The Evolutionarily Conserved Trimeric Structure of CutA1 Proteins Suggests a Role in Signal Transduction

Abstract

CutA1 are a protein family present in bacteria, plants, and animals, including humans. Escherichia coli CutA1 is involved in copper tolerance, whereas mammalian proteins are implicated in the anchoring of acetylcholinesterase in neuronal cell membranes. The x-ray structures of CutA1 from E. coli and rat were determined. Both proteins are trimeric in the crystals and in solution through an inter-subunit ␤-sheet formation. Each subunit consists of a ferredoxin-like (␤1␣1␤2␤3␣2␤4) fold with an additional strand (␤5), a C-terminal helix (␣3), and an unusual extended ␤-hairpin involving strands ␤2 and ␤3. The bacterial CutA1 is able to bind copper(II) in vitro through His 2 Cys coordination in a type II water-accessible site, whereas the rat protein precipitates in the presence of copper(II). The evolutionarily conserved trimeric assembly of CutA1 is reminiscent of the architecture of PII signal transduction proteins. This similarity suggests an intriguing role of CutA1 proteins in signal transduction through allosteric communications between subunits.CutA1 is a widespread protein of about 12 kDa found in bacteria, plants, and animals, including humans. The protein was originally identified in a gene locus of Escherichia coli called cutA involved in divalent metal tolerance (1). The cutA locus consists of two operons, one containing a single gene encoding a cytoplasmic protein, CutA1, and the other composed of two genes encoding a 50-kDa (CutA2) and a 24-kDa (CutA3) inner membrane proteins. Molecular genetics studies on the E. coli cutA locus showed that some mutations lead to copper sensitivity due to its increased uptake (1). However, the specific function of CutA1 in E. coli is still unknown. On the other hand recent studies from two independent groups highlighted a possible role of mammalian CutA1 in the anchoring of the enzyme acetylcholinesterase (AChE) 1 in neuronal cell membranes (2, 3). CutA1 does not directly interact with AChE (2), but the CutA1 gene is widely expressed in different regions of the brain with an expression pattern that parallels that of AChE (3). In addition CutA1 copurified with AChE from human caudate nucleus (3). CutA1, thus, might provide an intriguing link between copper tolerance in bacteria and a complex process in the brain of the most evolved organisms. The function of CutA1 in plants is still unknown.Copper is a transition metal essential to all organisms since it is involved in many redox reactions and in several biological processes (4). Although essential for cellular metabolism, copper is highly toxic when it exceeds cellular needs and accumulates in the cell. Proteins which bind copper are involved in several human neurological pathologies, such as the Menkes and Wilson diseases, the Alzheimer pathology, and the Creutzfeld-Jacob syndrome (5). For this reason, all organisms must have homeostatic mechanisms that allow the intake of the necessary amount of copper, thus preventing its accumulation beyond the level of toxicity (6, 7); these mechanisms are carried out by proteins t...

Crystallization of soluble proteins in vapor diffusion for x-ray crystallography

“…As revealed by the crystal structure, the p-hydroxy-mercury benzoate molecule protects the Cys sulfidryl group from reacting. At the same time, this mercury-protein adduct provides an excellent heavy atom derivative, which we used for the successful structure determination by multiwavelength anomalous diffraction measurements at the Hg edge 67 . The crystal structure (PDB code 1NAQ) revealed a trimeric quaternary structure for CutA1 with all Cys exposed to solvent covalently modified by the Hg-compound.…”

“…The protein was originally identified in a gene locus of Escherichia coli called cutA that is known to be involved in divalent metal ions tolerance, but its specific role in the different organisms was not clear. In order to shed light on the function of the protein, we started a structural determination project of one representative protein from the bacteria (E. coli CutA1) and one from mammals (rat CutA1) 67 . The E. coli protein used for the initial crystallization screenings was highly pure and well folded, as indicated by mass spectra and NMR spectroscopy, respectively, and the concentrated solutions appeared to be stable for several days.…”

“…A classical example of the usefulness of mercury compounds for obtaining high-quality crystals is given by the crystallization of human carbonic anhydrase 66 . A more recent example of the use of organomercury compounds is provided by the successful crystallization of both bacterial and mammalian CutA proteins 67 (see the ANTICIPATED RESULTS for the discussion). 17| Aliquot the protein sample into multiple small Eppendorf tubes: transfer 100 ml aliquots of protein solution for the initial screenings (Steps 25-52) and 20 ml aliquots of protein solution for optimization (Steps 53-98).…”

CutA Divalent Cation Tolerance Homolog (Escherichia coli) (CUTA) Regulates β-Cleavage of β-Amyloid Precursor Protein (APP) through Interacting with β-Site APP Cleaving Protein 1 (BACE1)

“…Additional studies showed that many CutA proteins have a copper binding capacity and that copper could induce reversible aggregation of the CutA protein (21,22). Furthermore, the trimeric core of CutA is also homologous to that of the intracellular signal transduction protein P-II, implying a role in signal transduction (21). However, the exact functions of the mammalian CUTA protein, especially human CUTA, remain largely unclear.…”

“…In bacteria, CutA is involved in copper tolerance, and some mutations in the cutA gene lead to copper sensitivity due to its increased uptake (20). Additional studies showed that many CutA proteins have a copper binding capacity and that copper could induce reversible aggregation of the CutA protein (21,22). Furthermore, the trimeric core of CutA is also homologous to that of the intracellular signal transduction protein P-II, implying a role in signal transduction (21).…”