Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that belong to the Cys-loop receptor superfamily. These receptors are allosteric proteins that exist in different conformational states, including resting (closed), activated (open), and desensitized (closed) states. The acetylcholine binding protein (AChBP) is a structural homologue of the extracellular ligand-binding domain of nAChRs. In previous studies, the degree of the C-loop radial extension of AChBP has been assigned to different conformational states of nAChRs. It has been suggested that a closed C-loop is preferred for the active conformation of nAChRs in complex with agonists whereas an open C-loop reflects an antagonist-bound (closed) state. In this work, we have determined the crystal structure of AChBP from the water snail Lymnaea stagnalis (Ls) in complex with dihydro-β-erythroidine (DHβE), which is a potent competitive antagonist of nAChRs. The structure reveals that binding of DHβE to AChBP imposes closure of the C-loop as agonists, but also a shift perpendicular to previously observed C-loop movements. These observations suggest that DHβE may antagonize the receptor via a different mechanism compared to prototypical antagonists and toxins.

This article describes the trimeric structure of the first PDZ domain of human PSD-93 at 2 Å resolution.

The crystal structure of the PDZ1 domain of human PSD-93 has been determined to 2.0 Å resolution. The PDZ1 domain forms a crystallographic trimer that is also predicted to be stable in solution. The main contributions to the stabilization of the trimer seem to arise from interactions involving the PDZ1–PDZ2 linker region at the extreme C-terminus of PDZ1, implying that the oligomerization that is observed is not of biological significance in full-length PSD-93. Comparison of the structures of the binding cleft of PSD-93 PDZ1 with the previously reported structures of PSD-93 PDZ2 and PDZ3 as well as of the closely related human PSD-95 PDZ1 shows that they are very similar in terms of amino-acid composition. However, the cleft is significantly narrower in PSD-95. This could be part of the basis of peptide selectivity between PSD-93 PDZ1 and PSD-95 PDZ1.

GluRδ2 is a member of the iGluR family, but despite a prominent role in cerebellar synaptic plasticity, this receptor does not appear to function as an ion channel. Endogenous ligands that modulate the activity of native GluRδ2 in the cerebellum have not been identified, but two candidate modulators are D-serine and extracellular calcium. Taking advantage of known crystal structures and spontaneously active GluRδ2 receptors containing the lurcher mutation (GluRδ2Lc), we investigated the mechanism by which calcium and D-serine regulate the activity of GluRδ2Lc. Our data suggest that calcium binding stabilizes the dimer interface formed between two agonist binding domains and increases GluRδ2Lc currents. The data further suggests that D-serine binding induces rearrangements at the dimer interface to diminish GluRδ2Lc currents by a mechanism that resembles desensitization at AMPA and kainate receptors. Thus, we propose that calcium and D-serine binding have opposing effects on the stability of the dimer interface. Furthermore, the effects of calcium are observed at concentrations that are within the physiological range, suggesting that the ability of native GluRδ2 to respond to ligand binding may be modulated by extracellular calcium. These findings place GluRδ2 among AMPA and kainate receptors, where the dimer interface is not only a biologically important site for functional regulation, but also an important target for exogenous and endogenous ligands that modulate receptor function.

The three-dimensional structure of a SARS coronavirus-derived peptide, VQQESSFVM, bound to the human major histocompatibility complex (MHC) class I antigen HLA-B*1501 is presented.

The human leukocyte antigen (HLA) class I system comprises a highly polymorphic set of molecules that specifically bind and present peptides to cytotoxic T cells. HLA-B*1501 is a prototypical member of the HLA-B62 supertype and only two peptide–HLA-B*1501 structures have been determined. Here, the crystal structure of HLA-B*1501 in complex with a SARS coronavirus-derived nonapeptide (VQQESSFVM) has been determined at high resolution (1.87 Å). The peptide is deeply anchored in the B and F pockets, but with the Glu4 residue pointing away from the floor in the peptide-binding groove, making it available for interactions with a potential T-cell receptor.

The structure of rat acidic fibroblast growth factor was determined and compared with those of human, bovine and newt origin. The rat and human structures were found to be very similar.

Fibroblast growth factors (FGFs) constitute a family of 22 structurally related heparin-binding polypeptides that are involved in the regulation of cell growth, survival, differentiation and migration. Here, a 1.4 Å resolution X-ray structure of rat FGF1 is presented. Two molecules are present in the asymmetric unit of the crystal and they coordinate a total of five sulfate ions. The structures of human, bovine and newt FGF1 have been published previously. Human and rat FGF1 are found to have very similar structures.

The structure of HLA-A*1101 in complex with a HBV peptide homologue is presented and discussed in the context of vaccine design.

A high-resolution structure of the human MHC-I molecule HLA-A*1101 is presented in which it forms a complex with a sequence homologue of a peptide that occurs naturally in hepatitis B virus DNA polymerase. The sequence of the bound peptide is AIMPARFYPK, while that of the corresponding natural peptide is LIMPARFYPK. The peptide does not make efficient use of the middle E pocket for binding, which leads to a rather superficial and exposed binding mode for the central peptide residues. Despite this, the peptide binds with high affinity (IC50 of 31 nM).

Mouse L1 modules Ig I–IV and F3 I–III were crystallized. The crystals diffracted X-rays to 3.5 and 2.8 Å resolution, respectively.

Four amino-terminal immunoglobulin (Ig) modules and three fibronectin type III (F3) modules of the mouse neural cell-adhesion molecule L1 have been expressed in Drosophila S2 cells. The Ig modules I–IV of L1 crystallized in a trigonal space group, with unit-cell parameters a = b = 239.6, c = 99.3 Å, and the crystals diffracted X-rays to a resolution of about 3.5 Å. The F3 modules I–III of L1 crystallized in a tetragonal space group, with unit-cell parameters a = b = 80.1, c = 131 Å, and the crystals diffracted X-rays to 2.8 Å resolution. This is a step towards the structure determination of the multimodular constructs of the neural cell-adhesion molecule L1 in order to understand the function of L1 on a structural basis.

Although amyloid fibrillation is generally believed to be a nucleation-dependent process, the nuclei are largely structurally uncharacterized. This is in part due to the inherent experimental challenge associated with structural descriptions of individual components in a dynamic multi-component equilibrium. There are indications that oligomeric aggregated precursors of fibrillation, and not mature fibrils, are the main cause of cytotoxicity in amyloid disease. This further emphasizes the importance of characterizing early fibrillation events. Here we present a kinetic x-ray solution scattering study of insulin fibrillation, revealing three major components: insulin monomers, mature fibrils, and an oligomeric species. Low-resolution three-dimensional structures are determined for the fibril repeating unit and for the oligomer, the latter being a helical unit composed of five to six insulin monomers. This helical oligomer is likely to be a structural nucleus, which accumulates above the supercritical concentration used in our experiments. The growth rate of the fibrils is proportional to the amount of the helical oligomer present in solution, suggesting that these oligomers elongate the fibrils. Hence, the structural nucleus and elongating unit in insulin amyloid fibrillation may be the same structural component above supercritical concentrations. A novel elongation pathway of insulin amyloid fibrils is proposed, based on the shape and size of the fibrillation precursor. The distinct helical oligomer described in this study defines a conceptually new basis of structure-based drug design against amyloid diseases.

Author Summary

Diseases associated with the presence of amyloid structures, such as Alzheimer and Parkinson disease, are characterized by the presence of protein aggregates in the form of highly ordered fibrils. This amyloid fibril formation is also commonly observed for a number of protein drugs, such as insulin. Detailed information on how and why these fibrils are formed will be very useful to design compounds and drugs that may reverse or even prevent fibril formation, but existing knowledge in this field is still limited. We have studied, in real time, the fibril formation of insulin using a technique based on scattering of x-rays (small-angle x-ray scattering [SAXS]). Using SAXS, we obtained hitherto unprecedented three-dimensional structural information on these fibrils in solution. Most importantly, we were able to describe the three-dimensional structure of a crucial intermediate, which is probably a structural starting point (nucleus) in the fibril formation process. These results suggest that under our experimental conditions this crucial intermediate serves both as the fibrillation nucleus, as well as the elongating species. We propose that the latter intermediate is an interesting target for small molecules in order to prevent or reduce amyloid fibril formation.

Insulin fibrillation in solution is studied using small-molecule x-ray scattering. This study provides insights into the early stages of amyloid fibrillation, revealing the amyloid fibril repeat unit and evidence of a helical oligomer.