Structure of the stand-alone RAM-domain protein from Thermus thermophilus HB8

Abstract

The stand-alone RAM (regulation of amino-acid metabolism) domain protein SraA from Thermus thermophilus HB8 (TTHA0845) was crystallized in the presence of zinc ions. The X-ray crystal structure was determined using a multiple-wavelength anomalous dispersion technique and was refined at 2.4 A resolution to a final R factor of 25.0%. The monomeric structure is a betaalphabetabetaalphabeta fold and it dimerizes mainly through interactions between the antiparallel beta-sheets. Furthermore, five SraA dimers form a ring with external and internal diameters of 70 and 20 A, respectively. This decameric structure is unique compared with the octameric and dodecameric structures found for other stand-alone RAM-domain proteins and the C-terminal RAM domains of Lrp/AsnC-family proteins.

Figure 1

Amino-acid sequence alignment of T. thermophilus SraA (TTHA0845) with SARD proteins and C-terminal RAM domains of Lrp/AsnC-family proteins (HTH-RAM). The sequences were aligned using ClustalW (Thompson et al., 1994 ▶). Secondary-structure elements of SraA, obtained using the DSSP program (Kabsch & Sander, 1983 ▶), are shown above the alignment. Red, green, blue and yellow represent β-strand, 3₁₀-helix, α-helix and π-helix, respectively. Species abbreviations are as follows: Therm.t., Thermus thermophilus; Rhiz.l., Rhizobium loti; Strep.a., Streptomyces avermitilis; Arch.f., Archaeoglobus fulgidus; Pyro.f., Pyrococcus furiosus; Pyro.sp., Pyrococcus sp.; Myco.t., Mycobacterium tuberculosis; Bacl.s., Bacillus subtilis; E.coli, Escherichia coli; Sulf.s., Sulfolobus solfataricus. The amino-acid sequences of the Q8U228_Pyro.f. and DM1_Pyro.sp proteins are the same as those of the P. furiosus PF1022 and P. horikoshii PHS023 proteins, respectively.

Figure 2

Purification and gel-filtration analysis of the recombinant SraA protein. (a) The recombinant protein (3 µg) was analyzed by SDS–PAGE (lane 2). Lane 1, molecular-weight markers (kDa). (b) The purified SraA protein (3.36 µg/1.7 µl) was analyzed by gel-filtration column chromatography in the presence of 0.4 µg thyroglobulin (669 kDa), 0.9 µg ferritin (440 kDa), 0.2 µg catalase (158 kDa) and 1.3 µg each of bovine serum albumin (BSA) (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (20.4 kDa) and RNase A (13.7 kDa). The elution profile of the sample was detected by measuring A ₂₁₅. The peak for each protein is indicated by an arrow.

Figure 3

Schematic representation of the overall fold of the T. thermophilus SraA protein. (a) The dimeric structure (subunits A and F). The colour coding is the same as in Fig. 1 ▶. (b) The decameric structure. Subunits A–J are indicated. Zinc ions in the asymmetric unit are coloured grey. (c) Stereoview of the zinc-binding site. A zinc ion interacts with Glu50 and Asp54 of helix α2 in subunit B and Glu20 of helix α1 in subunit A of the neighbouring ring. These figures were prepared using MOLSCRIPT (Kraulis, 1991 ▶) and RASTER3D (Merritt & Murphy, 1994 ▶).

Figure 4

(a) The Asp39 residues of SraA are coloured green on a ribbon diagram of the decameric ring. The side-chain atoms of Asp39 are represented by a ball-and-stick model. This figure was prepared with MOLSCRIPT (Kraulis, 1991 ▶). (b) The Asp39 residues are represented on the surface of the tetrameric SraA molecule composed of the AF (yellow) and BG (white) dimers. This figure was prepared using PyMOL (http://www.pymol.org).