Molecular assembly and function of Androglobin, a novel chimeric globin.

Androglobin (Adgb) is a multi-domain protein that contains a calpain-like and a circularly permuted globin-like domain. Adgb is the fifth mammalian globin to be discovered and is predominately expressed in the testes. Whilst Adgb is probably the least studied and understood globin member this project aims to provide further insight into the haem domain and overcome issues associated with generating the full-length protein by recombinant techniques. Comparing 362 Adgb sequences using multiple sequence alignments, the critical amino acids of the haem domain were identified as the proximal His F8, distal Gln E7, and CD1 Tyr residues. Consistent with the literature, Adgb does not have a close phylogenetic relationship with the other mammalian globin members. Despite being circularly permuted, the 3D model of the haem domain was capable of producing a globin-like fold, even though the haem domain does not resemble the customary secondary structure order, as observed in the other globin members. The 3D haem domain model suggests a pentacoordinate haem geometry. The purified haem domain protein partially supports this, revealing mixed penta/hexa-coordinated properties. Using 3D modelling it was predicted that Adgb contains two surface-exposed Cys in close proximity to form a disulfide bond in the CD region (in a similar position to that found in Ngb), which was confirmed by dithiodipyridine experiment. Adgb binds to NO as a pentacoordinate haem iron species, which is highly unusual for globins and similar to that observed for NO binding to cytochrome c’. Adgb exhibits a higher NiR activity than other globin members although, similar to that of Globin X. Therefore, it can be proposed that Adgb may have a specialised function in nitrite reduction that could be related to spermatogenesis. The ligation of the full Adgb gene into the pET28a vector and truncation of the full gene was accomplished by molecular cloning techniques something that has not been previously achieved however, challenges were still encountered during protein expression. Overall this study provides a deeper insight into the structure and biochemical properties of the haem domain of Adgb, an unusual globin.