Supplemental Data


Abstract
Full Text
The Human Nuclear Xenobiotic Receptor PXR: Structural Determinants of Directed Promiscuity
Ryan E. Watkins, G. Bruce Wisely, Linda B. Moore, Jon L. Collins, Millard H. Lambert, Shawn P. Williams, Timothy M. Willson, Steven A. Kliewer, Matthew R. Redinbo

Supplementary Material


Supplemental Table 1. Comparison of hPXR-LBD with nuclear receptor LBDs of known structure. The cavity volumes were calculated with GRASP (1) using a grid spacing of 0.2 Å, probe radius of 1.4 Å and the atomic radii of Bondi (H = 1.2 Å, C = 1.7 Å, N = 1.55 Å, O = 1.52 Å, S = 1.8 Å) (2), where hydrogens were treated explicitly. Openings in the PXR, PPARgamma and RXRalpha pockets were closed by covering these proteins with an external layer of water molecules prior to calculation of the molecular surface and cavities.
Number Residues SuperimposedSequence Identity (%)RMSD (Å)Ligand-Binding Cavity Volume (Å3)
PXR------1,150
Vit D Receptor225451.8871
RXRalpha178222.8687
PPARgamma203252.61,619
Progesterone Receptor201192.9557
Estrogen Receptor180182.8476

Supplemental Table 2. Calculated buried solvent-accessible surface area of amino acid residues that line the ligand-binding cavity of hPXR. Boldface indicates identity; italics indicates similarity. The buried surface area upon ligand binding was calculated using CNS (3). The four residues underlined were mutated to examine the ligand specificity of mouse and human PXR.
ResidueBuried surface (Å2)Contacts SR12813 Ligand?HumanRabbitRatMouse
2069.8YesLLRS
20815.8YesSTPP
20930.5YesLMMM
21116.3YesVLII
24014.8YesLLLL
24343.6YesMLLL
2441.9NoAAAA
2466.1YesMMVV
24730.4YesSSSS
25111.2YesFFFF
28129.3YesFLFF
2849.6YesCCCC
28523.0YesQLII
28829.6YesFFFF
29928.6YesWWWW
30612.9NoYYYY
3083.7NoLVFF
3214.2NoEDDD
32320.3YesMLLL
32412.0YesLLMM
3272.5NoHHHH
40741.6YesHHQQ
41014.7YesRRQQ
41133.1NoLLLL
41411.5NoIIII
4209.3YesFFFF
4256.2NoMMMM
4293.6NoFFFF


Supplemental Figure 1. Sequence alignment of the ligand binding domains of human PXR, VDR, RXR and ER. Alignment of the sequences of human PXR, the vitamin D receptor (VDR), the 9-cis retinoic acid receptor alpha (RXRA) and the estrogen receptor alpha (ERA) is shown, with alpha helical regions in light red, beta strands in light green, and regions not traced in the corresponding crystal structures in yellow. Amino acid residues are colored by chemical type, and the positions of hPXR secondary structural elements are indicated.


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Supplemental Figure 2. Schematic representation of SR12813 binding to hPXR-LBD. Interactions between the three SR12813 orientations and residues lining the PXR ligand binding cavity, with van der Waals contact indicated with solid arrows, and hydrogen bonds with dashed arrows. (A) Position 1 SR12813. (B) Position 2 SR12813. (C) Position 3 SR12813. Created with CS ChemDraw Pro (CambridgeSoft).


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References

1. A. Nicholls, K. A. Sharp, B. Honig, Proteins11, 281 (1991).

2. A. Bondi, J. Phys. Chem. 68, 441 (1964).

3. A. T. Br�nger et al., Acta Crystallogr. D54, 905 (1998).