N-methyl-d-aspartate receptors (NMDARs) are members of the ionotropic glutamate receptor family that mediate excitatory synaptic transmission in the central nervous system. The channels of NMDARs are permeable to Ca2+ but blocked by Mg2+, distinctive properties that underlie essential brain processes such as induction of synaptic plasticity. However, due to limited structural information about the NMDAR transmembrane ion channel forming domain, the mechanism of divalent cation permeation and block is understood poorly. In this paper we developed an atomistic model of the transmembrane domain (TMD) of NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors). The model was generated using (a) a homology model based on the structure of the NaK channel and a partially resolved structure of an AMPA receptor (AMPAR), and (b) a partially resolved X-ray structure of GluN1/2B NMDARs. Refinement and extensive Molecular Dynamics (MD) simulations of the NMDAR TMD model were performed in explicit lipid bilayer membrane and water. Targeted MD with simulated annealing was introduced to promote structure refinement. Putative positions of the Mg2+ and Ca2+ ions in the ion channel divalent cation binding site are proposed. Differences in the structural and dynamic behavior of the channel protein in the presence of Mg2+ or Ca2+ are analyzed. NMDAR protein conformational flexibility was similar with no ion bound to the divalent cation binding site and with Ca2+ bound, whereas Mg2+ binding reduced protein fluctuations. While bound at the binding site both ions retained their preferred ligand coordination numbers: 6 for Mg2+, and 7–8 for Ca2+. Four asparagine side chain oxygens, a back-bone oxygen, and a water molecule participated in binding a Mg2+ ion. The Ca2+ ion first coordination shell ligands typically included four to five side-chain oxygen atoms of the binding site asparagine residues, two water molecules and zero to two backbone oxygens of the GluN2B subunits. These results demonstrate the importance of high-resolution channel structures for elucidation of mechanisms of NMDAR permeation and block.
Foi realizada uma análise de QSAR-2D com três descritores sobre a afinidade de ligação a receptor no citosolo humano. Um conjunto de vinte e três progesteronas foi dividido em um conjunto de treinamento de dezesseis compostos e em um conjunto de teste de sete compostos. O método quântico semi-empírico RM1 foi usado para calcular a geometria e algumas propriedades moleculares. O software DRAGON também foi usado para produzir descritores. O software MobyDigs foi usado para selecionar descritores e construir modelos QSAR. O melhor modelo de QSAR foi construído para o conjunto de treinamento usando regressão linear múltipla com três descritores, PW2, Mor15m e GAP-10, resultando em r 2 = 0,886, q 2 = 0,805, q = 0,476. Usando a representação gráfica dos coeficientes de regressão de PLS, correspondendo às interações espacial e eletrostática, foi possível obter uma interpretação mecânica. Foi mostrado que QSAR-2D e 3D juntos satisfazem todos os seis requerimentos do Princípio de Setubal (Princípio de OECD). A partir das informações obtidas pelo QSAR-3D foram construídas quatro novas progesteronas. As atividades de afinidade de ligação ao receptor destes novos compostos foram várias vezes maiores que qualquer uma do conjunto de vinte e três progesteronas já estudado.A 2D QSAR analysis with three descriptors of binding affinity to human cytosol receptor was performed. The set of twenty-three progestins was divided into a training set of sixteen molecules and a test set of seven molecules. The quantum chemical RM1 semiempirical method was used to calculate geometry and some molecular properties. DRAGON software was also use to produce descriptors. MobyDigs software was used to select descriptors and build QSAR models. The best 2D QSAR model was constructed for the training set with multiple linear regression (MLR) using three descriptors , PW2, Mor15m, and GAP-10, resulting in r 2 = 0.866, q 2 = 0.805, q = 0.476. Based upon the graphical representation of PLS regression coefficients corresponding to steric and electrostatic interactions, it was possible to obtain a mechanistic interpretation. Thus the 2D and 3D QSAR together satisfy all the six Setubal Principles (OECD principles). Based upon the information obtained from the 3D QSAR analysis, the structures of four new progestins are proposed. Their receptor binding activities are estimated to be several times more potent than the most potent progestin of the twenty-three studied.
A wealth of high-resolution structural information regarding pentameric ligand-gated ion channels is now available, but less is known of the molecular details underlying complex allosteric mechanisms involved in channel gating and desensitization. Receptor allostery can be studied by identifying statedependent distance constraints that may be used in molecular modeling of these receptors. Systematically generated single Cys mutations of the human a 1 glycine receptor (GlyR) expressed in insect cells were labeled with a clickable methanethiosulfonate-benzophenone crosslinker. After covalent ligation to Cys, crosslinks may then be introduced in the resting, open, or desensitized states by photoactivation. Including an alkyne tag on the crosslinker permits click chemistry addition of biotin, which allows for enrichment by avidin chromatography. Mass spectrometry (MS) fingerprinting of monomeric and higher-order GlyR bands on SDS-PAGE using ESI-QTOF MS/MS then allows for determination of the site of crosslinking. Our initial proof-of-principle studies conducted on purified GlyR have provided state-dependent information on this receptor. This approach may be broadly applicable to studies of any allosteric complex.
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