Computational Docking to Sweet Taste Receptor Models - American

Chapter 11

Downloaded by UNIV OF MICHIGAN ANN ARBOR on February 18, 2015 | http://pubs.acs.org Publication Date: March 4, 2008 | doi: 10.1021/bk-2008-0979.ch011

Computational Docking to Sweet Taste Receptor Models D. Eric Walters Department of Biochemistry and Molecular Biology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064

Computer modeling tools are extremely useful in understanding the ways that sweeteners interact with their receptors. This chapter describes homolgy-based models of the N-terminal ligand binding domains of the sweet taste receptor, T1R2 + T1R3. These models are used in combination with docking calculations and structure-taste data to identify a likely binding mode for the sweet protein brazzein. The taste-modifying protein miraculin has been modeled, and possible modes of miraculin-taste receptor interaction are also identified.

Introduction The chemical structures of sweet tasting compounds are incredibly diverse. Sucrose and other sugars are the natural ligands for sweet receptors, but other compounds have been known to trigger sweet taste at least since the discovery of saccharin, reported in 1879. The list is long, and it includes polyols (sorbitol, maltitol, lactitol), heterocyclics (saccharin, acesulfame K ) ; amino acids (glycine, D-tryptophan), dipeptides (aspartame, neotame), sulfamates (cyclamate), halogenated sugars (sucralose), terpenes and terpene glycosides (hernandulcin, glycyrrhizin, stevioside, rebaudiosides), urea derivatives (dulcin, superaspartame, suosan), nitroanilines (P-4000), oximes (perillartine). In addition, a number of proteins also have a sweet taste. These include monellin, thaumatin, and brazzein. Curiously, miraculin, a glycoprotein, induces sweet 162

© 2008 American Chemical Society In Sweetness and Sweeteners; Weerasinghe, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


163 taste when it is applied to the tongue and then followed with acidic solutions. It would appear to bind to the sweet receptor and induce the appropriate conformational change when exposed to low pH. The diversity of sweet tasting structures long ago convinced most workers that there must be multiple receptors responsible for detecting sweetness. It was therefore surprising when a single receptor was shown to be responsible for all sweetness transduction (1-3). This receptor is a heterodimer formed by two members of the T1R taste receptor family, T1R2 and T1R3. Subsequently, it has been demonstrated that this receptor has several different binding sites (4-6). Sequence homology of the taste receptor proteins with a metabotropic glutamate receptor indicates that the T1R receptors are G protein coupled receptors of class C. A s illustrated in Figure 1, these proteins have a large extracellular N terminal domain that is linked to the 7-helix transmembrane domain by a small cysteine-rich domain. Since the N-terminal ligand binding domain of the brain metabotropic glutamate receptor, m G l u R l , has been studied using X-ray crystallography ( 7 ) , it has been possible to construct homology-based models of the N-terminal ligand binding domains of T1R2 and T1R3 (8-11).

Figure 1. Schematic representation of the T1R2/T1R3 sweet taste receptor, based upon homology to the mGluRl receptor (7). NTD = N-terminal domain; CR = cysteine-rich domain; TMD = 7 helix transmembrane domain. In the unliganded state (left), both NTDs are open. With ligand(s) bound (right), one of the subunits closes, and a conformational change occurs at the dimer interface, bringing the CR domains closer together.

Here I discuss the construction of homology-based models o f the T1R2/T1R3 N-terminal domains. These models have been used in docking calculations with the sweet protein brazzein. I also describe the homologybased modeling of miraculin and docking of a miraculin model with the receptor models.

In Sweetness and Sweeteners; Weerasinghe, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Receptor Models The T1R2 and T1R3 proteins have significant sequence homology to a brain metabotropic glutamate receptor, m G l u R l , which functions as a homodimer. The N-terminal domain of mGluRl has been expressed, purified, and crystallized both with and without bound glutamate (7). In the ligand-activated form, one monomer exists in a fairly open form, and the other in a closed conformation. In using this structure as a template for homology modeling of T1R2 + T1R3, it is possible to model two activated forms of the receptor. One, which is designated Form 1, has T1R2 closed and T1R3 open. The other, designated Form 2, has T1R2 open and T1R3 closed. Homology modeling of both forms has been described recently.

Brazzein Docking Brazzein is a potently sweet protein (54 amino acids) produced by the African plant Pentadiplandra brazzeana (12). It is 2,000 times as sweet as sucrose on a weight basis, and 37,500 times as sweet as sucrose on a molar basis. Its three-dimensional structure has been determined by N M R spectroscopy (13). We have used Vakser's G R A M M software (14, 15) to carry out docking of brazzein to both forms of our receptor model. Protein-protein docking is a challenging problem, and results are generally only approximate. In the case of docking brazzein to models of the T1R2/T1R3 N-terminal domains, G R A M M consistently places brazzein in the apparent binding site of the open subunit, but it does not consistently orient the brazzein in the same way. We made use of the extensive brazzein structure-taste results (16, 17) to assess 20 different docking orientations. We were able to identify one in which brazzein interacts with the T1R2 subunit in an orientation that is consistent with the structure-taste relationships of 21 of 23 brazzein mutants (11). This model is now being tested through the design and evaluation of additional brazzein variants.

Miraculin Model Miraculin is a glycoprotein produced by Richadella dulcifica, a plant native to West Africa (18-21). It is composed of 191 amino acids and two N-linked polysaccharides. Glycosylation occurs at Asn-42 and Asn-186. Miraculin is a homodimer, covalently linked by an intermolecular disulfide at Cys-138. Each miraculin monomer has 4 intramolecular disulfide linkages as well. The miraculin protein sequence was used to search the Pfam database (22). Miraculin is a member of a family of protease inhibitors that includes the Kunitz zoybean trypsin inhibitor. The Pfam database provided an alignment of miraculin with 337 related protein sequences. It also provided links to 15 related



165 crystal structures in the Protein Data Bank (23). The barley subtilisin inhibitor structure (24: P D B code 1AVA) was chosen as a template for homology modeling miraculin because it has 33% sequence identity and 1.9 A resolution. Homology modeling was carried out using the Homology Model module of Molecular Operating Environment, version 2005.06 (Chemical Computing Group, Montreal). Twenty models of miraculin (monomer) were generated. Miraculin has one more intramolecular disulfide than do the crystal structures in the P D B ; several of the miraculin models placed the two additional cysteines in close proximity, so it was possible to generate the fourth disulfide linkage and carry out minimization of the final model using the C H A R M M 2 2 force field.

Miraculin Docking G R A M M was again used to dock the miraculin model (as a monomer) to the two forms of the sweet receptor model. As in the case of brazzein, the apparent binding site of the open subunit was consistently found by G R A M M , but there is not a body of miraculin mutation data to use in evaluating the various docking orientations. Instead, we made the assumption that miraculin must dock in a way that leaves Cys-138 (site of dimerization) and Asn-42 and Asn-186 (sites of glycosylation) oriented away from the binding site. Two orientations of miraculin in the open form of T1R2 and two orientations of miraculin in the open form of T1R3 were identified. It would be necessary to carry out mutations of miraculin or of the receptor (or both) to definitively identify the way in which miraculin binds to the receptor; these models could facilitate design of such experiments.

Conclusion Computer modeling facilitates an understanding of the ways that sweet proteins may interact with taste receptors. It also assists in the design of new experiments that will further our knowledge of such interactions.

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Computational Docking to Sweet Taste Receptor Models - American

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