Glycan-independent binding and internalization of human IgG to FCMR, its cognate cellular receptory

IgM is the first antibody to be produced in immune responses and plays an important role in the neutralization of bacteria and viruses. Human IgM is heavily glycosylated, featuring five N-linked glycan sites on the μ chain and one on the J-chain. Glycosylation of IgG is known to modulate the effector functions of Fcγ receptors. In contrast, little is known about the effect of glycosylation on IgM binding to the human Fcμ receptor (hFCMR). In this study, we identify the Cμ, omain of IgM as the target of hFCMR, and show that binding and internalization of IgM by hFCMR is glycan-independent. We generated a homology-based structure for hFCMR and used molecular dynamic simulations to show how this interaction with IgM may occur. Finally, we reveal an inhibitory function for IgM in the proliferation of T cells

Modeling genome-wide replication kinetics reveals a mechanism for regulation of replication timing

We developed analytical models of DNA replication that include probabilistic initiation of origins, fork progression, passive replication, and asynchrony.We fit the model to budding yeast genome-wide microarray data probing the replication fraction and found that initiation times correlate with the precision of timing.We extracted intrinsic origin properties, such as potential origin efficiency and firing-time distribution, which cannot be done using phenomenological approaches.We propose that origin timing is controlled by stochastically activated initiators bound to origin sites rather than explicit time-measuring mechanisms

Identification of the minimal binding region of a Plasmodium falciparum IgM binding PfEMP1 domain

Binding of host immunoglobulin is a common immune evasion mechanism demonstrated by microbial pathogens. Previous work showed that the malaria parasite Plasmodium falciparum binds the Fc-region of human IgM molecules, resulting in a coating of IgM on the surface of infected erythrocytes. IgM binding is a property of P. falciparum strains showing virulence-related phenotypes such as erythrocyte rosetting. The parasite ligands for IgM binding are members of the diverse Plasmodium falciparum Erythrocyte Membrane Protein One (PfEMP1) family. However, little is known about the amino acid sequence requirements for IgM binding. Here we studied an IgM binding domain from a rosette-mediating PfEMP1 variant, DBL4ζ of TM284var1, and found that the minimal IgM-binding region mapped to the central region of the DBL domain, comprising all of subdomain 2 and adjoining parts of subdomains 1 and 3. Site-directed mutagenesis of charged amino acids within subdomain 2, predicted by molecular modelling to form the IgM binding site, showed no marked effect on IgM binding properties. Overall, this study identifies the minimal IgM binding region of a PfEMP1 domain, and indicates that the existing homology model of PfEMP1-IgM interaction is incorrect. Further work is needed to identify the specific interaction site for IgM within the minimal binding region of PfEMP1

Stochastic association of neighboring replicons creates replication factories in budding yeast

Peer reviewedPublisher PD

IgM, Fc mu Rs, and malarial immune evasion

IgM is an ancestral Ab class found in all jawed vertebrates, from sharks to mammals. This ancient ancestry is shared by malaria parasites (genus Plasmodium) that infect all classes of terrestrial vertebrates with whom they coevolved. IgM, the least studied and most enigmatic of the vertebrate Igs, was recently shown to form an intimate relationship with the malaria parasite Plasmodium falciparum. In this article, we discuss how this association might have come about, building on the recently determined structure of the human IgM pentamer, and how this interaction could affect parasite survival, particularly in light of the just-discovered Fc mu R localized to B and T cell surfaces. Because this parasite may exploit an interaction with IgM to limit immune detection, as well as to manipulate the immune response when detected, a better understanding of this association may prove critical for the development of improved vaccines or vaccination strategies

Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin

Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM) and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing

Self-assembling subnanometer pores with unusual mass-transport properties

A long-standing aim in molecular self-assembly is the development of synthetic nanopores capable of mimicking the mass-transport characteristics of biological channels and pores. Here we report a strategy for enforcing the nanotubular assembly of rigid macrocycles in both the solid state and solution based on the interplay of multiple hydrogen-bonding and aromatic π − π stacking interactions. The resultant nanotubes have modifiable surfaces and inner pores of a uniform diameter defined by the constituent macrocycles. The self-assembling hydrophobic nanopores can mediate not only highly selective transmembrane ion transport, unprecedented for a synthetic nanopore, but also highly efficient transmembrane water permeability. These results establish a solid foundation for developing synthetically accessible, robust nanostructured systems with broad applications such as reconstituted mimicry of defined functions solely achieved by biological nanostructures, molecular sensing, and the fabrication of porous materials required for water purification and molecular separations

DNA replication timing is deterministic at the level of chromosomal domains but stochastic at the level of replicons in Xenopus egg extracts

Replication origins in Xenopus egg extracts are located at apparently random sequences but are activated in clusters that fire at different times during S phase under the control of ATR/ATM kinases. We investigated whether chromosomal domains and single sequences replicate at distinct times during S phase in egg extracts. Replication foci were found to progressively appear during early S phase and foci labelled early in one S phase colocalized with those labelled early in the next S phase. However, the distribution of these two early labels did not coincide between single origins or origin clusters on single DNA fibres. The 4 Mb Xenopus rDNA repeat domain was found to replicate later than the rest of the genome and to have a more nuclease-resistant chromatin structure. Replication initiated more frequently in the transcription unit than in the intergenic spacer. These results suggest for the first time that in this embryonic system, where transcription does not occur, replication timing is deterministic at the scale of large chromatin domains (1–5 Mb) but stochastic at the scale of replicons (10 kb) and replicon clusters (50–100 kb)

Do replication forks control late origin firing in Saccharomyces cerevisiae?

Recent studies of eukaryotic DNA replication timing profiles suggest that the time-dependent rate of origin firing, I(t), has a universal shape, which ensures a reproducible replication completion time. However, measurements of I(t) are based on population averages, which may bias the shape of the I(t) because of imperfect cell synchrony and cell-to-cell variability. Here, we measure the population-averaged I(t) profile from synchronized Saccharomyces cerevisiae cells using DNA combing and we extract the single-cell I(t) profile using numerical deconvolution. The single cell I(t) and the population-averaged I(t) extracted from DNA combing and replication timing profiles are similar, indicating a genome scale invariance of the replication process, and excluding cell-to-cell variability in replication time as an explanation for the shape of I(t). The single cell I(t) correlates with fork density in wild-type cells, which is specifically loosened in late S phase in the clb5Δ mutant. A previously proposed numerical model that reproduces the wild-type I(t) profile, could also describe the clb5Δ mutant I(t) once modified to incorporate the decline in CDK activity and the looser dependency of initiation on fork density in the absence of Clb5p. Overall, these results suggest that the replication forks emanating from early fired origins facilitate origin firing in later-replicating regions

Author response

Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain 3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane