The discovery of covalent modification of eukaryotic proteins by the conjugation of ubiquitin to the ε-amino groups of target lysines has spawned some of the most exciting directions of research in current molecular biology [1
]. Ubiquitin (Ub) itself is a small polypeptide of 76 residues, and its crystal structure revealed a distinctive fold dominated by a β-sheet with 5 anti-parallel β-strands and a single helical segment [4
] (Figure ). Pioneering investigations of Kraulis, Overington and Murzin showed that this fold was not unique to Ub, but was also present in several other proteins with biologically distinct functions. These included the staphylococcal enterotoxin B, the streptococcal immunoglobulin (Ig)-binding protein G and 2Fe-2S ferredoxins [6
]. The common fold shared by these proteins was termed the β-grasp, because the β-sheet appears to grasp the helical segment in this domain [7
]. These early studies provided the first indications that, despite its small size, the β-grasp fold (β-GF) might serve as a multi-functional scaffold in diverse biological contexts.
Figure 1 Topology diagrams of selected β-GF members. A generalized representative is shown in (A) with the key structural features found in certain lineages of the fold labeled, while (B) depicts idealized versions of specific lineages, the names of which (more ...)
The centrality of Ub conjugation in eukaryotic molecular biology has led to numerous investigations on Ub and Ub-related domains [9
]. These studies have resulted in a large body of data on the properties of the Ub-like versions of the β-GF. The key emerging findings were that several other Ub-like proteins (Ubl), such as Urm1 [11
], Apg12 [12
], Nedd8 [13
], and SUMO [14
] are also covalently linked to target polypeptides, just as Ub itself. In contrast, some Ub-related domains, like the Ubx domain or Ub-like domains of IκB kinases, play adaptor roles in Ub-signaling [16
]. These studies also showed that eukaryotes possess a distinctive enzymatic apparatus for Ub-modification, comprised of a cascade of three enzymes: E1, E2 and E3. These enzymes successively activated Ub/Ubls for transfer using the free energy derived from ATP hydrolysis, relayed it via thiocarboxylate linkages involving the C-terminal residue of Ub/Ubls, and finally transferred it to lysines on target polypeptides [1
]. Eukaryotes were also shown to contain an elaborate apparatus for removal of covalently linked Ub/Ubls and proteasomal degradation of Ub-modified proteins [23
Concomitantly, structural studies also uncovered several new versions of the β-GF in a variety of domains, greatly widening its horizon of biological functions. Examples of such β-GF domains are: 1) the TGS domain, an RNA-binding domain found in aminoacyl tRNA synthetases and other translation regulators (PDB: 1QF6 [28
]). 2) The doublecortin (DCX) (PDB: 1MJD [30
]), RA (PDB: 1C1Y [31
]), PB1 (PDB: 1IPG [32
]), and FERM N-terminal domains (PDB: 1EF1 [33
]), which function as adaptors in animal signaling proteins and apoptosis regulators by mediating protein-protein interactions. 3) The soluble ligand-binding β-GF (SLBB) domain involved in binding vitamin B12
and other solutes in animals and bacteria (PDB: 2BBC, 2FUGS [34
]). 4) Various toxins related to the staphylococcal enterotoxin B including superantigens involved in the toxic shock syndrome (PDB: 1ESF [37
]). 5) Functionally obscure subunits of various enzymatic complexes, like TmoB of the aromatic monooxygenase oxygenase complex (PDB: 1T0S [38
]) and RnfH of the Rnf dehydrogenases [39
]. 6) Conserved domains, perhaps involved in RNA binding, in the archaeo-eukaryotic RNA polymerase RPB2 subunit [40
] and bacterial translation initiation factor IF3 (PDB: 1TIF [41
]). 7) Staphylokinases and streptokinases which are fibrinolytic enzymes of low GC Gram-positive bacteria (PDB: 2SAK [44
]). 8) MutT/nudix enzymes – a group of phosphohydrolases acting on diverse substrates [45
]. These observations suggested that the β-GF is indeed a widely utilized structural scaffold, with an underappreciated versatility and an evolutionary history rich in adaptive radiations.
One notable evolutionary question in this regard was the origin of eukaryotic Ub and its relationships to other domains with the β-GF. The first major advances in this direction came with the identification of the sulfur transfer proteins, ThiS and MoaD, respectively involved in thiamine and Molybdenum cofactor (MoCo) biosynthesis, which contained β-GFs closely related to Ub [46
]. Furthermore, it was demonstrated that their C-terminal residues formed thiocarboxylates, just like Ub, and this was catalyzed by enzymes (ThiF and MoeB), which are very similar to the E1 enzymes involved in Ub-conjugation [46
]. More recently, research from our group showed that the Ub-conjugation systems might not be an exclusive feature of eukaryotes. Proteins with Ub-like β-GF domains, and functionally linked enzymes related to E1, E2 and deubiquitinating peptidases of the JAB domain superfamily were found in several, phylogenetically diverse bacteria. We presented evidence that though some of these systems are likely to be involved in sulfur transfer reactions in metabolite biosynthesis, akin to ThiS and MoaD, others might potentially function as bona fide
conjugation systems that transfer β-GF proteins to target polypeptides [39
]. Hence, the eukaryotic Ub-conjugation system might have evolved from more ancient precursors that were present in bacteria prior to the origin of eukaryotes.
With some clarity emerging on issue of the origin of Ub/Ubls and the associated biochemical networks, we sought to investigate the broader issue of the adaptive radiations of the entire β-GF. In particular we were interested in a number of problems from structural and evolutionary stand points: 1) Establishing the entire gamut of structural and topological variations that have emerged in the β-GF. 2) Identifying any unifying structural themes that might exist across most or all functionally diverse versions of the fold. 3) Determination of the lineage-specific sequence-structure correlates for the varied functional adaptations of the β-GF. 4) Developing a higher order evolutionary classification for the β-GF and using it as a scaffold to identify the major temporal phases of adaptive radiation. 5) Identifying instances of drastic shifts in biological or biochemical functions in specific monophyletic lineages of the β-GF. One example of such a functional shift is seen in the evolution of the classical Ub-like proteins, where a unique post-translational modification system emerged from a core metabolic sulfur transfer system. 6) Identifying previously unrecognized members of the fold, if any, and thereby expanding the functional spectrum or providing a rationale for function prediction of uncharacterized members of the fold. 7) We also hoped that the β-GF might provide a model for understanding the more general problem of how certain small protein folds tend to be extensively deployed in a whole diversity of functional contexts.
In this article we present the results of our systematic analysis of the β-GF with the objective of addressing the above points.