Quantifying conformational changes in GPCRs: glimpse of a common functional mechanism

Background

G-protein-coupled receptors (GPCRs) are important drug targets and a better understanding of their molecular mechanisms would be desirable. The crystallization rate of GPCRs has accelerated in recent years as techniques have become more sophisticated, particularly with respect to Class A GPCRs interacting with G-proteins. These developments have made it possible for a quantitative analysis of GPCR geometrical features and binding-site conformations, including a statistical comparison between Class A GPCRs in active (agonist-bound) and inactive (antagonist-bound) states.

Results

Here we implement algorithms for the analysis of interhelical angles, distances, interactions and binding-site volumes in the transmembrane domains of 25 Class A GPCRs (7 active and 18 inactive). Two interhelical angles change in a statistically significant way between average inactive and active states: TM3-TM6 (by -9°) and TM6-TM7 (by +12°). A third interhelical angle: TM5-TM6 shows a trend, changing by -9°. In the transition from inactive to active states, average van der Waals interactions between TM3 and TM7 significantly increase as the average distance between them decreases by >2 Å. Average H-bonding between TM3 and TM6 decreases but is seemingly compensated by an increase in H-bonding between TM5 and TM6. In five Class A GPCRs, crystallized in both active and inactive states, increased H-bonding of agonists to TM6 and TM7, relative to antagonists, is observed. These protein-agonist interactions likely favour a change in the TM6-TM7 angle, which creates a narrowing in the binding pocket of activated receptors and an average ~200 ų reduction in volume.

Conclusions

In terms of similar conformational changes and agonist binding pattern, Class A GPCRs appear to share a common mechanism of activation, which can be exploited in future drug development.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0567-3) contains supplementary material, which is available to authorized users.     

Substituent effect on the preferred DNA binding mode and affinity of a homologous series of naphthalene diimides

A combination of isothermal titration calorimetry (ITC), topoisomerase I DNA unwinding assays, and ethidium bromide displacement studies were employed to investigate the binding of a homologous series of naphthalene diimides (NDI) to DNA. Our results suggest that the nature of the substituent plays a significant role in both the preferred binding mode and relative binding affinity of the compounds of this study. Only intercalative-type binding (K = 15 ± 3 × 10⁶ M⁻¹) was observed for the NDI with the smallest substituent (trimethyl-ethylamino), while larger members of the series (diethylmethyl-, dipropylmethyl- and dibutylmethyl-ethylamino substituents) adopted an additional binding mode of higher affinity (K₁ = 31 − 78 × 10⁶ M⁻¹).     

Molecular dynamics of anthraquinone DNA intercalators with polyethylene glycol side chains

The interactions of a homologous series of four anthraquinone (AQ) intercalators with increasing lengths of polyethylene glycol (PEG) side chains with DNA have been studied via molecular dynamics (MD) simulations. The geometry, conformation, interactions, and hydration of the complexes were examined. The geometries of the four ligands were similar with parallel stacking to the long axis of the adjacent DNA base pairs. Hydrogen bonding between the AQ amide and DNA led to a preference for the trans-syn conformer. As the side chain lengthened, binding to DNA reduced the conformational space, resulting in an increase in unfavorable entropy. Increased localization of the PEG side chain in the DNA groove, indicating some interaction of the side chain with DNA, also contributed unfavorably to the entropy. The changes in free energy of binding due to entropic considerations (-3.9 to -6.3 kcal/mol) of AQ I-IV were significant. The hydration of the PEG side chain decreased upon binding to DNA. Understanding of side chain conformations, interactions, and hydration changes that accompany the formation of a ligand-DNA complex may be important in the development of new applications of pegylated small molecules that target biological macromolecules.     

Responses of Dendroctonus brevicomis (Coleoptera: Curculionidae) in behavioral assays: implications to development of a semiochemical-based tool for tree protection

Currently, techniques for managing western pine beetle, Dendroctonus brevicomis LeConte (Coleoptera: Curculionidae, Scolytinae), infestations are limited to tree removals (thinning) that reduce stand density and presumably host susceptibility, and/or the use of insecticides to protect individual trees. There continues to be significant interest in developing an effective semiochemical-based tool for protecting trees from D. brevicomis attack, largely as an alternative to conventional insecticides. The responses of D. brevicomis to tree volatiles and verbenone were documented in eight experiments (trapping assays) conducted over a 4-yr period in which 88,942 individuals were collected. Geraniol, a tree volatile unique to Pinus ponderosa that elicits female-specific antennal responses in D. brevicomis, did not affect D. brevicomis behavior. Blends of two green leaf alcohols [hexanol + (Z)-3-hexen-1-ol] tested at two release rates (5.0 and 100.0 mg/d) had no effect on the response of D. brevicomis to attractant-baited traps. A nine-component blend [benzaldehyde, benzyl alcohol, guaiacol, nonanal, salicylaldehyde, (E)-2-hexenal, (E)-2-hexen-1-ol, (Z)-2-hexen-1-ol, and (-) -verbenone; NAVV] and subsequent revisions of this blend disrupted the response of D. brevicomis to attractant-baited traps in all experiments. The inhibitory effect of a revised five-component blend [nonanal, (E)-2-hexenal, (E)-2-hexen-1-ol, (Z)-2-hexen-1-ol, and (-)-verbenone; NAVV5] on the response of mountain pine beetle, D. ponderosae Hopkins, to attractant-baited traps was also documented. Acetophenone significantly reduced D. brevicomis attraction, but was not as effective as verbenone alone. Acetophenone increased the effectiveness of NAVV5 in one of two experiments. Furthermore, by adding acetophenone to NAVV5 we were able to remove the aldehydes from NAVV5 without compromising effectiveness, resulting in a novel four-component blend [acetophenone, (E)-2-hexen-1-ol + (Z)-2-hexen-1-ol, and (-)-verbenone; Verbenone Plus]. We discuss the implications of these and other results to development of Verbenone Plus as a semiochemical-based tool for management of D. brevicomis and D. ponderosae infestations.     

Sulfonated naphthyl porphyrins as agents against HIV-1

Sulfonated 5,10,15,20-tetra(1-naphthyl)porphyrin (T1NapS) and 5,10,15,20-tetra(2-naphthyl)porphyrin (T2NapS) and their copper and iron chelates show activity against the human immunodeficiency virus (HIV-1). The porphyrins were prepared by sulfonation of the parent structures with sulfuric acid. More highly sulfonated structures were prepared by sulfonation for longer times. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry showed species with as many as eight sulfonates. Some of the mass spectral peaks for the copper chelates were consistent with loss of water, apparently from intramolecular sulfone formation between two adjacent naphthalene rings that took place during copper insertion. The compounds could be separated using capillary electrophoresis; addition of beta- or gamma-cyclodextrin gave substantially better separation of the components. Activity against HIV was evaluated using an epithelial HeLa-CD4-CCR5 cell line; EC50 values for HIV-1 IIIB and HIV-1 JR-FL ranged from 1 to 15 microg/ml. The compounds exhibit low toxicity for human epithelial cells and have potential as microbicides which might be used to provide protection against sexual transmission of HIV.     

Amino acid sequence versus morphological data and the interordinal relationships of mammals

To a large extent, the mutual affinities of the mammalian orders continue to puzzle systematists, even though comparative anatomy and amino acid sequencing offer a massive data base from which these relationships could potentially be adduced. In the present paper the consistency index--the number of character states less the number of characters in a data set, divided by the total number of changes in the character states on a cladogram--was used to examine the relative resolving powers of recently published morphological and molecular- sequence data. Consistency indices were calculated for previously published alpha crystallin A chain and myoglobin amino acid-sequence cladograms and for four original amino acid-sequence cladograms (alpha crystallin A, myoglobin, and alpha and beta hemoglobin); these were found to be comparable to the consistency indices of morphologically based cladograms. Qualitative comparisons between the morphologically based and molecularly based trees were also made; only moderate congruence between the two was observed. Moreover, there was a general lack of congruence between the cladograms specified by each of the four proteins. Amino acid-sequence and morphological data agreed on the placement of edentates as an early eutherian offshoot and on the grouping of hyracoids, proboscideans, and sirenians. Otherwise there was only limited congruence: morphology strongly supported the grouping of lagomorphs and rodents and the alliance of pholidotes and edentates, but sequence analyses did not. The placement of tubulidentates differed widely among proteins. Morphology indicated the close association of sirenians with proboscideans; proteins suggested a pairing of sirenians with hyracoids. Sequence data did not identify many (morphologically well-diagnosed) orders as monophyletic (e.g., Lagomorpha).(ABSTRACT TRUNCATED AT 250 WORDS)      

Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).     

Effects of histamine and acetylcholine on equine digital lymph flow and composition.

We measured the flow rate and protein concentration of lymph collected from a digital lymphatic in eight anesthetized ponies. Additionally, we recorded systemic arterial pressure (Part), and small vein pressure (Psv). Control lymph flow averaged 0.068 ml/min, and contained 3.11 g/100 ml of protein with albumin/globulin ratio of 0.75. Twenty-minute local intra-arterial infusion of acetylcholine (10 mug/min.) elevated Psv but did not increase lymph flow rate or protein concentration. A 60-min local intra-arterial infusion of histamine (10 mug/min) produced a marked sustained increase in Psv and both lymph flow and protein concentration. Edema developed in the digit receiving histamine. These data support the conclusion that in the horse, as in other species, histamine edema is due primarily to a decreased transcapillary colloid osmotic pressure gradient rather than an increased transcapillary hydrostatic pressure gradient.