Insights into the fold organization of TIM barrel from interaction energy based structure networks

Abstract

There are many well-known examples of proteins with low sequence similarity, adopting the same structural fold. This aspect of sequence-structure relationship has been extensively studied both experimentally and theoretically, however with limited success. Most of the studies consider remote homology or "sequence conservation" as the basis for their understanding. Recently "interaction energy" based network formalism (Protein Energy Networks (PENs)) was developed to understand the determinants of protein structures. In this paper we have used these PENs to investigate the common non-covalent interactions and their collective features which stabilize the TIM barrel fold. We have also developed a method of aligning PENs in order to understand the spatial conservation of interactions in the fold. We have identified key common interactions responsible for the conservation of the TIM fold, despite high sequence dissimilarity. For instance, the central beta barrel of the TIM fold is stabilized by long-range high energy electrostatic interactions and low-energy contiguous vdW interactions in certain families. The other interfaces like the helix-sheet or the helix-helix seem to be devoid of any high energy conserved interactions. Conserved interactions in the loop regions around the catalytic site of the TIM fold have also been identified, pointing out their significance in both structural and functional evolution. Based on these investigations, we have developed a novel network based phylogenetic analysis for remote homologues, which can perform better than sequence based phylogeny. Such an analysis is more meaningful from both structural and functional evolutionary perspective. We believe that the information obtained through the "interaction conservation" viewpoint and the subsequently developed method of structure network alignment, can shed new light in the fields of fold organization and de novo computational protein design.

Figure 1. Canonical TIM fold.

The canonical TIM fold (αβ₈) is shown from two different view–points. (A) The three different interfaces namely the β/β encompassing the central β barrel, the α/β and α/α interfaces are highlighted. The face comprising of the C–term end of the β strands, the adjoining loops/turns and the N–term of the helices are broadly classified as the catalytic face of the TIM fold (B), since they feature the catalytic sites.

Figure 2. Schematic representation of the construction of family specific PEN at an energy cutoff ‘e’ and commonality coefficient ‘cc’ (f–PEN_e(cc)).

The steps are indicated with a simple example of two β–loop–β structural motifs, one structure with a short loop (structure 1) and another with a long loop (structure 2). (A) The PEN_es of the structures (1 and 2) are generated by connecting the residues based on their Cα–Cα distances (however the cutoff energy values (e) are chosen in the real cases to draw edges). (B) The structures are superimposed using MUSTANG . (C) The structure based sequence alignment (MSSA) is obtained where the strands are aligned forming a set of equivalent nodes (VLKY and LCIKV) and the non–aligned loops are compensated using gaps in the MSSA. (D) Remapping of PEN_es of structures 1 and 2 on matrices of the same size (27×27) in which the gaps are represented as virtual nodes (VN, highlighted using self-edges). The arrays of nodes in both the structure networks (red and blue) are equivalent (i.e. Y31 (position 1st row and 2nd column) of structure 1 is structurally equivalent to Y12 of structure 2). (E) The f–PEN_e is obtained by aligning both the remapped PEN_e and edges are introduced in the network if they are present in any of the remapped PENs. (F) In this specific case the cc = 1.0 (i e. X = 2), and the family specific network represents only edges that are common to both the structures in the MSSA. The residues involved in the interactions in f–PEN_e(1.0) are highlighted as green spheres and the matrix of size 10×10 below the cartoon represents the interactions (X = 2) among the highlighted residues in both the structures.

Figure 3. Different modes of stabilization of central β barrel in TIM Barrel families.

(a) The cartoon shows the residues involved in the conserved cluster of interactions (f–PEN_–30(1.0)) in the C1A family. The residues that are involved in these conserved interactions are highlighted in spheres with blue for basic and red for acidic residues (the representative protein being d1p1xa_). (b) The entropy based conservation indices (EC) (obtained from family specific MSSAs (see Methods section)) for residues involved in long range β/β interactions in f–PEN_–15(0.8) are given. (c) The cartoon shows the residues involved in low–energy contiguous β barrel interactions formed by the side–chains of residues from adjacent β strands in DC (c.1.2.3) family. The residues that are involved in these conserved interactions are highlighted in spheres with white for hydrophobic and green for polar residues (the representative protein being d1x1za1). Few polar/charged residues (blue) forms vdW interactions with other residues inside the barrel. (d) The ECs for the residues involved in contiguous hydrophobic stabilization of the β barrel (obtained from f–ljPEN_–7(1.0)) are given. The TIM barrels in (a) and (d) are depicted at an orientation similar to Figure 1a.

Figure 4. Functional significance of loop based conserved high–energy interactions.

The figure depicts the presence of conserved interactions involving loops around the catalytic site in TIM fold. (a) TIM domain from 2mnr was taken as the representative structure for DGDL (c.1.11.2, see Table S1) family. Ligands obtained from close homologues (Protein Data Bank IDs: 1DTN, 1FHV, 1JCT, 1KKR, and 1MDL) were mapped onto 2mnr (after structural alignment of the individual TIM domain and 2mnr) and depicted as vdW spheres colored according to the atom types. The conserved high–energy interactions (f–PEN_–20(0.8)) are represented as red spheres while the conserved low–energy interactions (f–ljPEN_–8(0.8)) are represented in blue. The important E317 residue which acts as a general acid catalyst in concerted acid–base catalyzed formation of a stabilized enolic tautomer of mandelic acid is highlighted in green. An alternate view of the barrel is given in (b). (c) shows the ternary complex of XIP–GH10–GH11 where the conserved high–energy loop interactions (f–PEN_–20(0.8); the involved residues are highlighted as spheres) in XIP1 (gray cartoon) involved in the inhibitory interactions of GH10 and GH11 (cartoons; cyan and green respectively) at the Enzyme Binding Sites (EBS) are presented.

Figure 5. Hub statistics for the different families of the TIM fold.

The EC scores (a and b), secondary structure type (c and d) and the amino–acid types (e and f) of the conserved hubs identified for the TIM fold families for PEN_–15(0.7) (a, c, and e) and ljPEN_–7(1.0) (b, d, and f).

Figure 6. Comparison of network and sequence based cladograms.

A comparison of interaction based and sequence based phylogenetic analysis. (a) The cladogram of the hierarchical clustering of the members from network similarity scores (Methods Section). (b) The cladogram of the sequence based phylogeny. For sequence based phylogeny a Maximum Likelihood based statistical method was used for phylogenetic reconstruction.

Figure 7. Comparison of f–PEN clusters and SCA sectors for β–glycanase family.

A comparison of the cluster residues obtained from f–PEN and the Sector residues identified from SCA of β–glycanase family. The residues are mapped on the backbone cartoon structure of bacterial cellulose catalytic domain (PDB id: 1EDG). The top four clusters are rendered in yellow, green, magenta and red color backbone representation. The yellow, green, magenta and red spheres are the Sector residues from SCA matching with residues in the clusters of corresponding color. The grey spheres are from Sector, which do not match with the cluster residues (similarly, there are cluster residues which do not match with the Sector residues).