108 



Journal of the Kentucky Academy of Science 66(2) 



Table 1. 



Accession numbers 



for opioid receptor se- 





LlJ>trLl 111 LIII3 oLLlLIV. 







Secjuencx' accession 



Chain 



Receptor 



number 



I 



11 Llllld.il iVl LI 



NP 000905 



0 



X/frMic/^ \/Tn 





3 



Rilf Mil 



NP n'^79n'^ 



4 



Human Kappa 



NF_000903 



5 



Mouse Kappa 



NP_035141 



6 



Rat Kappa 



NP_058863 



7 



Human Delta 



NP_000902 



8 



Mouse Delta 



NP_038650 



9 



Rat Delta 



NP-036749 



10 



EDGl Model 





were compared to experimental literature to 

 verify the accuracy of the models. Finally, the 

 docking results were used to predict what res- 

 idues were needed for binding, helical pack- 

 ing, and binding profiles. 



MATERIALS AND METHODS 

 Alignment 



All modeling in this study was completed 

 using the Molecular Operating Environment 

 (MOE) software package developed by the 

 Chemical Computing Group Inc. (MOE 

 2003). The sequences downloaded from Na- 

 tional Center for Biotechnology and Infor- 

 mation (NCBI) are fisted in Table 1 and rep- 

 resent human, mouse, and rat sequences for 

 the mu, kappa, and delta receptors. The se- 

 quences were aligned with the template struc- 

 ture, the previously published EDCl model 

 (Bautista et al. 2000; Fischer et al. 2001; Par- 

 rill, Baker et al. 2000; Parrill, Wang et al. 2000; 

 Sardar et al. 2002; Wang et al. 2001) using the 

 default settings in MOE. Homologies of the 

 opioid sequences, based on identity, ranged 



from 94% to 47%. The homology of the tem- 

 plate was much lower and ranged from 22% 

 to 20% (Table 2). This low homology would 

 generally be of concern, but GPCR homology 

 models have been successfully developed with 

 homology in this range (Klabunde and Hessler 

 2002). The most conserved residue was 

 aligned manually for each helix (van Rhee and 

 Jacobson 1996) (Figure 1). 



Models 



Homology models of the human mu, kappa, 

 and delta receptors were developed using the 

 default settings in MOE. The template selec- 

 tion is critical for model development. The 

 template should share significant sequence ho- 

 mology and have similar function (Klabunde 

 and Hessler 2002). Two main template se- 

 quences often used are the crystal structure of 

 bovine rhodopsin [PDB ID 1F88] (Palczewski 

 et al. 2000) and a theoretical model of bovine 

 rhodopsin based on electron microscopy 

 [PDB ID IBOJ] (Pogozheva et al. 1997). The 

 crystal structure is considered an inactive tem- 

 plate because it was generated in the dark 

 (Bissantz et al. 2003; Palczewski et al. 2000). 

 A third template, endothefial differentiation 

 gene 1 (EDGl), was used in our study. EDGl 

 is a GPCR whose function is to act as a re- 

 ceptor for phospholipids (Bautista et al. 2000). 

 The EDGl model was developed based on 

 the IBOJ template with modifications (see 

 Bautista et al. 2000). This template was se- 

 lected since it has the extracellular and intra- 

 cefiular loops unlike IBOJ that is compro- 

 mised of only the transmembrane helices and 

 the model was experimentally validated. 



The models were minimized to a root mean 

 squared gradient (RMSG) of 0.1 Kcal/mol-A 



Table 2. Sequence homology for alignment of the human, mouse, and rat opioid sequences. Chains are listed in Table 1. 



Chains 



1 



2 



3 



4 



5 



6 





8 



9 



10 



1 





90.5 



94.0 



60.5 



59.7 



60.3 



58.6 



58.3 



59.1 



20.6 



2 



78.6 





95.0 



60.0 



59.5 



59.7 



58.3 



57.8 



58.6 



19.9 



3 



81.0 



94.3 





60.3 



59.7 



60.0 



58.1 



57.8 



58.6 



19.9 



4 



49.8 



56.9 



57.5 





93.7 



94.2 



58.9 



58.1 



58.3 



19.6 



5 



49.1 



56.4 



57.0 



93.7 





98.9 



57.8 



57.3 



57.5 



19.9 



6 



49.6 



56.6 



57.3 



94.2 



98.9 





58.3 



57.8 



58.1 



19.9 



7 



47.2 



54.1 



54.3 



57.6 



56.6 



57.1 





93.0 



93.5 



22.3 



8 



47.0 



53.6 



54.0 



56.8 



56.1 



56.6 



93.0 





96.5 



22.3 



9 



47.6 



54.4 



54.8 



57.1 



56.3 



56.8 



93.5 



96.5 





22.3 



10 



13.4 



15.0 



15.1 



15.5 



15.8 



15.8 



18.0 



18.0 



18.0 





