Modulating the Intrinsic Disorder in the Cytoplasmic Domain Alters the Biological Activity of the N-Methyl-d-aspartate-sensitive Glutamate Receptor

The NMDA-sensitive glutamate receptor is a ligand-gated ion channel that mediates excitatory synaptic transmission in the nervous system. Extracellular zinc allosterically regulates the NMDA receptor by binding to the extracellular N-terminal domain, which inhibits channel gating. Phosphorylation of the intrinsically disordered intracellular C-terminal domain alleviates inhibition by extracellular zinc. The mechanism for this functional effect is largely unknown. Proline is a hallmark of intrinsic disorder, so we used proline mutagenesis to modulate disorder in the cytoplasmic domain. Proline depletion selectively uncoupled zinc inhibition with little effect on receptor biogenesis, surface trafficking, or ligand-activated gating. Proline depletion also reduced the affinity for a PDZ domain involved in synaptic trafficking and affected small molecule binding. To understand the origin of these phenomena, we used single molecule fluorescence and ensemble biophysical methods to characterize the structural effects of proline mutagenesis. Proline depletion did not eliminate intrinsic disorder, but the underlying conformational dynamics were changed. Thus, we altered the form of intrinsic disorder, which appears sufficient to affect the biological activity. These findings suggest that conformational dynamics within the intrinsically disordered cytoplasmic domain are important for the allosteric regulation of NMDA receptor gating.     

Effect of Src kinase phosphorylation on disordered C-terminal domain of N-methyl-D-aspartic acid (NMDA) receptor subunit GluN2B protein

NMDA receptors are ligand-gated ion channels with a regulatory intracellular C-terminal domain (CTD). In GluN2B, the CTD is the largest domain in the protein but is intrinsically disordered. The GluN2B subunit is the major tyrosine-phosphorylated protein in synapses. Src kinase phosphorylates the GluN2B CTD, but it is unknown how this affects channel activity. In disordered proteins, phosphorylation can tip the balance between order and disorder. Transitions can occur in both directions, so it is not currently possible to predict the effects of phosphorylation. We used single molecule fluorescence to characterize the effects of Src phosphorylation on GluN2B. Scanning fluorescent labeling sites throughout the domain showed no positional dependence of the energy transfer. Instead, efficiency only scaled with the separation between labeling sites suggestive of a relatively featureless conformational energy landscape. Src phosphorylation led to a general expansion of the polypeptide, which would result in greater exposure of known protein-binding sites and increase the physical separation between contiguous sites. Phosphorylation makes the CTD more like a random coil leaving open the question of how Src exerts its effects on the NMDA receptor.     

A network-based approach to identify substrate classes of bacterial glycosyltransferases

Background

Bacterial interactions with the environment- and/or host largely depend on the bacterial glycome. The specificities of a bacterial glycome are largely determined by glycosyltransferases (GTs), the enzymes involved in transferring sugar moieties from an activated donor to a specific substrate. Of these GTs their coding regions, but mainly also their substrate specificity are still largely unannotated as most sequence-based annotation flows suffer from the lack of characterized sequence motifs that can aid in the prediction of the substrate specificity.

Results

In this work, we developed an analysis flow that uses sequence-based strategies to predict novel GTs, but also exploits a network-based approach to infer the putative substrate classes of these predicted GTs. Our analysis flow was benchmarked with the well-documented GT-repertoire of Campylobacter jejuni NCTC 11168 and applied to the probiotic model Lactobacillus rhamnosus GG to expand our insights in the glycosylation potential of this bacterium. In L. rhamnosus GG we could predict 48 GTs of which eight were not previously reported. For at least 20 of these GTs a substrate relation was inferred.

Conclusions

We confirmed through experimental validation our prediction of WelI acting upstream of WelE in the biosynthesis of exopolysaccharides. We further hypothesize to have identified in L. rhamnosus GG the yet undiscovered genes involved in the biosynthesis of glucose-rich glycans and novel GTs involved in the glycosylation of proteins. Interestingly, we also predict GTs with well-known functions in peptidoglycan synthesis to also play a role in protein glycosylation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-349) contains supplementary material, which is available to authorized users.     

Mitochondrial haplotype diversity in the tortoise species <Emphasis Type="Italic">Testudo graeca</Emphasis> from North Africa and the Middle East

Background  

To help conservation programs of the endangered spur-thighed tortoise and to gain better insight into its systematics, genetic variation and evolution in the tortoise species Testudo graeca (Testudines: Testudinidae) was investigated by sequence analysis of a 394-nucleotide fragment of the mitochondrial 12S rRNA gene for 158 tortoise specimens belonging to the subspecies Testudo graeca graeca, Testudo graeca ibera, Testudo graeca terrestris, and a newly recognized subspecies Testudo graeca whitei. A 411-nucleotide fragment of the mitochondrial D-loop was additionally sequenced for a subset of 22 T. graeca, chosen because of their 12S gene haplotype and/or geographical origin.     

State-dependent changes in the electrostatic potential in the pore of a GluR channel

The M2 loop and the M3 segment are the major pore-lining domains in the GluR channel. These domains determine ion permeation and channel block processes and are extensively involved in gating. To study the distribution of the membrane electric potential across the GluR channel pore, we recorded from alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptors containing M2 and M3 cysteine substitutions in the GluR-A subunit and measured the voltage dependence of the modification rate of these substituted cysteines by methanethiosulfonate reagents either in the presence or absence of glutamate. In the presence of glutamate, the voltage dependence became gradually stronger for positions located deeper in the pore suggesting that the electrostatic potential drops fairly uniformly across the pore in the open state. In contrast, in the absence of glutamate, the voltage dependence was biphasic. The difference in the electrostatic potential in the presence and absence of glutamate had an apparent maximum in the middle of the extracellular vestibule. We suggest that these state-dependent changes in the membrane electric potential reflect a reorientation of the dipoles of the M2 loop alpha-helices toward and away from the center of the channel pore during gating.     

Molecular determinants of caste differentiation in the highly eusocial honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis>

Background  

In honeybees, differential feeding of female larvae promotes the occurrence of two different phenotypes, a queen and a worker, from identical genotypes, through incremental alterations, which affect general growth, and character state alterations that result in the presence or absence of specific structures. Although previous studies revealed a link between incremental alterations and differential expression of physiometabolic genes, the molecular changes accompanying character state alterations remain unknown.     

A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

<Emphasis Type=Italic>Pseudomonas aeruginosa</Emphasis> inhibits <Emphasis Type=Italic>in-vitro Candida</Emphasis> biofilm development

Background  

Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.