Crystal structures of 2OG oxygenases involved in ribosomal protein hydroxylation

Post-translational modifications play diverse biological functions. Hydroxylation of collagen proteins has long been a recognised post-translational modification in eukaryotes. In the case of collagen, hydroxylation of prolyl residues, by 2-oxoglutarate and iron dependent enzymes (2OG oxygenases), in collagen proteins allows for the stabilisation of the collagen triple helix structure through conformational restraint and through the addition of a hydrogen bond donor. Additionally, hydroxylation of lysine side chains of collagen is required for cross-linking collagen (and possibly other proteins) in the extra-cellular matrix. Post-translational prolyl hydroxylation also plays a pivotal role in transcriptional regulation of the hypoxic response, as catalyzed by the hypoxia inducible factor / HIF prolyl hydroxylases (PHDs or EGLN enzymes). Recently, ribosomal protein hydroxylation catalyzed by 2OG- and Fe(II)-dependent oxygenases has been found to be a highly conserved post-translational modification in eukaryotes and prokaryotes (Ge et al and Loenarz et al). We present several crystal structures of 2OG oxygenases involved in ribosomal protein hydroxylation.

"From one seed a whole handful": homologous proteins as seeds in crystallisation

Protein structures have significantly impacted and aided drug discovery efforts. However, it is not enough to know the structure of a protein; it must be the right structure. Small alteration in sequence can lead to different conformations and oligomerization states, cause changes which lead to different active site architecture and also which modify function. Protein crystallization is an essential prerequisite for the determination of protein structures by X-ray crystallography. We have obtained encouraging initial results for a hitherto unexplored crystallization method with the enzyme arylamine N-acetyltransferase from M. tuberculosis (TBNAT). Despite prolonged and varied trials to crystallize TBNAT, an important anti-tubercular drug target, no crystals were obtained. In an alternative approach, cross-seeding of TBNAT protein with micro-crystalline seeds from a homologous NAT from M. marinum (74 % sequence identity (SID)) surprisingly resulted in a single 20 micron sized TBNAT crystal that diffracted to 2.1 Å and allowed for TBNAT structure determination (Abuhammad et al., 2013). To our knowledge, cross-seeding crystallisation using homologous proteins has only been previously successful in cases with more than 85% SID. In this study, we have explored the effect of low sequence homology on cross seeding using β-lactamases with SID as low as 30%. Despite the low SIDs, the results show cross seeding leads to an increase in hits obtained, the identification of new crystallization conditions, shortening of crystallization time and an improvement in the quality of the crystals obtained.

Structures of human m⁶A RNA demethylases: FTO and ALKBH5

Methylation at the N6 position of adenosine in mRNA (m⁶A) was first observed around 40 years ago but little was known about the function of this modification at the time of discovery. Research over the last ten years has led to the identification of methyltransferases that produce or `write' the m<sup>6</sup>A RNA mark and recently demethylases that remove or `erase' m⁶A marks, thus indicating that the m⁶A modification is reversible. Recent analyses have identified the motif and regions where m⁶A marks are present within RNA transcripts as well as m⁶A binding protein `readers'. These studies have revealed a role for m⁶A methylation in mRNA stability and demonstrated that sequence specific subsets of transcripts can be regulated in this manner. Like the histone lysine JmjC demethylases, nucleic acid demethylases are members of the 2-oxoglutarate and iron dependent oxygenase superfamily. The human nucleic acid oxygenases (NAOXs) with known structures include the TET enzyme that produces 5-hydroxymethylcytosine in DNA, ALKBH2 and 3 that demethylate 3-methylcytosine and 1-methyladenine in DNA, and ALKBH8 that modifies tRNA. We identified the fat mass and obesity associated protein, FTO, as a NAOX¹ and m⁶A in RNA was recently reported to be a bona-fide substrate of FTO in in-vivo. Subsequently, ALKBH5 was also shown to be an m⁶A demethylase. Here we present structural studies of FTO² and ALKBH5³ as well as biochemical work that has led to structurally informed inhibitor design of the m⁶A demethylases for the development of small molecules to probe biological function and for potential use as therapeutics.