Aconitase

The Aconitase - IRP1 Switch

So how does cytosolic aconitase switch between its enzymatic activity and its mRNA-binding regulatory role?

It all depends upon its binding to a [4Fe-4S] cluster. When the iron-sulphur cluster is bound, the protein functions as a cytosolic aconitase; but when the cell lacks iron, leading to the disassembly of iron-sulphur clusters, cAcn converts to IRP1. Therefore, factors that affect the assembly and disassembly of iron-sulphur clusters can help regulate cAcn and IRP1 activities - such as iron availability or oxidative stress. Iron excess promotes cAcn and iron starvation promotes IRP1.

cAcn/IRP1 Structures

cAcn/IRP1 is composed of four domains, but their structure and relative organisation changes between the two functional forms of this protein. The three N-terminal domains have a similar a/b/a structure, while the C-terminal domain 4 is a mobile ‘swivel’ domain with a b/b/a structure (the domain architecture is similar in mAcn, but in bacterial AcnB the domains are swapped around, with the ‘swivel’ domain 4 near the N-terminal, preceded by a distinct HEAT-like domain).

In cAcn, the four domains come together to form a compact globular protein with the [4Fe-4S] cluster embedded in the centre at the interface of the four domains, the cluster being bound by residues in domains 1 and 2 (which form the core of the protein). Residues required for RNA-binding are inaccessible, being buried within the cAcn protein and blocked by binding to the iron-sulphur cluster. As such, the RNA-binding sites of IRP1 overlap with the enzyme active site of cAcn.

Upon disassembly of the [4Fe-4S] cluster, cAcn undergoes a rearrangement of its domains, as well as some internal conformational changes. Whereas domains 3 and 4 abut one another in cAcn, these domains swivel apart from one another to form an L-shaped open structure with domains 1 and 2 forming the central core. This movement mainly concerns the ‘swivel’ domain 4. The result is to open up a central cavity, allowing the IRE stem-loop of mRNA access to two newly formed RNA-binding sites in IRP1 (all four domains make contact with the IRE sequences in the mRNA).

mRNA binding

The IRE stem-loop structure makes several contacts with IRP1, resulting in high affinity and specificity for certain mRNA transcripts. Furthermore, the wide separation of the two RNA binding sites prevents interactions with non-specific RNAs. This 2-point recognition system may account for the different responses observed for different IRE-containing mRNAs. In addition, the variations in IRE stem-loop structures found among different mRNAs coincide with graded responses to iron signals and different IRP binding stabilities.

Next: What InterPro Tells Us

Previous: Iron Regulatory Protein 1, How It Works