The aim of this study was to generate a reliable model for the homotetrameric structure of the human TRPM8 cation channel, a temperature sensor involved in innocuous cold perceptions. The described model was generated using a fragmental strategy and its interaction capacities were explored by docking a representative set of ligands. The analysis of the quaternary structure suggests that the N-terminus possesses a solenoidal topology which could be involved in tetramerization due to its electrostatic characteristics. Again, the tetramer model unveils a precise fitting between the segments of neighbouring monomers affording attractive suggestions for the multifaceted mechanism of channel gating. Docking results are in convincing agreement with mutational analyses and confirm that S4 and S4-S5 linker play a key role in channel activation. Overall, the proposed model could find fertile applications to further investigate the gating mechanism and to design truly selective ligands able to clarify the pathophysiological roles of the TRPM8 channel.

Homology model for hPepT1The Figure shows the hPepT1 model, colored by segments, unveiling its typical folding with 12 transmembrane segments (TM1-12) and a large extracellular loop (EL5). The structural quality of the model is assessed by the significant percentage of residues which fall in the allowed regions of the Ramachandran's plot (70.62%) with a marked preponderance of helix motifs.The TM bundle assumes an elliptical truncated conic shape, which is due to fact that the TM segments are far from being parallel and some segments are staggered with an angle of 30°in respect to the adjacent heli ces. The TMs arrangement does not agree the numerical order, but it is possible to recognize an internal group of helices (i.e. TM1, TM4, TM5, TM7, TM10), which line the central pore and bear the key residues for the binding, and an external set of TM segments (TM2, TM3, TM6, TM8, TM11, TM12), which define the boundary of TM bundle. Notably, the helices facing the central pore are clearly more hydrophilic than the external TM segments.The extracellular loop EL5 (red segment) fully covers the extracellular side and consists of two large domains connected by two hinge loops. The hinges may confer flexibility to the domains, which could assume closed or open conformations modulating the accessibility of the binding cavity. Such a flexibility is confirmed by the used template (sucrose phosphatase) which can assume two different states. Such template is a metalloenzyme which selectively recognizes some sugars. This suggests that also EL5 may bind sugars and/or metal ions involved in modulatory effects on hPePT1, as reported by experimental studies. Docking analysesAsn22 Glu23 Ile331 Trp294Phe293 Trp294 Glu291 Thr327The ammonium head probably plays the most critical role since it realizes a reinforced Hbond with Tyr588 as well as ion-pairs with Glu23 (TM1) and/or Glu26 (TM1).Notably, the contact between Tyr588 and ammonium head characterizes the most affinitive ligands.The carboxy terminus appears less involved in ligand recognition, since it stabilizes only H-bonds with the backbone of Ala295 (TM7), Leu296 (TM7), and Phe297 (TM7) without forming strong ionic interactionsThe residues which interact with the side chains are heterogeneous, justifying the ability of hPepT1 to interact with structurally diverse substrates. It is possible to recognize a set of residues involved in the interaction with the N-terminal side chain (SC1) such as Asn22 (TM1), Glu23, and Phe293, while the Cterminal side chain (SC2) contact Trp294, Ile331 (TM8), and Glu291 (TM7) and Thr327 (EL4).The central peptide bond can stabilize H-bonds with backbone atoms of Phe293 and Trp294. Such interactions can be hindered by bulky side chains, and, thus, one can conclude that the contacts of the peptide groups could partially counterbalance the reduced interactions stabilized by small side chains.

Three-dimensional models of the five human muscarinic receptors were obtained from their known sequences. Homology modelling based on the crystallographic structure of bovine rhodopsin yielded models compatible with known results from site-directed mutagenesis studies. The only exceptions were the cytoplasmic loop 3 (CL3) in the five receptors, and the large C-terminal domain in M(1). Here, homology modelling with other closely related proteins allowed to solve these gaps. A detailed comparative discussion of the five models is given. The second part of the work involved docking experiments with the physiological ligand acetylcholine, again yielding results entirely compatible with results from mutagenesis experiments. The study revealed analogies and differences between the five receptors in the residues, and interactions leading to the recognition and binding of acetylcholine.

The homology modeling of GPCRs has benefitted vastly from the availability of some resolved structures, which allow the generation of many reliable GPCR models. However, the dynamic behavior of such receptors has been only minimally examined in silico, although several pieces of evidence have highlighted some conformational switches that can orchestrate the activation mechanism. Among such switches, Pro-containing helices play a key role in determining bending in TM helices and thereby the width of the TM bundle. The approach proposed herein involves the generation of a set of possible models (conformational chimeras) by exhaustively combining the two main conformations (straight and bent) that a Pro-containing helix can assume. This approach was validated by generating conformational chimeras for the Cys-LTR1 receptor, which is involved in contractile and inflammatory processes. The generated chimeras were then used for docking a small set of representative ligands. The results revealed the flexibility mechanisms of Cys-LTR1, showing how the docked agonists vary their stabilizing interactions, shifting from the open to closed state, and how the examined antagonists are able to block the receptor in an open and inactive conformation, thus behaving as inverse agonists. This study emphasizes the promising potential of chimera modeling, confirms the key role of proline residues in receptor activation, and suggests that docking results can be improved by considering the often-overlooked flexibility of receptors.

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