Reproduction cannot take place without the proper functioning of the lutropin/choriogonadotropin receptor (LHR). When the LHR does not work properly, ovulation does not occur in females and Leydig cells do not develop normally in the male. Also, because the LHR is essential for sustaining the elevated levels of progesterone needed to maintain pregnancy during the first trimester, disruptions in the functions of the LHR during pregnancy have catastrophic consequences. As such, a full understanding of the biology of the LHR is essential to the survival of our species. In this review we summarize our current knowledge of the structure, functions, and regulation of this important receptor.

Wordom is a versatile, user-friendly, and efficient program for manipulation and analysis of molecular structures and dynamics. The following new analysis modules have been added since the publication of the original Wordom paper in 2007: assignment of secondary structure, calculation of solvent accessible surfaces, elastic network model, motion cross correlations, protein structure network, shortest intra-molecular and inter-molecular communication paths, kinetic grouping analysis, and calculation of mincut-based free energy profiles. In addition, an interface with the Python scripting language has been built and the overall performance and user accessibility enhanced. The source code of Wordom (in the C programming language) as well as documentation for usage and further development are available as an open source package under the GNU General Purpose License from http://wordom.sf.net. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011

In this study, a quantitative approach was used to investigate the role of D142, which belongs to the highly conserved E͞DRY sequence, in the activation process of the ␣ 1B -adrenergic receptor (␣ 1B -AR). Experimental and computer-simulated mutagenesis were performed by substituting all possible natural amino acids at the D142 site. The resulting congeneric set of proteins together with the finding that all the receptor mutants show various levels of constitutive (agonist-independent) activity enabled us to quantitatively analyze the relationships between structural͞dynamic features and the extent of constitutive activity. Our results suggest that the hydrophobic͞hydrophilic character of D142, which could be regulated by protonation͞deprotonation of this residue, is an important modulator of the transition between the inactive (R) and active (R*) state of the ␣ 1B -AR. Our study represents an example of quantitative structureactivity relationship analysis of the activation process of a G protein-coupled receptor.The ␣ 1B -adrenergic receptor (AR) belongs to the superfamily of G protein-coupled receptors (GPCRs). The seven transmembrane domains (TMDs) common to all GPCRs contribute to the formation of the ligand binding pocket, whereas aa sequences of the intracellular loops (i) appear to mediate receptor-G protein coupling (1, 2). However, how binding of the extracellular signals is converted into receptor activation remains largely unknown.Recently, we have investigated the activation process of the ␣ 1B -AR linked to phospholipase C-mediated activation of polyphosphoinositide hydrolysis. By a combination of site-directed mutagenesis of the ␣ 1B -AR with computational simulations of receptor dynamics we explored the potential molecular mechanisms underlying the process of receptor activation, by focusing on a number of constitutively active receptor mutants (3). We identified a series of molecular changes that appear to be correlated with the transition from the inactive (R) to the active (R*) state, independently of the presence of the agonist. We proposed that the equilibrium between R and R* of the ␣ 1B -AR depends, at least in part, on the prototropic equilibrium between the deprotonated (anionic) and protonated (neutral) forms of D142, the negatively charged residue present in the E͞DRY motif, which is highly conserved among different GPCRs (Fig. 1A). As a result, we found that replacement of D142 with the nonpolar aa alanine conferred high constitutive (agonist-independent) activity to the ␣ 1B -AR. According to our analysis, a series of intramolecular interactions that might be of fundamental importance in the process of receptor activation depends on the protonation state of D142. In particular, our model pointed to a conserved ''polar pocket'' formed near the cytosol via a network of Hbonding interactions among N63, D91, N344, and Y348 (Fig. 1 A). This set of interactions constrains the receptor in its inactive state by exerting control on the degree of cytosolic exposure of the arginine resid...

5CINECA Supercomputing Centre. 6University of Modena and Reggio Emilia.7Christian-Albrechts-University Kiel.8Michigan State University. International course and report were conceived by Pietro Cozzini and Glen E. Kellogg. * To whom correspondence should be addressed. For G.E.K.: Department of Medicinal Chemistry, Virginia Commonwealth University, Box 980540, Richmond, VA 23298-0540; (phone) 804-828-6452; (fax) 804-827-3664; (e-mail) glen.kellogg@vcu.edu. For P.C.: Department of General and Inorganic Chemistry, University of Parma, Via G.P. Usberti 17/A 43100, Parma, Italy; (phone) +39-0521-905669; (fax) +39-0521-905556; (e-mail) pietro.cozzini@unipr.it. NIH Public Access IntroductionStructure-based drug discovery has played an important role in medicinal chemistry 1 beginning nearly when the first X-ray crystal structure of the myoglobin and hemoglobin proteins at nearatomic resolution were described by Perutz, Kendrew and colleagues. 2-5 Even though only static structures were (and still generally are) used for most Structure-Based Drug Design (SBDD), and indeed most molecular modeling, the importance of flexibility was recognized immediately: hemoglobin has two rather different structures, "tense" and "relaxed", depending on its oxygenation, although in recent years a family of relaxed hemoglobin structures with different tertiary structure conformations have been reported. 6 In fact, all proteins are inherently flexible systems. This flexibility is frequently essential for function (e.g., as in hemoglobin). Proteins have an intrinsic ability to undergo functionally relevant conformational transitions under native state conditions, 7,8 on a wide range of scales, both in time and space. 9 In adenylate kinase large conformational changes due to movements of the nucleotide 'lids'-rate-limiting for overall catalytic turnover 10,11 -are 'linked' with relatively small-amplitude atomic fluctuations on the ps timescale such that changes in the local backbone conformation are required for lid closure. 12 Nuclear receptors are modular proteins where a significant degree of conformational flexibility is essential to biological function. Most of the pharmacology of nuclear receptor ligands has been discussed on the basis of their ability to stabilize (or displace) a short α-helix segment (known as H12 or AF-2) localized at the carboxy terminus of the receptor in (or from) its conformation in the protein "active" form. 13-15 Available X-ray crystal structures show a surprisingly wide range of structural diversity in ligands binding to, and inhibiting, nuclear receptor proteins such as the farnesoid X-receptor (FXR). 16,17 Protein dynamics is also a key component of intramolecular and intermolecular communication/signaling mechanisms and an essential requirement for the function of Gprotein coupled receptors (GPCRs), which are the largest known superfamily of membrane proteins. GPCRs regulate cell activity by transmitting extracellular signals to the inside of cells and respond to these signals by catalyzing nucleotide e...

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The Automatic Rhodopsin Modeling (ARM) protocol has recently been proposed as a tool for the fast and parallel generation of basic hybrid quantum mechanics/molecular mechanics (QM/MM) models of wild type and mutant rhodopsins. However, in its present version, input preparation requires a few hours long user's manipulation of the template protein structure, which also impairs the reproducibility of the generated models. This limitation, which makes model building semiautomatic rather than fully automatic, comprises four tasks: definition of the retinal chromophore cavity, assignment of protonation states of the ionizable residues, neutralization of the protein with external counterions, and finally congruous generation of single or multiple mutations. In this work, we show that the automation of the original ARM protocol can be extended to a level suitable for performing the above tasks without user's manipulation and with an input preparation time of minutes. The new protocol, called a-ARM, delivers fully reproducible (i.e., user independent) rhodopsin QM/MM models as well as an improved model quality. More

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Cysteine351 is the site for pertussis toxin-catalyzed ADP-ribosylation in the G protein Gi1 alpha. Alteration of this residue, or the equivalent cysteine in other Gi-family G proteins, has been used to examine specific interactions between receptors and these G proteins. However, no systematic analysis has been performed to determine the quantitative effect of such alterations. To address this we mutated cysteine351 of Gi1 alpha to all other possible amino acids. Each of the G protein mutants was transiently coexpressed along with the porcine alpha 2A-adrenoceptor in HEK 293/T cells. Following pertussis toxin treatment of the cells, membranes were prepared and the capacity of the agonist UK14304 to stimulate the binding of [35S]GTP gamma S to the modified G proteins was measured. A spectrum of function was observed. The presence of either a charged amino acid or a proline at this position essentially attenuated agonist regulation. The wild-type G protein did not result in maximal stimulation by agonist. The presence of certain branched chain aliphatic amino acids or bulky aromatic R groups at amino acid351 resulted in substantially greater maximal stimulation by the alpha 2A-adrenoceptor than that achieved with the wild-type sequence. The degree of activation of the forms of Gi1 alpha correlated strongly with the octanol/water partition coefficient of the amino acid at residue351. Variation in EC50 values for agonist-induced stimulation of binding of [35S]GTP gamma S to the mutant G proteins also correlated with the octanol/water partition coefficient. These results define a central role for hydrophobicity of this residue in defining productive receptor-G protein interactions.