MBI –researchers reveal the molecular structure of an enzyme involved in organization of the cell.
In the recent July issue of Structure, Svein Isungset Støve, Håvard Foyn, Bengt Erik Haug og Thomas Arnesen from the NAT-group at the Department of molecular biology and the Department of chemistry, UIB, together with researchers at University of Pennsylvania, USA, publish the structure of the human golgi-associated N-terminal acetyltransferase (NAT) Naa60. Naa60 is one of 6 different human NATs that catalyze N-terminal acetylation (Nt-acetylation), and has previously been shown to be important both for the subcellular organization of the golgi apparatus, and for processes involved in cell division. The golgi apparatus is an organelle that is present in most eukaryotes where it organize and prepare proteins for secretion. If Naa60 is removed from cells, the organization of this organelle is disrupted, most likely due to loss of Nt-acetylation of one or more Naa60 substrates. A previous study has also shown that Naa60 is important for normal chromosome segregation, a process that is very important for both meiotic and mitotic cell division.
Structural studies reveal unique features with Naa60
- For a long time Naa60 has been a bit of a mystery to us working with these enzymes, but in the recent 2-3 years a lot of new exiting data on Naa60 has been gathered in our group, says Svein. In the beginning of 2015, Henriette Aksnes and colleagues in the NAT group together with members of Mathias Zieglers group at MBI published a ground breaking study among other showing that Naa60 associate with golgi membranes and that it mainly Nt-acetylate membrane proteins with a N-termini facing the cytosol. - When we finally got a structure of Naa60, the first thing we saw was that the acetyltransferase domain of Naa60 closely resembles the acetyltransferase domain of the human orthologue Naa50, and that Naa60 most likely consist of two domains, the acetyltransferase domain and a membrane association domain says Svein. We also saw that Naa60 has two unique extended loop regions which are involved in peptide substrate binding and an until now unknown dimerization mechanism.
In order to study how Naa60 bind its substrates, Svein and his colleagues crystallized Naa60 bound to a bisubstrate analogue that has been developed in collaboration between the NAT-group at MBI and Bengt Erik Haug at the department of chemistry. The bisubstrate analogue consists of the acetyl-donor CoA covalently bound to a polypeptide representing the N-terminus of a known Naa60 substrate. In an attempt to crystallize the protein in a ternary complex with both substrates (CoA and a peptide substrate) Svein and his colleagues obtained unexpected data. – When we solved the structure of what we initially thought was Naa60 bound to both CoA and a peptide substrate it turned out that the peptide substrate was not there, instead one of the uniquely extended loops of Naa60 had shifted slightly and mediated homodimerization of the enzyme. - Further studies showed that Naa60 also dimerize in solution and that it shifts between a dimeric state and a monomeric state in order to acetylate its substrates.
- This was really interesting, Svein says, but we are still uncertain about the in vivo relevance of this dimerization, and this is a question we are currently studying further in the lab.
Svein Isungset Støve defended his PhD thesis at the department of Molecular Biology in 2015. Much of the work described in this article was carried out on a research stay in Ronen Marmorsteins lab at the University of Pennsylvania, Philadelphia, USA. Håvard Foyn is currently a post-doc in the Lab of Thomas Arnesen at the Department of Molecular Biology, and has synthesized bisubstrate analogues in collaboration with Bengt Erik Haug at the department of chemistry. Robert S. Magin is a PhD student in the Marmorstein lab at the University of Pennsylvania. The project was funded by Kreftforeningen, Helse Vest, BFS and NIH.