Supplementary Content

Crystal structure of a hypothetical protein, TTHA0829 from Thermus thermophilus HB8, composed of cystathionine-b-synthase (CBS) and aspartate-kinase -chorismate- mutase tyrA (ACT) domains

Makoto Nakabayashi*1,2 · Naoki Shibata*1,3 · Emi Ishido-Nakai3 · Mayumi Kanagawa3 · Yota Iio4 · Hirofumi Komori1 · Yasufumi Ueda1 · Noriko Nakagawa4 · Seiki Kuramitsu4 · Yoshiki Higuchi1,3

1. Graduate School of Life Science, University of Hyogo

2. Graduate School of Natural Science and Technology, Okayama University

3. RIKEN SPring-8 Center, Harima Institute

4. Department of Biological Sciences, Graduate School of Science, Osaka University

Figure S1

Ribbon representation of two consecutive CBS motifs (CBS-1 and CBS-2) of TTHA0829

Table S1 and Figure S2

Result of gel-filtration

Figure S32

Stereo diagram representing domain swapping and tetramer formation of TTHA0829

Figure S43

Sequence alignment of several ACT domains

Figure S5

Sequence alignment of TTHA0829 and a putative acetoin dehydrogenase from Symbiobacterium thermophilum

Figure S6

Sequence alignment of TTHA0829 and an IMPDH from Pseudomonas aeruginosa

(Figure S1)

Supplementary Figure S1

Ribbon representation of two consecutive CBS motifs (CBS-1 and CBS-2) of TTHA0829 (stereo view)

CBS-1 contains a2 (amino acid residues 20-30), b1 (34-39), b2 (42-48), a3 (49-54), a4 (60-74) and a5 (77-80). CBS-2 contains a1 (3-6), a6 (94-104),b3 (108-113), b4 (116-122) and a7 (123-134). The a-helices, b-strands and loop regions are shown by red, green and cyan, respectively. The N- and C-termini are labeled.

Molecular Mass
(Da) / log (Molecular
Mass) / Elution Volume
(ml)
Aldolase / 158,000 / 5.1987 / 47.5
BSA / 67,000 / 4.8261 / 54.2
Ovalbumin / 43,000 / 4.6335 / 60.2
Chymotrypsinogen / 25,000 / 4.3979 / 71.5
Ribonuclease A / 13,700 / 4.1367 / 79.0
TTHA0829 / 88,064 / 4.9448 / 53.0

(Table S1)

(Figure S2)

Supplementary Table S1 and Figure S2

Result of gel-filtration

Elution volumes obtained from gel-filtration profiles of the aldolase (of which the molecular mass is 158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), ribonuclease A (13.7 kDa) are 47.5, 54.2, 60.2, 71.5 and 79.0 ml, respectively. Molecular mass of the TTHA0829 estimated from the elution volume (53 ml) is 88 kDa (table S1 and figure S2), suggesting that the TTHA0829 takes tetrameric structure.

HiLoad 16/60 Superdex 75 pg (GE Healthcare) and buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl were employed in this experiment.

(Figure S32A)

(Figure S32B)

Supplementary Figure S32

Stereo diagram representing domain swapping and tetramer formation of TTHA0829

Two CBS-2 domains interact mainly through hydrophobic amino acid residues. Val 123, Leu 127, Phe 130 and Leu 134 are arranged on the interface. In addition, the side chain of Arg 184 of the C-terminal domain interacts with the Glu 95 side chain of CBS-2 of the other subunit. A salt bridge between the Arg 184 and Glu 95 side chains is shown as a magenta dotted line. One of the two monomers is shown as a ribbon model and the other is shown as a chain-trace model (Fig. S32A).

Lys 53 and Asp 54 of CBS-1 in one subunit interact with Asp 54 and Lys 53 of the other subunit, respectively. The two dimer-units contact each other through hydrophobic interactions between Trp 66 at the interface of the CBS-1 region to make a tetramer. The four subunits are equivalent crystallographically. One of the four monomers is shown as a ribbon model and the other three are shown as chain-trace models (Fig. S32B).

(Figure S43)

Supplementary Figure S43

Sequence alignment of several ACT domains

Sequences of the C-terminal ACT domains of TTHA0829 (from Thermus thermophilus), PGDH (from Escherichia coli and Mycobacterium tuberculosis) and AK (from Escherichia coli and Arabidopsis thaliana) are shown. Residues forming a-helices and b-strands are colored red and blue, respectively. Amino acid residue deletions are indicated by ‘–’ symbols. The three black arrows point to, Leu and Gly consensus residues. The glycine residues found characteristically in side-by-side type ACT domains are framed by black rectangles and are conserved in the C-terminal domain of TTHA0829.

(Figure S5)

Supplementary Figure S5

Sequence alignment of TTHA0829 and a putative acetoin dehydrogenase from Symbiobacterium thermophilum

Amino acid residue deletions are indicated by ‘–’ symbols. One-letter codes colored in red mean the consensus amino acid residues. Sequence identity between the two proteins is 33 % (= 68 identical residues / 205 overlapped residues).

(Figure S6A)

(Figure S6B)

Supplementary Figure S6

Sequence alignment of TTHA0829 and an IMPDH from Pseudomonas aeruginosa

Amino acid residue deletions are indicated by ‘–’ symbols. One-letter codes colored in red mean the consensus amino acid residues. Sequence identity between the two proteins is 25 % (= 53 identical residues / 209 overlapped residues) (Fig. S6A).

Positions of the consensus residues are highlighted by red and the other part are colored in gray in three-dimensional structure of the IMPDH from Pseudomonas aeruginosa (stereo view, PDB entry 4DQW chain A). ATP molecules bound to the IMPDH are shown as blue stick models. More than half of the consensus residues concentrate adjacent to its ATP-binding site composed of two CBS domains (Fig. S6B).

S2