Sterol Carrier Protein-2 and Anandamide Cellular Uptake
Supplementary Information: Mol. Neurobiol.
Elizabeth SabensLiedhegner, Caleb D. Vogt, Daniel S. Sem, Christopher W. Cunninghamand Cecilia J. Hillard
Neuroscience Research Center and Departments of Pharmacology and Toxicology,Medical College of Wisconsin, Milwaukee, WI 53226
Department of Pharmaceutical Sciences, Concordia University Wisconsin School of Pharmacy, Mequon, WI 53097
Please send correspondence and proofs to:
Cecilia J. Hillard, Ph.D.
Neuroscience Research Center
Medical College of Wisconsin
8701 Watertown Plank Road
Milwaukee, WI 53226
Phone: 414-955-8493
Fax: 414-955-6545
Table of Contents:
TitlePage
Comparison of NMR structure of SCP-2 with crystal structure of an SCP-2-like domainS2
Detailed molecular docking of 16-doxyl-stearic acid within the SCP-2 binding siteS3
Comparison of NMR structure of SCP-2 with crystal structure of an SCP-2-like domain:While there is no structure of human SCP-2 with a ligand bound, there is an NMR structure of substrate-free SCP-2. While there is no structure of a substrate-bound form of human SCP-2, there is a structure of a homologous protein, human peroxisomal multifunctional enzyme type 2 (MFE-2), with a lipid substrate (Triton X-100). MFE-2 contains an SCP-2-like domain and was co-crystalized with lipid analogue, Triton X-100 (1ikt)(1). Before using theSCP-2 NMR structure of human SCP-2(1qnd, pdb.org)(2)as a basis for automated docking, we first attempted to identify the substrate binding pocket by measuring the ability to dock the known reference substrate ligand (Triton X-100) into the active site cavity of MFE-2. The Triton X-100 ligand was removed, and protein structures aligned. Triton X-100 was then subjected to docking and scored for free energy (ΔG) of binding within the lipophilic cavity of each protein. The mean free energy conformations for the lowest energy binding scores were then selected and overlaid, and are shown in Figure S1. Triton X-100 engaged in similar molecular modes of binding within both proteins, suggesting the relevant binding site in the NMR structure of SCP-2 is similar to that in MFE-2, and is viable for targeted docking in the current study. Also, the ability to reproduce the crystal structure binding pose in MFE-2 provides validation for the docking method (using Autodock 4.0) and scoring function that was used.
Fig. S1Overlap of SCP-2 NMR structure (1qnd, magenta) and MFE-2 SCP-2-like domain crystal structure (1ikt, yellow). Automated docking of Triton X-100 into SCP-2 (cyan) and co-crystalized with MFE-2 (green) shows a high degree of overlap between predicted and experimentally determined molecular modes of action, respectively.
Detailed molecular docking of 16-doxyl-stearic acid within the SCP-2 binding site.The putative substrate binding site of SCP-2 was first proposed based on NMR studies using 16-doxyl-stearic acid, which completely broadened Thr85 and Gly86 NMR signals(2). The lowest relative free energy binding conformation of 16-doxyl-stearic acid is shown in Fig S2. 16-Doxyl-stearic acid engaged in electrostatic interactions with the side-chain of Lys122, similar to arachidonic acid. The lipophilic tail was found to preferentially maximize hydrophobic interactions with the backbone extending from Asn120-Pro118, thus binding in an alternate orientation than other arachidonate analogues modeled here. The close proximity between the free radical N-O functionality and Thr85-Gly86 in Fig S2 supports NMR studies described earlier.
Fig. S2 Targeted docking of 16-doxyl-stearic acid (light blue) with SCP-2 (1qnd, cyan). The anionic carboxylate of 16-doxyl-stearic acid forms a salt bridge with the cationic sidechain of Lys122. The flexible, saturated C14 sidechain linking the carboxylate head group with the 3-oxazolidinyloxy terminal group allows for a preferential binding mode that orients the radical quenching NO toward Thr85 and Gly86. This is in agreement with the original NMR study by Wüthrich(2), which showed the NMR peaks ofthese two amino acids were significantly broadened when bound by 16-doxyl-stearic acid. Figure produced using PyMOL.
References
1.Haapalainen AM, van Aalten DM, Merilainen G, Jalonen JE, Pirila P, Wierenga RK, Hiltunen JK, Glumoff T (2001) Crystal structure of the liganded SCP-2-like domain of human peroxisomal multifunctional enzyme type 2 at 1.75 Å resolution. J. Mol. Biol. 313:1127-1138.
2.Garcia FL, Szyperski T, Dyer JH, Choinowski T, Seedorf U, Hauser H, Wuthrich K (2000) NMR structure of the sterol carrier protein-2: Implications for the biological role. J. Mol. Biol. 295:595-603.
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