Cross-species analyses identify the BNIP-2 and Cdc42GAP homology (BCH) domain as a distinct functional subclass of the CRAL_TRIO/Sec14 superfamily

Abstract

The CRAL_TRIO protein domain, which is unique to the Sec14 protein superfamily, binds to a diverse set of small lipophilic ligands. Similar domains are found in a range of different proteins including neurofibromatosis type-1, a Ras GTPase-activating Protein (RasGAP) and Rho guanine nucleotide exchange factors (RhoGEFs). Proteins containing this structural protein domain exhibit a low sequence similarity and ligand specificity while maintaining an overall characteristic three-dimensional structure. We have previously demonstrated that the BNIP-2 and Cdc42GAP Homology (BCH) protein domain, which shares a low sequence homology with the CRAL_TRIO domain, can serve as a regulatory scaffold that binds to Rho, RhoGEFs and RhoGAPs to control various cell signalling processes. In this work, we investigate 175 BCH domain-containing proteins from a wide range of different organisms. A phylogenetic analysis with ~100 CRAL_TRIO and similar domains from eight representative species indicates a clear distinction of BCH-containing proteins as a novel subclass within the CRAL_TRIO/Sec14 superfamily. BCH-containing proteins contain a hallmark sequence motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs ('h' is large and hydrophobic residue and 's' is small and weekly polar residue) and can be further subdivided into three unique subtypes associated with BNIP-2-N, macro- and RhoGAP-type protein domains. A previously unknown group of genes encoding 'BCH-only' domains is also identified in plants and arthropod species. Based on an analysis of their gene-structure and their protein domain context we hypothesize that BCH domain-containing genes evolved through gene duplication, intron insertions and domain swapping events. Furthermore, we explore the point of divergence between BCH and CRAL-TRIO proteins in relation to their ability to bind small GTPases, GAPs and GEFs and lipid ligands. Our study suggests a need for a more extensive analysis of previously uncharacterized BCH, 'BCH-like' and CRAL_TRIO-containing proteins and their significance in regulating signaling events involving small GTPases.

Figure 1. Phylogenetic tree of CRAL_TRIO and BCH domains from the Sec14 superfamily.

This bootstrapped Neighbor Joining tree includes 175 BCH domains and 98 CRAL_TRIO/BCH-like domains from multiple organisms. The tree is displayed in a circular mode and different groups are marked by colored stripes. The clades with branch length <0.05 are collapsed and the number against each collapsed clade gives the number of collapsed branches. Branch lengths are ignored in order to maintain clarity. Against each branch the domain architectures of individual protein are shown as identified by the Pfam database (release 25) with a cut-off e-value ≤0.1. The Pfam database does not differentiate between CRAL_TRIO and BCH domains and thus both are indicated by yellow colored rectangles. However, if no such domain was identified by the Pfam database, we marked the annotations for BCH domains as determined by our analyses and they are indicated by an orange colored rectangle. The protein length is scaled. Eexcept when there is more than one protein from one genus (for these NCBI accessions are also given with name initials) only the generic names are given. The accession codes for remaining species/branches can be found in the Table S1. The abbreviations used are as follows; Dr: Danio rerio, Tn: Tetraodon nigroviridis, Ci: Ciona intestinalis, Dd: Dictyostelium discoideum, At: Arabidopsis thaliana, Rc: Ricinus communis, Pt: Populus trichocarpa, Gm: Glycine max, Mt: Medicago truncatula, Ps: Picea sitchensis, Zm: Zea mays, Os: Oryza sativa. This phylogenetic tree shows the distinct clustering of BCH domains from CRAL_TRIO domains. The three BCH subgroups are group I, group II and group III respectively and distinct groups within the CRAL_TRIO domain are also marked accordingly. Each cluster represents a distinct domain architecture. Pfam does not recognize the complete domain in BCH groups. The CRAL_TRIO_N domain, which is characteristically associated with CRAL_TRIO domains, is also missing in BCH and BCH-like (NF1 and RhoGEF) proteins. Similar to NF1 protein, Dictyostelium discoideum has an ancestral BCH sequence, which is associated with a RasGAP domain.

Figure 2. Sequence logos of CRAL_TRIO and BCH domains.

The sequence logos derived from 175 BCH and 78 CRAL_TRIO domain sequences are shown in this figure. The conserved residues are marked with arrows and the numbering is given according to the yeast Sec14p protein (NCBI accession: NP_013796) for CRAL_TRIO domains and the human BNIP-2 protein (NCBI accession: NP_004321) for BCH domains. The approximate positions of α-helices and β-beta strands are indicated at the bottom by blue cylinders and red arrows. In order to avoid any biased data, the ‘BCH-like’ groups (NF1 and RhoGEFs) were excluded from the logo calculation. These logos reveal characteristic differences between BCH and CRAL_TRIO domains. Unique positions within the two groups are marked by arrows. BCH domains have a unique signature motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs in which ‘h’ refers to any large and hydrophobic residue and ‘s’ is a small and weekly polar residue (A, T, G, S). This motif is missing in CRAL_TRIO domains. The motif contains a patch of positively charged residues referred to as an Arg/Lys patch. Similarly, as exemplified by the aromatic residue in the middle of three α-helices, many of the hydrophobic residues (shown in grey) are conserved at various positions. The conservation of long and hydrophobic residues in the β-strands provides a hydrophobic surface.

Figure 3. Gene structure of BCH domains.

The gene structures of BCH domains are shown for four representative organisms, Homo sapiens, Drosophila melanogaster, Arabidopsis thaliana, Dictyostelium discoideum. Their accessions are NP_056040 (Hs group-I), CAQ06715 (Hs group-II), NP_060156 (Hs group-III), ABY20545 (Dm group-I), NP_724599 and NP_724597 (Dm group-II), NP_648552 (Dm group-III), NP_564960 and NP_195300 (At group-II), XP_638573 (Dd group-II), XP_645940 (Dd group-III). The positions of introns are marked by arrows on the secondary structure (not scaled) of the BCH domain. With few exceptions, BCH domains of other organisms within the same group exhibit similar intron insertion patterns. Plants and lower organisms have no group-I BCH representatives. Except the ‘BCH-only’ gene, which has two introns, the BCH domain genes of Dictyostelium discoideum (XP_645456: primitive-type, with RasGAP domain and XP_640612) are intronless. Similar insertion positions in plant group-II genes suggest that they might have evolved from ‘BCH-only’ genes through intron insertions and association with macro domain later. These introns were preferentially inserted in the three alpha-helices and in loops (in plants).

Figure 4. Predicted three dimensional structures of BCH domains.

(a) A predicted three dimensional structure of the HsBNIP-2 BCH domain is displayed in this figure. The highly conserved proline residues are shown in yellow in a sphere representation. They are positioned in loops connecting the β-strands and α-helices. The patch of positively charged residues (called as Arg/Lys patch) is highlighted in blue color and the highly conserved residues H248, K271 are marked. (b) The side-chain of K271 comes in close contact with the backbone oxygen of R238 in the Arg/Lys patch (shown in zoomed box). This predicted interaction could provide added stability to the helical loop, which likely gates a lipid-binding cavity. (c) The side-chain of N189 from the Rho-binding region interacts with the side-chain of D143 of N-terminus α-helix (distance: 2.7 Å). This indicates that the N-terminus helix might be involved in Rho binding activity.

Figure 5. Diversification of BCH domains.

BCH domains evolved from a CRAL_TRIO like ancestor and diverged into three subgroups with distinct protein domain architectures. This figure displays the predicted path of divergence for each of the three BCH subgroups. Plant and animal BCH subgroups diverged independently. The ‘BCH-only’ subgroups of plants and insects also descended from different ancestors. This is evident from their phylogenetic clustering and their gene-structure. Group-I BCH proteins/genes might have arisen after domain swapping events. Nematodes have only group-III BCH domain proteins, which are associated with a RhoGAP domain. The divergence into three distinct subgroups in the following lineages is therefore the result of either another domain swapping event or an unknown intermediate ancestor.