinc finger (BTB-ZF), BTB-BACK-kelch (BBK), voltage-gated potassium channel T1 (T1-Kv), MATH-BTB, BTB-NPH3 and BTB-BACK-PHR (BBP) families of proteins, among others. In contrast, the Skp1 and ElonginC proteins consist almost exclusively of the core BTB fold. There are numerous lineage-specific expansions of BTB proteins, as seen by the relatively large number of BTB-ZF and BBK proteins in vertebrates, MATH-BTB proteins in Caenorhabditis elegans, and BTB-NPH3 proteins in Arabidopsis thaliana. Using the structural homology between Skp1 and the PLZF BTB homodimer, we present a model of a BTB-Cul3 SCF-like E3 ubiquitin ligase complex that shows that the BTB dimer or the T1 tetramer is compatible in this complex.

Conclusion: Despite widely divergent sequences, the BTB fold is structurally well conserved. The fold has adapted to several different modes of self-association and interactions with non-BTB proteins.

Figure 1

Comparison of structures containing the BTB fold. (a) Superposition of the BTB core fold from currently known BTB structures. The BTB core fold (approximately 95 residues) is retained across four sequence families. The BTB-ZF, Skp1, ElonginC and T1 families are represented here by the domains from Protein Data Bank (PDB) structures 1buo:A, 1fqv:B, 1vcb:B, 1t1d:A. (b) Schematic of the BTB fold topology. The core elements of the BTB fold are labeled B1 to B3 for the three conserved β-strands, and A1 to A5 for the five α-helices. Many families of BTB proteins are of the 'long form', with an amino-terminal extension of α1 and β1. Skp1 proteins have two additional α-helices at the carboxyl terminus, labeled α7 and α8. The dashed line represents a segment of variable length that is often observed as strand β5 in the long form of the domain, and as an α-helix in Skp1. (c) Structure-based multiple sequence alignment of representative BTB domains from each of the BTB-ZF, Skp1, ElonginC and T1 families. The core BTB fold is boxed. Secondary structure is indicated by red shading for α-helices and yellow for β-strands, with the amino- and carboxy-terminal extensions shaded in gray. The low complexity sequences, which are disordered in the Skp1 structures, are indicated by open triangles. See Figure 3 for the PDB codes for the corresponding sequences.

Figure 2

Sequence conservation in BTB domains. The most probable sequences (majority-rule consensus sequences) from each of seven different family-specific hidden Markov models (HMMs) were generated with HMMER hmmemit. Residue positions with a probability score (P(s)) of less than 0.6 are variable and are indicated by dots, residues with 0.6 < P(s) < 0.8 have intermediate levels of sequence conservation and are indicated by lower case letters, and residues with a P(s) > 0.8 are highly conserved and are indicated by capital letters. Gray shading indicates positions that are similar in at least four of the seven families shown, and selected 'signature sequences' that are particular to a specific family are boxed in blue. Gaps are indicated by blank spaces. Residue positions that are buried in the core of the BTB fold are indicated with black circles, and contact sites for four known protein-protein interaction surfaces are shown in the grid below the alignment. The secondary structure elements β1, α1, α4, β5, α7 and α8 occur only in some of the families, and are discussed in the text. Additional Data File 1 includes multiple sequence alignments for these families.

Figure 3

Pairwise sequence and structure comparisons of BTB structures. Cells contain the percentage identity and root mean square deviation (Å) value for each structure pair. Representative structures from the Protein Data Bank are labeled as follows: ^a1buo:A and 1cs3:A; ^b1nex:a; ^c1ldk:D, 1p22:b, 1fqv:B, 1fs1:B, 1fs2:B; ^d1hv2:a; ^e1vcb:B, 1lm8:C, 1lqb:B; ^f1a68:_, 1eod:A, 1eoe:A, 1eof:A, 1t1d:A, 1exb:E (rat Kv1.1); ^g1s1g:A; ^h1r28:A, 1r29:A, 1r2b:A. The T1 domains from Kv1.2, Kv3.1 and Kv4.2 were omitted for clarity. El.C, ElonginC. Ac, Aplysia californica; Hs, Homo sapiens; Sc, Saccharomyces cerevisiae.

Figure 4

Protein-protein interaction surfaces in BTB domains. Left column: the BTB monomer is shown in the same orientation for each of four structural families with the core fold in black, and the amino- and carboxy-terminal extensions in blue. Middle column: the monomers are shown with the protein-protein interaction surfaces shaded. Right column: the monomers are shown in their protein complexes.

Figure 5

Distribution of BTB proteins in eukarytoic genomes. (a) Representation of BTB proteins in selected sequenced genomes. Twelve of the seventeen genomes we searched are represented, showing each type of BTB protein architecture as bar segments. Data for Apis mellifera, Canis familiaris, Gallus gallus, Pan troglodytes and Xenopus tropicalis are available at [35]. Several lineage-specific expansions are evident: BTB-ZF and BBK proteins in the vertebrates; the MATH-BTB proteins in the worm; the BTB-NPH3 proteins in the plant; the Skp1 proteins in the plant and worm; and the T1 proteins in worm and vertebrates. In the Dictyostellium discoideum genome, a single star indicates five BTB-kelch proteins that do not contain the BACK domain, and a double star indicates two MATH-BTB proteins that also contain ankyrin repeats. (b) Phylogenetic relationship of analyzed genomes. Adapted from [39]. The total number of BTB proteins is shown above each genome.

Figure 6

BTB sequence clusters and protein architectures. Family-specific amino- and carboxy-terminal extensions to the core BTB fold are indicated. Regions with no predicted secondary structure are indicated by dashed lines, and ordered regions are indicated with either domain notations or thick solid lines. The Uniprot code for a representative protein is indicated. Clustering by BLASTCLUST was based on the average pairwise sequence identity for all BTB domain sequences from our database, except for the RhoBTB proteins, where only the carboxy-terminal BTB domain was used. Domain names are from Pfam [44].

Figure 7

Structural model of the ubiquitin-E2-Cul3-Rbx1-BBK complex. The complex forms a dimer by the self-association of the BTB domain in the BBK protein. The approximate position of the two-fold axis is indicated. Each full-length BBK protein is shown in red, with the BTB dimer shown in the darkest shading in surface representation, the two BACK domains in pink surface, and the two Kelch β-propellers shown in pink cartoon representation. The Cul3 homology model is shown in green cartoon representation, Rbx1 is in gray cartoon representation, E2 Ubch7 is in yellow cartoon representation, and ubiquitin is shown as a blue surface. Stars indicate the position associated with substrate binding [114]. Depth cuing is used to indicate distances in the plane of the page, such that the diffuse colors are most distant to the viewer than the intense colors.