Detailed analysis of function divergence in a large and diverse domain superfamily: toward a refined protocol of function classification

Abstract

Some superfamilies contain large numbers of protein domains with very different functions. The ability to refine the functional classification of domains within these superfamilies is necessary for better understanding the evolution of functions and to guide function prediction of new relatives. To achieve this, a suitable starting point is the detailed analysis of functional divisions and mechanisms of functional divergence in a single superfamily. Here, we present such a detailed analysis in the superfamily of HUP domains. A biologically meaningful functional classification of HUP domains is obtained manually. Mechanisms of function diversification are investigated in detail using this classification. We observe that structural motifs play an important role in shaping broad functional divergence, whereas residue-level changes shape diversity at a more specific level. In parallel we examine the ability of an automated protocol to capture the biologically meaningful classification, with a view to automatically extending this classification in the future.

Figure 1

HUP domain core: (a) three-dimensional representation of the HUP domain core, using CATH domain 2ielA00; (b) TOPS diagram (Westhead et al., 1999) illustrating the core secondary structure elements, which are numbered as referred to in the text. The N- and C-termini are represented as blue squares marked N and C, respectively. Both (a) and (b) illustrate the reference orientation referred to throughout the text. All three-dimensional molecular graphics were generated using Molscript (Kraulis, 1991) and rendered with Raster3D (Merritt et al., 1997).

Figure 2

Sequence space in the HUP superfamily. Each dot represents a domain and domains are coloured according to the FSG to which they are likely to belong (ETFs: red, AAtRSs: green, ATP-PPases: greenblue, C-DNAPs: violet, NTs: yellow, PSs: cyan, PAPSs: brown, tRMUs: light-blue, USPAs and unclassified: white). Captions in the Figure represent different functional specificities within these FSGs.

Figure 3

2DSEC diagram (Reeves et al., 2006) showing an alignment of the secondary structure elements of representatives from the nine FSGs in the HUP superfamily. Each line in the plot represents a protein. Elements of secondary structure are represented by circles for α-helices and triangles for β-sheets. Conserved and non-conserved α-helices are coloured pink and magenta, respectively. Conserved and non-conserved β-sheets are coloured yellow and brown, respectively. The size of circles and triangles reflects the size of the corresponding secondary structures. The conserved elements constitute the common core of the superfamily.

Figure 4

Examples of HUP embellishment being involved in different aspects of molecular function. HUP domains are displayed in cartoons. HUP domain cores are coloured in grey and presented in the reference orientation (see Figure 1) in all subfigures. Embellishments to the HUP domain cores are coloured in different shades of green and blue. (a) tRNA 2-thiouridylase (PDB 2deu) in complex with a tRNA fragment (displayed in wireframe and coloured orange) and an AMP molecule (displayed and coloured in CPK). The light blue and dark green embellishments are important for binding the tRNA. (b) Carbapenam synthetase (PDB 1q15) contains two domains. The N-terminal domain is coloured orange, and the C-terminal domain is the HUP domain. All four embellishments to the HUP domain (coloured in light blue, light green, dark green and dark blue, respectively) are involved in contacts with the extra N-terminal domain. (c) Tyrosyl-tRNA synthetase (PDB 1h3f) is a homodimeric enzyme. One subunit is represented by a light-pink α-trace, whereas the HUP domain of the other subunit is displayed in cartoons. A major HUP domain embellishment (coloured light blue) is important for contacts between the two subunits. For clarity reasons, not all residues are shown in this figure. (d) Electron transfer flavoproteins (PDB 1efv) consist of two different chains, designated α (coloured light gold and displayed in cartoons) and β. Embellishments to the HUP domain in subunit β (coloured light blue and light green) are very important for stabilising the complex with subunit α.

Figure 5

Functional role of motifs detected by FLORA and embellishments in HUP domains. In all three subfigures, the HUP domain is displayed in cartoons, with the core of the domain coloured grey, the embellishments coloured green, the motifs identified by FLORA coloured blue, and the residues that are identified by FLORA and that are also in embellishments are coloured in yellow. For clarity, extra domains are not shown in this figure. (a) Human Electron Transfer Flavoprotein subunit β (PDB structure 1efv). FLORA detects motifs in two embellishments that are very important for mediating the interaction of subunit β with the other subunit in the complex (displayed as a pink coil). (b) Yeast ATP sulfurylase (PDB 1r6x); a sulfate ion displayed as CPK indicates the location of the active site. One single motif is detected by FLORA in ATP sulfurylase; it is centred on a helix that is part of a large C-terminal embellishment that is specific to the FSG of nucleotidyltransferases. In addition, this helix is located on the top of the main active site, and several of its residues have been shown to be important for substrate binding in other members of this FSG such as phosphopantetheine adenylyltransferase (Izard, 2002). (c) Human tyrosyl-tRNA synthetase (PDB 1n3l). Here, only two very small motifs are detected by FLORA. One lies within a large embellishment that is common to all aminoacyl-tRNA synthetases, and both are located in the interface with the other subunit (displayed as a pink coil) of the tyrosyl-tRNA synthetase homodimer.