Structural basis of hereditary coproporphyria -- Lee et al. 102 (40): 14232 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Lee et al. 10.1073/pnas.0506557102.

Supporting Information

Files in this Data Supplement:

Supporting Text
Supporting Table 1
Supporting Figure 6
Supporting Figure 7
Supporting Table 2
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10

Supporting Figure 6

Fig. 6.
Residue interactions across the CPO dimer interface. Hydrogen bonds between any two residues are shown as blue lines. Each line represents one H bond. Nonbonded contacts are shown as red-striped lines whose width is proportional to the number of atomic contacts. The identity of the secondary structural element that harbors each residue is also shown.

Supporting Figure 7

Fig. 7.
Human CPO is a homodimer in solution. Equilibrium scans at 280 nm are shown in B (gray circles) with the fitted model (monomer-dimer-tetramer, solid lines) overlaid. The best fit curve from a global analysis of two different starting concentrations at five different speeds yields a K_D = 0.5 mM. The combined residuals to all fitted scans are shown in A. The relative monomeric molecular mass of 39,370 determined from the equilibrium analysis is in excellent agreement with that calculated from the sequence, 39,248.

Supporting Figure 8

Fig. 8.
A three-dimensional view of citrate bound at the active site. Dotted lines (cyan) indicate hydrogen bonds.

Supporting Figure 9

Fig. 9.
Configuration of the CPO active site. Residues identified in the decarboxylation corridor are invariant in >500 unique CPO sequences.

Supporting Figure 10

Fig. 10.
Structural consequences of H327R, R328C, R331W, and R391W. Residues altered by HCP mutations are shown in red.

Table 1. Data collection and crystallographic statistics

X-ray data
Source	BL 9-2, SSRL	MAD phasing, BL 5.0.2, ALS
Wavelength, Å	0.9536Å	0.9795Å	0.9796Å	0.9537Å
Space group	P23	P23
Cell dimensions, Å	a = b = c = 112.720	a = b = c = 112.696
Resolution, Å	1.58	1.9	1.9		1.9
Measured reflections	415,420	455,649	455,312		455,407
Unique reflections	65,081	37,254	37,201		37,188
Average I / σ(I) (last shell)	41.9 (2.5)	32.8 (2.2)	30.3(2.1)		27.4 (1.9)
Completeness I / σ(I) > 0, % (last shell)	99.5 (98.3)	99.6 (96.9)	98.9 (96.3)		98.6 (96.3)
R_sym*(last shell)	0.050 (0.670)	0.047 (0.563)	0.049 (0.593)		0.051 (0.592)
No. of selenium sites	7
Figure of merit (sharp)	0.48
Refinement
Resolution range, Å	32.0–1.58
Reflections used, \|F \| / σ\|F\| > 0, working/test	61,774/3,293
R_cryst^† buster and refmac5 (outer shell)	0.186 (0.230)
R_free^‡ buster and refmac5 (outer shell)	0.210 (0.245)
Average B factors, Å²
Protein (2,880 atoms)	28.3^§
Citrate (12 atoms)	34.2
rms deviation from ideality
Bond lengths, angle distances, Å	0.011, 0.024
Bond, torsion, and improper torsion angles, °	1.36, 22.3, 0.87
Bonded B factors, Å² (main chain, side chain)	1.1, 1.6
Ramanchandran plot, % (most favored, allowed)	93.8, 6.2

SSRL, Stanford Synchotron Radiation Laboratory; ALS , Advanced Light Source.

*R_sym = Σ½I – <I>½ ¤ Σ<I>, where I and <I> are the measured and averaged symmetry-related intensities, respectively, of the same reflection.

^†R_cryst = Σ½F_o – F_c½ ¤ ΣF_o, where F_o and F_c are the observed and calculated structure factors, respectively. ^‡R_free is computed with 5% of the total reflections chosen randomly and unused throughout the refinement. ^§Wilson plot of the observed intensities versus resolution yields a B factor of 24.0.

Table 2. Equilibrium fitting results

Model	Variance	Monomer MM	Kd (1-2)	Kd (1-4)
Single ideal species	2.04 ´ 10^–5	77.7	N/A	N/A
Monomer-dimer	1.72 ´ 10^–5	49.5 (42.5, 56.5)	6.71 ´ 10^–6 (1.55 ´ 10^–6, 2.92 ´ 10^–5)	N/A
Monomer-dimer-tetramer	1.31 ´ 10^–5	37.6 (32.6, 42.6)	4.31 ´ 10^–7 (1.13 ´ 10^–7, 1.25 ´ 10^–6)	1.39 ´ 10^–17 (4.36 ´ 10^–19, 4.44 ´ 10^–16)

Eight equilibrium scans were globally fitted to the indicated models. Variance, monomer molecular mass (MM in kilodaltons), and dissociation constants are reported. Values in parentheses represent 95% confidence intervals from a 5,000-iteration Monte Carlo analysis of the fit. The single ideal species model did not fit well enough to warrant Monte Carlo analysis. Kd (1-2) refers to the monomer-dimer dissociation constants, which is reported in molar concentration units, whereas Kd (1-4) refers to the monomer-tetramer dissociation constant, which is reported in molar³ concentration units.