X‐ray crystal structure of CutA from Thermotoga maritima at 1.4 Å resolution

The structure of the CutA protein from Thermotoga maritima (tmCutA) was determined at 1.4 A resolution using the Se-Met multiwavelength anomalous diffraction (MAD) technique. This protein (TIGR annotation - TM1056, DNA bases 1,069,580–1,069,885) is conserved in numerous bacteria, archaea and eucarya, including plants and mammals (COG1324, Fig. 1). The CutA Escherichia coli homolog—CutA1 (35% ID) is involved in divalent cation homeostasis,1 while the mammalian homolog—mCutA (40% ID) was found to be associated with cell surface acetylcholinesterase.2 However, the biological function of the CutA proteins is yet to be determined. Fig. 1 Multiple sequence alignment of CutA from Thermotoga maritima (Thema) against other periplasmic divalent cation tolerance proteins from bacteria (Aquae = Aquifex aeolicus, Deira = Deinococcus radiodurans) and archaea (Pyrho = Pyrococcus horikoshii, Arcfu . The tmCutA monomer assumes a two-layer α/β α/β sandwich fold (β1β2α1β3β4α3β5β6α5) shared by a large number of proteins (CATH number 3.30.70.120). Two short (β2β5 and two long (β3β4) β-strands arranged in an antiparallel fashion β5↑β2↓β3↑β4↓ form a curved sheet. Three α-helices cover one side of the molecule (Fig. 2a). Each monomer is elongated with overall dimensions approximately 25 × 30× 60 A (Fig. 2a). The monomers are assembled into a trimer with dimensions 55 × 55 × 35 A through interaction of the edges of three β-strands (β3 on the top and β5β6 on the bottom) (Fig. 2b). In the trimer, the subunits are related by crystallographic three fold axes. The same oligomeric state was observed for tmCutA in solution by size exclusion experiments (see Methods) and appears to be functionally relevant. In the trimer, there is a cavity that is accessible from outside via three solvent accessible channels. The inner surface of the cavity is lined with several conserved residues including Cys35, which may serve as a potential metal binding site. Fig. 2 Ribbon representations of the tmCutA monomer (a), trimer (b) and superimposed structures of tmCutA and GlnK’ complex with ATP (c). a: The α-helixes and β-strains are colored in red and blue, respectively. b: Each monomer in the . Structural homologues of tmCutA were identified using Dali search3 and SSAP structure comparison program 4. The closest structural matches, with a Dali score of 9.4 Z, were the E. coli signal transducer proteins GlnB (PII)5 and GlnK6 (Fig. 2c). These two proteins are 67% identical to each other but have insignificant sequence similarity (less than 15% ID) to tmCutA. GlnB and GlnK, which are also trimeric, are involved in maintaining nitrogen homeostasis7–9 and have been shown to cooperatively bind ATP and α-ketoglutarate.6,10 The ATP binding sites of GlnB and GlnK are located in clefts between the momoners,11 but most of the conserved amino acids that mediate ATP binding in PII proteins are not found in tmCutA. The cavity on the side of tmCutA is formed by a number of conserved aromatic (Tyr45, Trp47, Tyr81, and Trp101) and charged residues (Asp54, Glu56, Glu82) (Figs. 1 and ​and2c),2c), which makes this cleft a strong candidate for a conserved function. In PII-type proteins, the two central β-strains β3 and β4 are joined by a flexible loop called the T-loop, which is composed of 17 amino acids. The T-loop plays a key role in protein-protein interactions with downstream effector proteins.12 The β3 and β4 strands in tmCutA, while longer than the corresponding strands in the PII proteins (12 residues in the β3 strand of tmCutA compared with 9 in the β2 strand of PII proteins), are joined by only a two-amino acid (Lys48Gly49) turn and form a hairpin. Although the residues in the C-terminal extension of β3-strand in tmCutA proteins (Tyr45, Trp46, and Trp47, Fig. 1) are conserved among tmCutA homologs, the structure of this region in tmCutA is different than in PII proteins and thus this region of tmCutA may not participate in protein-protein interactions. CutA is annotated as being involved in metal homeostasis. Several other structural homologs of tmCutA, including the PII proteins, metallochaperone Atx1 (5.9 Z score) and the metal-binding domain of the Menkes copper-transporting ATPase (4.5 Z score) have metal-binding properties.13,14 These proteins coordinate the metal through a CXXC sequence motif,15,16 but this motif is not found in CutA. Therefore, tmCutA may represent a new branch of this family that differs from PII and metal binding proteins. The biochemical experiments are under way to define possible ligands and functional partners of CutA proteins.