The structures of the CutA1 proteins

What is it about?

CutA1 (copper tolerance A1) is a widespread cytoplasmic protein found in archaea, bacteria, plants and animals, including humans. In Escherichia coli it is implicated in divalent metal tolerance, while the mammalian CutA1 homologue has been proposed to mediate brain enzyme acetylcholinesterase activity and copper homeostasis. The X-ray structures of CutA1 from the thermophilic bacterium Thermus thermophilus (TtCutA1) with and without bound Na+ at 1.7 and 1.9 A ̊ resolution, respectively, and from the hyperthermophilic archaeon Pyrococcus horikoshii (PhCutA1) in complex with Na+ at 1.8 A ̊ resolution have been determined. Both are short and rigid proteins of about 12 kDa that form intertwined compact trimers in the crystal and solution. The main difference in the structures is a wide-type -bulge on top of the TtCutA1 trimer. It affords a mechanism for lodging a single-residue insertion in the middle of 2 while preserving the interprotomer main-chain hydrogen-bonding network. The liganded forms of the proteins provide new structural information about the metal-binding sites and CutA1 assembly. The Na+–TtCutA1 structure unveils a dodecameric assembly with metal ions in the trimer–trimer interfaces and the lateral clefts of the trimer. For Na+–PhCutA1, the metal ion associated with six waters in an octahedral geometry. The structures suggest that CutA1 may contribute to regulating intracellular metal homeostasis through various binding modes. In order to reveal the molecular determinants of metal-binding sites in TtCutA1 and PhCutA1, we report the crystal structures of metal-free TtCutA1 in the hexagonal space group P3221 and sodium-ion-liganded TtCutA1 in the cubic space group I23 at 1.9 and 1.7 A ̊ resolution, respectively, and of PhCutA1 in the orthorhombic space group P212121 with Na+ at 1.8 A ̊ resolution. The structures provide clues about how CutA1 binds and/ or segregates metal ions, which may help to maintain the proper amount of available ions in the cell. The study is of general interest, as the coordination chemistry used by a protein for metal management should be distinctive from that used to carry out catalytic and/or stability functions.

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Why is it important?

The CutA1 structures present several ion-binding sites with little effect on the local structural conformation. The Na+–TtCutA1 structure reveals Na+ inside of the lateral clefts of the trimers and at trimer–trimer interfaces. It is likely that ion binding to the surface of the trimer affects the dodecameric assembly of the protein. In Na+– PhCutA1, the hexa-aquated ion represents an effective response mechanism for regulating ions as the metal–water cluster in the pore and thus prevents direct contact of essential cellular components with the metal. The isolation of ions by binding inside of the lateral clefts, trimer–trimer interfaces and full hydration by the hexa-aquated arrangement may allow CutA1 to control the damaging effects of metal ions by sequestering them away from other cellular compo- nents. Owing to its compact fold, CutA1 has functionally important areas distributed over a large portion of its structure. The presenta- tion of different positions for ion binding may be specific to CutA1 crystals. It appears that CutA1 undergoes dynamic trimeric, dode- cameric or other associations depending on the metal status of the cellular compartment in which it is contained and a variety of other factors. Modifications of assembly states may allow CutA1 to operate in different parts of the cell in various environments for a range of challenges.

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