Structural and functional studies of the nicotinic acetylcholine receptor by solid-state NMR

Over the last seven years, solid-state NMR has been widely employed to study structural and functional aspects of the nicotinic acetylcholine receptor. These studies have provided detailed structural information relating to both the ligand binding site and the transmembrane domain of the receptor. Studies of the ligand binding domain have elucidated the nature and the orientation of the pharmacophores responsible for the binding of the agonist acetylcholine within the agonist binding site. Analyses of small transmembrane fragments derived from the nicotinic acetylcholine receptor have also revealed the secondary structure and the orientation of these transmembrane domains. These experiments have expanded our understanding of the channels structural properties and are providing an insight into how they might be modulated by the surrounding lipid environment. In this article we review the advances in solid-state NMR applied to the nicotinic acetylcholine receptor and compare the results with recent electron diffraction and X-ray crystallographic studies.Presented at the Biophysical Society Meeting on Ion channels – from structure to disease held in May 2003, Rennes, France     

Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analog

Sialic acid terminates oligosaccharide chains on mammalian and microbial cell surfaces, playing critical roles in recognition and adherence. The enzymes that transfer the sialic acid moiety from cytidine-5'-monophospho-N-acetyl-neuraminic acid (CMP-NeuAc) to the terminal positions of these key glycoconjugates are known as sialyltransferases. Despite their important biological roles, little is understood about the mechanism or molecular structure of these membrane-associated enzymes. We report the first structure of a sialyltransferase, that of CstII from Campylobacter jejuni, a highly prevalent foodborne pathogen. Our structural, mutagenesis and kinetic data provide support for a novel mode of substrate binding and glycosyl transfer mechanism, including essential roles of a histidine (general base) and two tyrosine residues (coordination of the phosphate leaving group). This work provides a framework for understanding the activity of several sialyltransferases, from bacterial to human, and for the structure-based design of specific inhibitors.     

Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade

The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals. In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation. Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the Galpha subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system. By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl cyclase. Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.     

Past and current gene flow in the selfing, wind-dispersed species Mycelis muralis in western Europe

The distribution of genetic diversity in Mycelis muralis, or wall lettuce, was investigated at a European scale using 12 microsatellite markers to infer historical and contemporary forces from genetic patterns. Mycelis muralis has the potential for long-distance seed dispersal by wind, is mainly self-pollinated, and has patchily distributed populations, some of which may show metapopulation dynamics. A total of 359 individuals were sampled from 17 populations located in three regions, designated southern Europe (Spain and France), the Netherlands, and Sweden. At this within-region scale, contemporary evolutionary forces (selfing and metapopulation dynamics) are responsible for high differentiation between populations (0.34 < F(ST) < 0.60) but, contrary to expectation, levels of within-population diversity, estimated by Nei's unbiased expected heterozygosity (H(E)) (0.24 < H(E) < 0.68) or analyses of molecular variance (50% of the variation found within-populations), were not low. We suggest that the latter results, which are unusual in selfing species, arise from efficient seed dispersal that counteracts population turnover and thus maintains genetic diversity within populations. At the European scale, northern regions showed lower allelic richness (A = 2.38) than populations from southern Europe (A = 3.34). In light of postglacial colonization hypotheses, these results suggest that rare alleles may have been lost during recolonization northwards. Our results further suggest that mutation has contributed to genetic differentiation between southern and northern Europe, and that Sweden may have been colonized by dispersers originating from at least two different refugia.     

Bicyclic peptidomimetic tetrahydrofuro[3,2-b]pyrrol-3-one and hexahydrofuro[3,2-b]pyridine-3-one based scaffolds: synthesis and cysteinyl proteinase inhibition

A stereoselective synthesis of (3aS,6aR)-tetrahydrofuro[3,2-b]pyrrol-3-ones and (3aS,7aR)-hexahydrofuro[3,2-b]pyridine-3-ones has been developed through Fmoc protected scaffolds 12 and 13. A key design element within these novel bicyclic scaffolds, in particular the 5,5-fused system, was the inherent stability of the cis-fused geometry in comparison to that of the corresponding trans-fused. Since the bridgehead stereocentre situated beta to the ketone was of a fixed and stable configuration, the fact that cis ring fusion is both kinetically and thermodynamically stable with respect to trans ring fusion provides chiral stability to the bridgehead stereocentre that is situated alpha to the ketone. To exemplify this principle, building blocks 12 and 13 were designed, prepared and utilised in a solid phase combinatorial synthesis of peptidomimetic inhibitors 10, 45a-e, 11 and 46. Both series were chirally stable with 5,5-series 10 and 45a-e exhibiting potent in vitro activity against a range of CAC1 cysteinyl proteinases. Compound 10, a potent and selective inhibitor of cathepsin K, possessed good primary DMPK properties along with promising activity in an in vitro cell-based human osteoclast assay of bone resorption.     

A fungal metallothionein is required for pathogenicity of Magnaporthe grisea

The causal agent of rice blast disease, the ascomycete fungus Magnaporthe grisea, infects rice (Oryza sativa) plants by means of specialized infection structures called appressoria, which are formed on the leaf surface and mechanically rupture the cuticle. We have identified a gene, Magnaporthe metallothionein 1 (MMT1), which is highly expressed throughout growth and development by M. grisea and encodes an unusual 22-amino acid metallothionein-like protein containing only six Cys residues. The MMT1-encoded protein shows a very high affinity for zinc and can act as a powerful antioxidant. Targeted gene disruption of MMT1 produced mutants that show accelerated hyphal growth rates and poor sporulation but had no effect on metal tolerance. Mmt1 mutants are incapable of causing plant disease because of an inability to bring about appressorium-mediated cuticle penetration. Mmt1 appears to be distributed in the inner side of the cell wall of the fungus. These findings indicate that Mmt1-like metallothioneins may play a novel role in fungal cell wall biochemistry that is required for fungal virulence.     

Voltage-sensitive dye mapping in Langendorff-perfused rat hearts

An imaging system suitable for recordings from Langendorff-perfused rat hearts using the voltage-sensitive dye 4-[beta-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS) has been developed. Conduction velocity was measured under hyper- and hypokalemic conditions, as well as at physiological and reduced temperature. Elevation of extracellular [K(+)] to 9 mM from 5.9 mM caused a slowing of conduction velocity from 0.66 +/- 0.08 to 0.43 +/- 0.07 mm/ms (35%), and reduction of the temperature to 32 degrees C from 37 degrees C caused a slowing from 0.64 +/- 0.07 to 0.46 +/- 0.05 mm/ms (28%). Ventricular activation patterns in sinus rhythm showed areas of early activation (breakthrough) in both the right and left ventricle, with breakthrough at a site near the apex of the right ventricle usually occurring first. The effects of mechanically immobilizing the preparation to reduce motion artifact were also characterized. Activation patterns in epicardially paced rhythm were insensitive to this procedure over the range of applied force tested. In sinus rhythm, however, a relatively large immobilizing force caused prolonged PQ intervals as well as altered ventricular activation patterns. The time-dependent effects of the dye on the rat heart were characterized and include 1) a transient vasodilation at the onset of dye perfusion and 2) a long-lasting prolongation of the PQ interval of the electrocardiogram, frequently resulting in brief episodes of atrioventricular block.     

Comparison of time- and voltage-dependent K+ currents in myocytes from left and right atria of adult mice

Consistent differences in K+ currents in left and right atria of adult mouse hearts have been identified by the application of current- and voltage-clamp protocols to isolated single myocytes. Left atrial myocytes had a significantly (P < 0.05) larger peak outward K+ current density than myocytes from the right atrium. Detailed analysis revealed that this difference was due to the rapidly activating sustained K+ current, which is inhibited by 100 muM 4-aminopyridine (4-AP); this current was almost three times larger in the left atrium than in the right atrium. Accordingly, 100 muM 4-AP caused a significantly (P < 0.05) larger increase in action potential duration in left than in right atrial myocytes. Inward rectifier K+ current density was also significantly (P < 0.05) larger in left atrial myocytes. There was no difference in the voltage-dependent L-type Ca2+ current between left and right atria. As expected from this voltage-clamp data, the duration of action potentials recorded from single myocytes was significantly (P < 0.05) shorter in myocytes from left atria, and left atrial tissue was found to have a significantly (P < 0.05) shorter effective refractory period than right atrial tissue. These results reveal similarities between mice and other mammalian species where the left atrium repolarizes more quickly than the right, and provide new insight into cellular electrophysiological mechanisms responsible for this difference. These findings, and previous results, suggest that the atria of adult mice may be a suitable model for detailed studies of atrial electrophysiology and pharmacology under control conditions and in the context of induced atrial rhythm disturbances.