Molecular docking has become an increasingly important tool for drug discovery. In this review, we present a brief introduction of the available molecular docking methods, and their development and applications in drug discovery. The relevant basic theories, including sampling algorithms and scoring functions, are summarized. The differences in and performance of available docking software are also discussed. Flexible receptor molecular docking approaches, especially those including backbone flexibility in receptors, are a challenge for available docking methods. A recently developed Local Move Monte Carlo (LMMC) based approach is introduced as a potential solution to flexible receptor docking problems. Three application examples of molecular docking approaches for drug discovery are provided.

The detection of sweet-tasting compounds is mediated in large part by a heterodimeric receptor comprised of T1R2؉T1R3. Lactisole, a broad-acting sweet antagonist, suppresses the sweet taste of sugars, protein sweeteners, and artificial sweeteners. Lactisole's inhibitory effect is specific to humans and other primates; lactisole does not affect responses to sweet compounds in rodents. By heterologously expressing interspecies combinations of T1R2؉T1R3, we have determined that the target for lactisole's action is human T1R3. From studies with mouse/ human chimeras of T1R3, we determined that the molecular basis for sensitivity to lactisole depends on only a few residues within the transmembrane region of human T1R3. Alanine substitution of residues in the transmembrane region of human T1R3 revealed 4 key residues required for sensitivity to lactisole. In our model of T1R3's seven transmembrane helices, lactisole is predicted to dock to a binding pocket within the transmembrane region that includes these 4 key residues.Taste is a primal sense that enables diverse organisms to identify and ingest sweet-tasting nutritious foods and to reject bitter-tasting environmental poisons (1). Taste perception can be categorized into five distinct qualities: salty, sour, bitter, umami (amino acid taste), and sweet (1). Salty and sour depend on the actions of ion channels. Bitter, umami, and sweet depend on G-protein-coupled receptors (GPCRs) 1 and coupled signaling pathways. Sweet taste in large part depends on a heterodimeric receptor comprised of T1R2ϩT1R3 (2-5).The T1R taste receptors (T1R1, T1R2, and T1R3) are most closely related to metabotropic glutamate receptors (mGluRs), Ca 2ϩ -sensing receptors (CaSRs), and some pheromone receptors (6 -10). All of these receptors are class-C GPCRs, with the large clam shell-shaped extracellular amino-terminal domain (ATD) characteristic of this family. Following the ATD is a cysteine-rich region that connects the ATD to the heptahelical transmembrane domain (TMD); following the TMD is a short intracellular carboxyl-terminal tail. The solved crystal structure of the ATD of mGluR1 identifies a "Venus flytrap module" (VFTM) involved in ligand binding (11). The canonical agonist glutamate binds within the VFTM in a cleft formed by the two lobes of this module to stabilize a closed active conformation of the mGluR1 ATD. In contrast, several positive and negative allosteric modulators of class-C GPCRs have been identified and shown to act via binding not within the VFTM but instead within the TMD (12-16).Over the past few decades, multiple models of the sweet receptor's hypothetical ligand binding site have been generated based on the structures of existing sweeteners but without direct knowledge of the nature of the sweet receptor itself. A consensus feature of these models is the presence of A-H-B groups, in which the AH group is a hydrogen donor and the B group is an electronegative center. These models have explanatory and predictive value for some, but not all sweeteners, suggesting th...

The artificial sweetener cyclamate tastes sweet to humans, but not to mice. When expressed in vitro, the human sweet receptor (a heterodimer of two taste receptor subunits: hT1R2 ؉ hT1R3) responds to cyclamate, but the mouse receptor (mT1R2 ؉ mT1R3) does not. Using mixed-species pairings of human and mouse sweet receptor subunits, we determined that responsiveness to cyclamate requires the human form of T1R3. Using chimeras, we determined that it is the transmembrane domain of hT1R3 that is required for the sweet receptor to respond to cyclamate. Using directed mutagenesis, we identified several amino acid residues within the transmembrane domain of T1R3 that determine differential responsiveness to cyclamate of the human versus mouse sweet receptors. Alanine-scanning mutagenesis of residues predicted to line a transmembrane domain binding pocket in hT1R3 identified six residues specifically involved in responsiveness to cyclamate. Using molecular modeling, we docked cyclamate within the transmembrane domain of T1R3. Our model predicts substantial overlap in the hT1R3 binding pockets for the agonist cyclamate and the inverse agonist lactisole. The transmembrane domain of T1R3 is likely to play a critical role in the interconversion of the sweet receptor from the ground state to the active state.Taste is a primal sense that is essential for humans and other organisms to detect the nutritive quality of a potential food source while avoiding environmental toxins (1-3). Taste perception can be categorized into five distinct qualities: sweet, bitter, salty, sour, and umami (amino acid taste) (1-3). Sweet, bitter, and umami tastes are mediated in large part by G-protein-coupled receptors (GPCRs) 2 and their linked signaling pathways. Sour and salty tastes are thought to be mediated by direct effects on specialized ion channels (1-3).The detection of sweet taste is mediated by two GPCR subunits, T1R2 and T1R3, which are specifically expressed in taste receptor cells (4 -13). When expressed in vitro, T1R2 ϩ T1R3 heterodimer responds to a broad spectrum of chemically diverse sweeteners, ranging from natural sugars (sucrose, fructose, glucose, and maltose), sweet amino acids (D-tryptophan, D-phenylalanine, and D-serine), and artificial sweeteners (acesulfame-K, aspartame, cyclamate, saccharin, and sucralose) to sweet tasting proteins (monellin, thaumatin, and brazzein) (10, 11, 14 -16). To date, all sweeteners tested in vitro activate the T1R2 ϩ T1R3 heterodimer. In vivo, genetic ablation in mice of T1R2, T1R3, or both either reduces or eliminates responses to sweet compounds (12, 13). Thus, the T1R2 ϩ T1R3 heterodimer is broadly tuned and functions as the principal or sole sweet taste receptor in vivo.T1R2 and T1R3 are class C GPCR subunits (4 -9), members of a group that also includes T1R1 (a component of the umami taste receptor), metabotropic glutamate receptors, the calcium-sensing receptor, ␥-aminobutyric acid type B receptors, and vomeronasal receptors. Like most other GPCRs, each class C receptor has a heptahelic...

Tandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as they result in maximal preservation of posttranslational modifications. Here we describe the histidine-biotin (HB) tag, a new tandem affinity tag for twostep purification under denaturing conditions. The HB tag consists of a hexahistidine tag and a bacterially derived in vivo biotinylation signal peptide that induces efficient biotin attachment to the HB tag in yeast and mammalian cells. HB-tagged proteins can be sequentially purified under fully denaturing conditions, such as 8 M urea, by Ni 2؉ chelate chromatography and binding to streptavidin resins. The stringent separation conditions compatible with the HB tag prevent loss of protein modifications, and the high purification grade achieved by the tandem affinity strategy facilitates mass spectrometric analysis of posttranslational modifications. Ubiquitination is a particularly sensitive protein modification that is rapidly lost during purification under native conditions due to ubiquitin hydrolase activity. The HB tag is ideal to study ubiquitination because the denaturing conditions inhibit hydrolase activity, and the tandem affinity strategy greatly reduces nonspecific background. We tested the HB tag in proteome-wide ubiquitin profiling experiments in yeast and identified a number of known ubiquitinated proteins as well as so far unidentified candidate ubiquitination targets. In addition, the stringent purification conditions compatible with the HB tag allow effective mass spectrometric identification of in vivo cross-linked protein com- Mass spectrometric analysis of proteins has tremendously contributed to our understanding of biological systems. Mapping of covalent protein modifications by mass spectrometric approaches has made it possible to identify and rapidly evaluate the biological significance of modifications. In addition, identification of protein complexes by mass spectrometry has allowed investigators to connect cellular pathways and to describe the dynamics of protein complexes (1, 2). These approaches typically require a high degree of purification of proteins or protein complexes. Importantly to get a genuine picture of the in vivo situation it is essential to avoid any changes in protein modification or protein complex composition that might occur during the purification procedure. Two-step purification strategies have been proven to be very effective in reducing nonspecific background, which is particularly important for the analyses of complex protein samples (3). The first widely and successfully used tandem affinity tag was the TAP 1 tag, which consists of the immunoglobulin-interacting domain of Protein A and a calmodulinbinding peptide (CBP) a...