Crystal structure of YbaK protein fromHaemophilus influenzae (HI1434) at 1.8 ? resolution: Functional implications

Abstract

Structural genomics of proteins of unknown function most straightforwardly assists with assignment of biochemical activity when the new structure resembles that of proteins whose functions are known. When a new fold is revealed, the universe of known folds is enriched, and once the function is determined by other means, novel structure-function relationships are established. The previously unannotated protein HI1434 from H. influenzae provides a hybrid example of these two paradigms. It is a member of a microbial protein family, labeled in SwissProt as YbaK and ebsC. The crystal structure at 1.8 A resolution reported here reveals a fold that is only remotely related to the C-lectin fold, in particular to endostatin, and thus is not sufficiently similar to imply that YbaK proteins are saccharide binding proteins. However, a crevice that may accommodate a small ligand is evident. The putative binding site contains only one invariant residue, Lys46, which carries a functional group that could play a role in catalysis, indicating that YbaK is probably not an enzyme. Detailed sequence analysis, including a number of newly sequenced microbial organisms, highlights sequence homology to an insertion domain in prolyl-tRNA synthetases (proRS) from prokaryote, a domain whose function is unknown. A HI1434-based model of the insertion domain shows that it should also contain the putative binding site. Being part of a tRNA synthetases, the insertion domain is likely to be involved in oligonucleotide binding, with possible roles in recognition/discrimination or editing of prolyl-tRNA. By analogy, YbaK may also play a role in nucleotide or oligonucleotide binding, the nature of which is yet to be determined.

Restoring species-specific posttransfer editing activity to a synthetase with a defunct editing domain

“…However, freestanding proteins with homology to the INS domain are found in the genomes of representative species from all three kingdoms of life (25,28 (25,31). In addition to these free-standing ProRS-like editing proteins, a module with homology to the INS domain is appended to the N terminus of yeast and other lower eukaryotic ProRSs (27,28). Previously, Plasmodium falciparum ProRS, a lower eukaryotic enzyme with an N-terminal INS-like domain, was shown to catalyze editing of Ec Ala-tRNA Pro , whereas Sc ProRS lacked this activity (28).…”

“…The unique ProRS editing domain is found in at least three different structural contexts: as an insertion (INS) between motifs 2 and 3 of the aminoacylation active site of bacterial enzymes (16,25,26), as an N-terminal extension in ProRSs of lower eukaryotes (27,28), and as a free-standing editing module (25,28). A highly conserved lysine (K279 in Ec ProRS) found in most INS domain homologs is critical for posttransfer editing function (16,29).…”

“…Sequence alignments have revealed that yeast and other lower eukaryotic ProRSs contain an ''extra'' N-terminal domain with weak homology to the bacterial ProRS INS domain ( Fig. 1 A) (15,27,28). Although the extra N-terminal domain common to lower eukaryotes is absent from higher eukaryotes, both share a C-terminal extension as a common feature (in addition to the class-defining active-site core and anticodon binding domain).…”

“…The N terminus of Sc ProRS also shares weak sequence similarity to the free-standing Hi YbaK protein (27). Although sequence identity is relatively low (13%), to explore structural similarity, we prepared a homology model of the Sc ProRS N-terminal extension based on the known structure of Hi YbaK (27).…”

“…Although sequence identity is relatively low (13%), to explore structural similarity, we prepared a homology model of the Sc ProRS N-terminal extension based on the known structure of Hi YbaK (27). A model of the Ec INS domain was generated in a similar manner.…”