PNNL

Abstract

A superfamily of proteins with a "Domain of Unknown Function", DUF3349, is present predominately in Mycobacterium and Rhodococcus bacterial species suggesting that these proteins may have a biological function unique to these bacteria, and consequently, prove to be an attractive drug target to combat tuberculosis. Following upon our inaugural structure of a DUF3349 superfamily member, Mycobacterium tuberculosis Rv0543c, we determined the structures of three additional DUF3349 proteins: Mycobacterium abscessus MAB_3403c and Mycobacterium smegmatis MSMEG_1063 and MSMEG_1066. Like Rv0543c, the NMR solution structure of MSMEG_1063 revealed a monomeric five ?-helix bundle with a similar overall topology. On the other hand, the crystal structure of MSMEG_1066 showed a five ?-helix protein with a strikingly different topology and a tetrameric quaternary structure. The tetrameric structure is not a by-product of crystal packing, as size exclusion chromatography indicates MSMEG_1066 is also a tetramer in solution. The NMR solution structure of a fourth member of the DUF3349 superfamily, MAB_3403c, with 18 residues missing at the N-terminus, revealed a monomeric alpha-helical protein with a folding topology similar to the three C-terminal helices in the protomer of the MSMEG_1066 tetramer. However, the MAB_3403c construct cannot form a tetramer likely due to the removal of the N-terminal 18 residues that eliminates ?1, an helix at the interface of two of the four interfaces in the tetramer. These structures, together with a more detailed bioinformatics analysis of the DUF3349 primary amino acid sequences, suggest two subfamilies within the DUF3349 family. The proteins in each DU3349 subfamily contain five ?-helices, but, they adopt different folds. Moreover, one fold initiates the assembly of a unique tetrameric quaternary structure. The division of the DUF3349 into two distinct subfamilies would have been lost if structure solution had stopped with the first structure in the DUF3349 family, highlighting the potential insights generated by solving many structures in a protein family. Moreover, a set of three-dimensonal protein “blueprints” for structure-based discovery of new drugs targeting tuberculosis would have been lost as well.