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GTPases of immunity-associated proteins (GIMAPs) are a distinctive family of GTPases, which control apoptosis in lymphocytes and play a central role in lymphocyte maturation and lymphocyte-associated diseases. To explore their function and mechanism, we determined crystal structures of a representative member, GIMAP2, in different nucleotide-loading and oligomerization states. Nucleotide-free and GDP-bound GIMAP2 were monomeric and revealed a guanine nucleotide-binding domain of the TRAFAC (translation factor associated) class with a unique amphipathic helix [alpha]7 packing against switch II. In the absence of [alpha]7 and the presence of GTP, GIMAP2 oligomerized via two distinct interfaces in the crystal. GTP-induced stabilization of switch I mediates dimerization across the nucleotide-binding site, which also involves the GIMAP specificity motif and the nucleotide base. Structural rearrangements in switch II appear to induce the release of [alpha]7 allowing oligomerization to proceed via a second interface. The unique architecture of the linear oligomer was confirmed by mutagenesis. Furthermore, we showed a function for the GIMAP2 oligomer at the surface of lipid droplets. Although earlier studies indicated that GIMAPs are related to the septins, the current structure also revealed a strikingly similar nucleotide coordination and dimerization mode as in the dynamin GTPase. Based on this, we reexamined the relationships of the septin- and dynamin-like GTPases and demonstrate that these are likely to have emerged from a common membrane-associated dimerizing ancestor. This ancestral property appears to be critical for the role of GIMAPs as nucleotide-regulated scaffolds on intracellular membranes. G protein | protein structure doi/ 10.1073/pnas.1010322107

His-tagged full-length human MxA (Fig. 1a) was recombinantly expressed in bacteria and purified to homogeneity (Methods and Supplementary Fig. 1). In crystallization trials, small needle-shaped protein crystals were obtained that [...], The interferon-inducible dynamin-like myxovirus resistance protein 1 (MxA; also called MX1) GTPase is a key mediator of cell-autonomous innate immunity against pathogens such as influenza viruses (1). MxA partially localizes to COPI-positive membranes of the smooth endoplasmic reticulum-Golgi intermediate compartment (2). At the point of infection, it redistributes to sites of viral replication and promotes missorting of essential viral constituents (3,4). It has been proposed that the middle domain and the GTPase effector domain of dynamin-like GTPases constitute a stalk that mediates oligomerization and transmits conformational changes from the G domain to thetargetstructure (5-7); however, the molecular architecture of this stalk has remained elusive. Here we report the crystal structure of the stalk of human MxA, which folds into a four-helical bundle. This structure tightly oligomerizes in the crystal in a criss-cross pattern involving three distinct interfaces and one loop. Mutations in each of these interaction sites interfere with native assembly, oligomerization, membrane binding and antiviral activity of MxA. On the basis of these results, we propose a structural model for dynamin oligomerization and stimulated GTP hydrolysis that is consistent with previous structural predictions and has functional implications for all members of the dynamin family.

The exact binding sites that are responsible for the interaction of the amyloid precursor protein (APP) and sorLA in neurons are delineated and their mode of action are characterized as a first approach toward elucidating this novel molecular mechanism that controls APP trafficking and processing the brain. The results help in understanding the molecular determinants of neuronal APP trafficking and processing and on possible targets for intervention with senile plaque formation in patients with Alzheimer's disease (AD).

sorLA (sorting protein-related receptor) is a type-1 membrane protein of unknown function that is expressed in neurons. Its homology to sorting receptors that shuttle between the plasma membrane, endosomes, and the Golgi suggests a related function in neuronal trafficking processes. Because expression of sorLA is reduced in the brain of patients with Alzheimer's disease (AD), we tested involvement of this receptor in intracellular transport and processing of the amyloid precursor protein (APP) to the amyloid [beta]-peptide (A[beta]), the principal component of senile plaques. We demonstrate that sorLA interacts with APP in vitro and in living cells and that both proteins colocalize in endosomal and Golgi compartments. Overexpression of sorLA in neurons causes redistribution of APP to the Golgi and decreased processing to A[beta], whereas ablation of sorLA expression in knockout mice results in increased levels of A[beta] in the brain similar to the situation in AD patients. Thus, sorLA acts as a sorting receptor that protects APP from processing into A[beta] and thereby reduces the burden of amyloidogenic peptide formation. Consequently, reduced receptor expression in the human brain may increase A[beta] production and plaque formation and promote spontaneous AD. endocytic receptors | knockout mouse | neurodegeneration | Vps10p-domain receptors

The oxygen-evolving photosystem II core complexes (PSIIcc) from the thermophilic cyanobacterium Thermosynechococcus elongatus (PSIIccTe) and the higher plant Spinacia oleracea (PSIIccSo) are isolated from the thylakoid membrane by solubilization with n-dodecyl-beta-D-maltoside, purified and characterized by gel permeation chromatography (GPC), dynamic light scattering (DLS), and analytical centrifugation (AUC). The results are discussed from a crystallization point of view.

Photon and fluorescence correlation spectroscopic studies yield the same apparent hydrodynamic radii measurement for the hexameric RepA DNA-helicase dimer and its complex with the single-stranded DNA. Data reveal that the RepA DNA-helicase binds to single-stranded DNA optimally under acidic conditions leading to a structural change in the former.

Research demonstrates that the monomeric form of hexokinase 2 of Saccharomyces cerevisiae exhibits high-affinity to its substrates mediated by substitution of serine-14 with glutamate and subsequent pseudophosphorylation of the enzyme. Data further point out that phosphorylation at serine-14 may be involved in glucose signaling.

Research was conducted to examine the water-soluble de novo beta-sheet peptides which self-assemble into fibrillar structures. Conformational analyses suggest differing potentials of the various peptides to adopt the beta-sheet conformation. Both the influence of D-amino acid substitution and the length of the secondary structure domain were correlated with the beta-sheet stability and the association behavior. Results demonstrate that strong association of beta-strands leads to fibrillar structures which can bind Congo Red and exhibit birefringence.

Research was conducted to demonstrate that serine-14 phosphorylation in Saccharomyces cerevisiae influences the oligomeric state of hexokinase through site-directed mutagenesis and sedimentation analysis. It was also noted that its substitution by glutamate caused complete dissociation while glutamate exchange for serine-157 did not. It is concluded that the in vivo phosphorylation at serine-14 may offer a mechanism to enhance glucose utilization from low level.

Analytical centrifugation was performed to understand the quatenary structures and interaction in solution of the GroE chaperonin system. The heterogenous association between GroEL and GroEs oligomers under different conditions was analyzed by studying the sedimentation equilibrium in the analytical centrifuge. Low amounts of asymmetrical GroEL- GroES2 complexes were observed under in vivo conditions.

Enzyme assay, peptide sequencing and size determination by mass spectrometry were done to identify the site of autophosphorylative inactivation of hexokinase 2 derived from Saccharomyces cerevisiae. The results confirmed that the site is localized at serine-158. This was verified by mutation analysis wherein serine-158 was replaced by alanine and cysteine which resulted in decreased enzyme activity. Mutation of the same amino acid to glutamate, on the other hand, led to complete loss of catalytic activity. It was also found that phosphoserine/phosphothreonine-specific protein phosphatase-2A can cause complete reversion of the autophosphorylative inactivation of hexokinase 2.