Molecular mechanism of thioredoxin regulation in photosynthetic A2B2-glyceraldehyde-3-phosphate dehydrogenase -- Fermani et al. 104 (26): 11109 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Fermani et al. 10.1073/pnas.0611636104.

Supporting Information

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SI Figure 7
SI Table 1
SI Figure 8
SI Table 2
SI Table 3
SI Table 4

SI Figure 7

Fig. 7. Amino acid sequence alignment of spinach GAPDH subunit A (GapA) and subunit B (GapB). Amino acids numbering corresponds to that used throughout the text and in the figures. Color codes: the light-gray background indicates the coenzyme-binding domain; the dark-gray background indicates the CTE (with redox-sensitive cysteines written in black). Amino acids on white background belong to the catalytic domain. The sequence of the S-loop is underlined. Site-specific mutations are reported below GapB sequence as white letters on a black background.

Fig. 8. Detail of the final [2F_o-F_c] electron-density map contoured at 1 s, showing the portion of the CTE belonging to chain O. Atom color codes: carbon, yellow; oxygen, red; nitrogen, blue; sulfur, green. Residues not belonging to the CTE are dark gray for the sake of clarity. The image was produced by Xtalview (1) and rendered by Raster3D (2).

McRee DE (1992) J Mol Graphics 10:44-47.

2. Merritt EA, Bacon DJ (1997) Methods Enzymol 277:505-524.

Table 1. Thermal parameters (B values) of important residues (Arg77, Arg183, Arg191, CTE, and sulfate ions in P_s and P_i sites) of oxidized A₂B₂-GAPDH and [A+CTE]₄-GAPDH, both complexed with NADP

Â	A₂B₂-GAPDH				[A+CTE]₄-GAPDH
Â	A subunit		B subunit		A subunit
Â	Mean B, Ã…²	B range, Ã…²	Mean B, Ã…²	B range, Ã…²	Mean B, Ã…²	B range, Ã…²
Arg77	50.4	49.1-51.9	48.5	47.7-49.0	47.1	37.6-63.2
Arg183	19.4	17.6-20.6	26.0	25.2-26.8	29.1	22.9-37.1
Arg191	33.5	33.2-33.8	38.5	37.1-39.5	39.9	31.5-46.0
CTE	/	/	71.8	64.0-83.6	/	/
SO₄ (P_s site)*	35.6	18.4-48.1	52.0	31.3-65.3	57.5	36.7-65.5
SO₄ (P_i site)^Â†	50.6	42.7-64.0	51.3	/	54.2	43.1-71.0

^Â

Mean B values were calculated on the basis of B values of corresponding residues in each subunit of the asymmetric unit. The B value of each residue was derived from both main- and side-chain atoms. The lowest and the highest B value of each residue are reported in the "B range" column.

*The occupancy (q) values for sulfate ions in the P_s site varied between 0.75 and 1 for subunits A, between 0.5 and 0.75 for subunits B of A₂B₂-GAPDH, and between 0.75 and 1 for [A+CTE]₄-GAPDH.

^Â†

The occupancy (q) values for sulfate ions in the P_i site varied between 0.5 and 0.75 for subunits A of A₂B₂-GAPDH and between 0.5 and 1.0 for [A+CTE]₄-GAPDH. Only one subunit B (chain T) showed a sulfate ion in the P_i site with a q value of 0.5.

Table 2. Shortest distances (<5 Ã…) in A₂B₂-GAPDH structure between the final region of CTE and protein residues or ligand atoms

Chain	CTE residues	Protein (chain) residues or ligand atoms	Distances, Ã…
O	Glu362-OE2	Arg183 (O)-NH2	2.6
O	Glu362-OE1	Arg183 (O)-NH1	4.3
O	Glu362-OE1	NDP (R)-AOP3	3.7
O	Glu362-OXT	Arg77 (O)-NH2	4.7
O	Tyr361-O	Arg183 (O)-NH1	4.2
O	Tyr361-OH	SO₄ P_s site (O)-O4	4.5
Q	Glu362-OE2	Arg183 (Q)-NH1	4.6
Q	Glu362-OXT	Arg183 (Q)-NH1	4.6
Q	Glu362-O	Arg183 (Q)-NH2	4.5
Q	Glu362-O	NDP (P)-AOP3	3.0
Q	Tyr361-O	Arg183 (Q)-NH1	3.5
Q	Tyr361-OH	SO₄ P_s site (Q)-O2	2.8
T	Glu362-OE1	Arg183 (T)-NH1	3.5
T	Glu362-OXT	Arg183 (T)-NH2	4.6
T	Tyr361-O	Arg183 (T)-NH1	3.1
T	Tyr361-OH	SO₄ P_s site (T)-O2	3.6

Table 3. Site-specific mutants of B₄-GAPDH were obtained as described in ref. 1 with the following PCR primers

Primers

R183A (left) 5'-TACACTGGTGACCAGGCGCTGTTGGATGCTTCTCAC-3'

R183A (right) 5'-TGAGAAGCATCCAACAGCGCCTGGTCACCAGTGTAG-3'

R191A (left) 5'-GTTGGATGCTTCTCACGCGGACTTGAGAAGAGCCAGG-3'

R191A (right) 5'-ctggctcttctcaagtccgcgtgagaagcatccaac-3'

D345N (left) 5'-GAAGATTTCTGCAAGAACAACCCTGCTGATGAGG-3'

D345N (right) 5'-CCTCATCAGCAGGGTTGTTCTTGCAGAAATCTTC-3'

E356Q (left) 5'-CCTGCTGATCAGGAGTGCAAACTTTACGAGTAAGG-3'

E356Q (right) 5'-CCTTACTCGTAAAGTTTGCACTCCTGATCAGCAGG-3'

E357Q (left) 5'-CCTGCTGATGAGCAGTGCAAACTTTACGAGTAAGG-3'

E357Q (right) 5'-CCTTACTCGTAAAGTTTGCACTGCTCATCAGCAGG-3'

1. Sparla F, Fermani S, Falini G, Zaffagnini M, Ripamonti A, Sabatino P, Pupillo P, Trost P (2004) J Mol Biol 340:1025-1037.

Table 4. Unit-cell parameters and data collection, refinement, and geometry statistics for oxidized A₂B₂-GAPDH and [A+CTE]₄-GAPDH, both complexed with NADP

Â	A₂B₂-GAPDH	[A-CTE]₄-GAPDH
Space group	C2	P2₁
Unit cell, Ã…, Â°	α = 186.15, b = 215.69, c = 81.47, β = 102.52	α = 105.38, b = 217.71, c = 114.79, β = 90.04
Resolution	91.2-3.6 (3.73-3.60)	72.5-2.4 (2.49-2.40)
Measured reflections	492,368	217,944
Unique reflections	36,699 (3,647)	154,012 (12,396)
Completeness, %	99.9, 99.3	76.6, 62.0
I/σ, I	4.5, 1.5	6.5, 1.5
R _sym	0.359 (0.694)	0.049 (0.336)
Solvent content a.u., %	65	56
R -factor/R_free	0.261/0.292	0.249/0.258
Reflection used in refinement*	36,271	153,938
Ramachandran plot, %^Â†	72.1, 25.2, 2.1, 0.7	88.4, 10.3, 0.9, 0.4
rmsd bond lengths and angles, Ã…, Â°	0.014, 1.9	0.018, 2.5
Overall estimated coordinate error, Ã…^Â‡	0.5	0.3
Mean B-value, Ã…²	30.9	24.6

^{*All observed reflections were used in refinement
^Â†The values refer to the percentage of residues in most favored, additionally allowed, generously allowed, and disallowed regions. The Ramachandran plot was calculated by PROCHECK (1).
^Â‡The overall estimated coordinate error was calculated by applying the Luzzati method (2).
Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) J Appl Crystallogr 26:283-291.
2. Luzzati V (1952) Acta Crystallogr 5:802-810.}