Daly TK et al. (2013), Beyond BLASTing: tertiary and quaternary struct...

ECB-ART-42699

Genome Biol Evol 2013 Jan 01;51:217-32. doi: 10.1093/gbe/evs135.

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Beyond BLASTing: tertiary and quaternary structure analysis helps identify major vault proteins.

Daly TK , Sutherland-Smith AJ , Penny D .

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We examine the advantages of going beyond sequence similarity and use both protein three-dimensional (3D) structure prediction and then quaternary structure (docking) of inferred 3D structures to help evaluate whether comparable sequences can fold into homologous structures with sufficient lateral associations for quaternary structure formation. Our test case is the major vault protein (MVP) that oligomerizes in multiple copies to form barrel-like vault particles and is relatively widespread among eukaryotes. We used the iterative threading assembly refinement server (I-TASSER) to predict whether putative MVP sequences identified by BLASTp and PSI Basic Local Alignment Search Tool are structurally similar to the experimentally determined rodent MVP tertiary structures. Then two identical predicted quaternary structures from I-TASSER are analyzed by RosettaDock to test whether a pair-wise association occurs, and hence whether the oligomeric vault complex is likely to form for a given MVP sequence. Positive controls for the method are the experimentally determined rat (Rattus norvegicus) vault X-ray crystal structure and the purple sea urchin (Strongylocentrotus purpuratus) MVP sequence that forms experimentally observed vaults. These and two kinetoplast MVP structural homologs were predicted with high confidence value, and RosettaDock predicted that these MVP sequences would dock laterally and therefore could form oligomeric vaults. As the negative control, I-TASSER did not predict an MVP-like structure from a randomized rat MVP sequence, even when constrained to the rat MVP crystal structure (PDB:2ZUO), thus further validating the method. The protocol identified six putative homologous MVP sequences in the heterobolosean Naegleria gruberi within the excavate kingdom. Two of these sequences are predicted to be structurally similar to rat MVP, despite being in excess of 300 residues shorter. The method can be used generally to help test predictions of homology via structural analysis.

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Species referenced: Echinodermata
Genes referenced: LOC100887844 LOC584143 mvp

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	Fig. 1.—. Vault ribonucleoprotein structure. (A) Rat MVP quaternary structure showing half a vault colored by monomer (PDB: 2ZUO, 2ZU4, and 2ZV5). A full vault will have at the lower left a copy of the upper half vault related by a 2-fold rotation axis. (B) Three rat MVP monomers colored by secondary structure (PDB 2ZUO stripped down to three monomers). This figure highlights the extensive lateral association required to dock into the vault quaternary structure.
	Fig. 2.—. MVP monomer comparison. (A) I-TASSER-modeled structure for the full-length rat MVP sequence (Q62667). Residues not observed in the crystal structure (PDB:2ZUO*b) are circled (shown by arrows). (B) I-TASSER-modeled structure for the sea urchin MVP monomer (Q5EAJ7). (C) I-TASSER-modeled structure for the kinetoplasts Trypanosome cruzi (Q4CUM2) and (D) Leishmania major (Q4QJJ7) MVPs.
	Fig. 3.—. Structural effect of the 2ZUOb constraint. (A) Structural comparison of the shoulder and cap-helix region of two rat MVP models either constrained by 2ZUOb (red) or unconstrained (blue). The kink in the unconstrained cap-helix modeled by I-TASSER results in poor docking in RosettaDock. The rat MVP sequence constrained by 2ZUOb (red) entirely aligns with 2ZUOb (obscured), and this model docks readily in RosettaDock. (B) Urchin MVP shoulder and cap-helix region structural comparison between models either constrained by 2ZUOb (red) or unconstrained (blue) relative to 2ZUOb (green). In this case, the unconstrained urchin MVP model docks more readily than the constrained model.
	Fig. 4.—. RosettaDock results from the crystal structure cap-helix. (A) Score graph depicting RosettaDock energy score versus RMSD (Å) of the docked monomers compared with their starting positions. The funnel shape of the score graph indicates a high confidence in the structure of the models with lowest energy score. (B) Cartoon of the lowest energy model (energy score −264) shaded by monomer. (C) Surface rendering of the lowest energy model.
	Fig. 5.—. RosettaDock results from the rat MVP shoulder region. (A) Score graph representing the RosettaDock energy scores versus RMSD (Å) for the 1,000 models generated by RosettaDock for the shoulder region of MVP (residues 520–646). The energy score for the shoulder region docking is higher than for the cap-helix (table 1). (B) Cartoon of the shoulder domain from the lowest energy model of the two docked monomers (energy score −12) shaded by chain. (C) Surface rendering of the lowest energy docked monomers.
	Fig. 6.—. I-TASSER modeling results for the negative control sequences. (A) Randomized rat MVP. (B) Rat myosin 1A. (C) Human merlin unconstrained. (D) Human merlin constrained by 2ZUO*b. Insert is the stomatin core from Pyrococcus horikoshii.
	Fig. 7.—. Naegleria gruberi MVP I-TASSER structural modeling. (A) D2V5B9, 559 residues. (B) D2W0Z9, 530 residues both identified from a BLASTp search of the UniProtKB database and submitted to I-TASSER without constraint. (C–F) Models derived from sequences retrieved via a PSI-BLAST of the National Center for Biotechnology Information (NCBI) database and submitted to I-TASSER constrained by the rat crystal structure 2ZUO*b. (C) DZUF7, 845 residues. (D) D2VSY6, 833 residues. (E) D2VC38, 694 residues. (F) D2VH38, 418 residues. UniProt accession numbers are provided for consistency. See also table 2.

References [+] :

Altschul, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. 1997, Pubmed