Crystal structure of a novel single‐stranded DNA binding protein from <i>Mycoplasma pneumoniae</i>

Abstract

Mycoplasmas represent the smallest genomes and are associated with pathogenicity in humans including autoimmune and immune system conditions, macrophage activation, cytokine induction, accessory factor in AIDS activation,1 and primary atypical pneumoniae in children and young adults caused by M. pneumoniae. Investigations on novel proteins from these organisms are therefore valuable in dissecting the molecular machinery of minimal genomes. The MPN554 protein (gi:1673959) from M. pneumoniae is a 104 amino acid basic protein of unknown function that is highly conserved in two other members of the mycoplasma family (73% sequence identity to M. genitalium MG376, gi:3844964 and 55% in M. gallisepticum, gi:31541222) and has been shown to be an essential gene in this organism by retrotransposon analysis.2 Apart from this, the MPN554 protein does not have any significant overall sequence similarity to other proteins as judged by PSI-BLAST3 and does not return any hits in PFAM.4 However, multiple rounds of iterative PSI-BLAST with evolving position-specific scoring matrices indicate sequence identities approaching almost 18% to segments of SSBs from numerous organisms. Since this protein is conserved in mycoplasmas, it is likely to be involved in some cellular function that is yet to be elucidated. The crystal structure of this protein has been determined in an effort to explore its structure-function relationships. The structure reveals that MPN554 belongs to the OB protein fold (oligonucleotide/oligosaccharide/oligopeptide binding fold) family5, 6 and is most similar in structure to the Escherichia coli PriB and the E. coli and human mitochondrial single-stranded DNA binding proteins (SSBs). SSBs are involved in a number of cellular processes including DNA replication, recombination, repair, transcription, translation, cold shock response and maintenance of telomeres.6-11 They bind and stabilize transiently formed single-stranded DNA (ssDNA) during biological processes, thereby sheltering them from chemical and nuclease breakdown and preventing the formation of unproductive secondary structures. Despite their structural resemblances even with relatively low sequence similarities, there are variations in their quaternary assemblies (tetrameric, trimeric, and dimeric) as well as sizes which range from about 140 to 300 amino acids. Approximately the first 120 N-terminal residues comprise the OB/SSB fold with conserved structure whereas the rest of the protein has been implicated in interacting with other protein partners and in some cases has been disordered in the crystal structures. Numerous prior efforts have reported in detail the crystal structures of functionally annotated SSBs,12-23 presented comparative analysis of homologous SSBs20, 24 and reported on structures of their protein-DNA complexes.25, 26 An electrophoretic mobility shift assay confirmed that MPN554 binds to a 39-mer single-stranded DNA. It is distinct from the 166 amino acid M. pneumoniae SSB which is homologous to bacterial SSBs and there is no annotated homolog of PriB in M. pneumoniae. Some of the E. coli SSB amino acid residues involved in ssDNA binding are conserved in MPN554. This is the first crystal structure of an SSB from one of the smallest replicating organisms and provides a structural framework for further biochemical investigations of this protein thereby allowing for the continued exploration of novel SSBs, their distributions in genomes and the roles they may play in the life processes of different organisms. The MPN554 gene was amplified by PCR using M. pneumoniae genomic DNA template and primers designed for Ligation-Independent Cloning (LIC).27 The amplified PCR product was prepared for vector insertion by purification, quantitation and treatment with T4 DNA polymerase (New England Biolabs, Beverley, MA) in the presence of 1 mM dTTP. The prepared insert was annealed into the LIC expression vector pB3, a derivative of pET21a (Novagen, Madison, WI)