An approach combining small-angle X-ray solution scattering (SAXS) data with coarse-grained (CG) simulations is developed to characterize the assembly states of Hck, a member of the Srcfamily kinases, under various conditions in solution. First, a basis set comprising a small number of assembly states is generated from extensive CG simulations. Second, a theoretical SAXS profile for each state in the basis set is computed by using the Fast-SAXS method. Finally, the relative population of the different assembly states is determined via a Bayesian-based Monte Carlo procedure seeking to optimize the theoretical scattering profiles against experimental SAXS data. The study establishes the concept of basis-set supported SAXS (BSS-SAXS) reconstruction combining computational and experimental techniques. Here, BSS-SAXS reconstruction is used to reveal the structural organization of Hck in solution and the different shifts in the equilibrium population of assembly states upon the binding of different signaling peptides.T yrosine kinases of the Src-family are large multidomain allosteric enzymes implicated in the signaling pathways regulating cell growth and proliferation (1). The key role that Src kinases play in the onset of many human diseases, particularly cancer, makes them important targets for therapeutic intervention (2).All Src kinases share a common structural organization comprising the SH3 and SH2 binding domains followed by a highly conserved catalytic domain connected by flexible linkers (1). Crystal structures have offered a detailed view of the down-regulated forms of Hck and c-Src (3, 4), characterized by a compact assembled form stabilized by auto-inhibitory intramolecular interactions between the N and C terminus of the catalytic domain with the SH3 and SH2 modules, respectively. The SH2 and SH3 modules of the Src-family tyrosine kinases play dual roles: both are involved in the auto-inhibitory intramolecular interactions down-regulating kinase activity, but can also serve as possible binding receptors for cellular signals leading to kinase activation.In a broadly accepted paradigm, Src inactivation/activation is pictured in terms of a simple two-state process involving an assembled (inactive) and a disassembled (active) conformation, i.e., once any of the intramolecular interaction is released, the kinase domain switches to a disassembled catalytically active state targeting down-stream cellular substrates (5-8). Crystal structures (3, 4) and mutational studies complemented by molecular dynamics (MD) simulations (9) are consistent with this view. However, one crystal structure of c-Src in a partially activated state in which the SH2-SH3 modules are reassembled in a different orientation with respect to the catalytic domain (10), suggests that the situation could be considerably more complex. In this regard, it seems likely that different signals could promote different assembly states, leading to differently configured activated kinases. While it is understood that Src increases its activity in respon...

Abstract-To determine whether T cells and B cells influence lipid metabolism and atherosclerosis, we crossed apolipoprotein E-deficient (apoE°) mice with recombination activating gene 2-deficient (RAG2°) mice. Total plasma cholesterol levels were Ϸ20% higher in male apoE°mice compared with the apoE°RAG2°mice at 8 weeks of age, and plasma triglyceride levels were 2.5-fold higher in the apoE°mice even when plasma cholesterol levels were similar. Male mice with plasma cholesterol levels between 400 and 600 mg/dL at 8 weeks of age were euthanized at 27 and 40 weeks of age. The aortic root lesion area in the apoE°RAG2°mice, compared with that in the immune-competent apoE°m ice, was 81% and 57% smaller at 27 and 40 weeks of age, respectively. In contrast, there was no difference in the size of the brachiocephalic trunk lesions. Similar results were obtained with mice euthanized at 40 weeks of age that had 8-week cholesterol levels between 300 and 399 mg/dL. In apoE°RAG2°mice, aortic root atherosclerosis was more profoundly suppressed at lower cholesterol levels. Thus, T and B cells and their products differentially influence the development of atherosclerosis at different sites. We also demonstrate a profound effect of the immune system on plasma lipid homeostasis. Key Words: atherosclerosis Ⅲ apolipoprotein E Ⅲ immune deficiency Ⅲ T cells Ⅲ lipoproteins T here is strong circumstantial evidence indicating that the immune response participates in the evolution of atherosclerosis in humans and experimental animals. Many components involved in this response have been detected in atherosclerotic vessels (see review 1 ). T cells are present in human 2 and murine 3 atherosclerotic lesions. Atherosclerotic plaques of apoE-deficient (apoE°) mice contain T cells reactive with oxidized lipoproteins and heat shock proteins. 4 ApoE°mice on a chow diet develop complex lesions throughout the vascular tree, and this occurrence is accelerated by a Western diet. 5,6 These mice have been crossed with recombination activating gene (RAG)1-deficient (RAG1°) and RAG2-deficient (RAG2°) mice. Both of these RAG proteins are necessary for recombination of the T-cell receptor and immunoglobulin genes; thus, RAG°mice lack mature T and B cells. ApoE°RAG1°mice fed a chow diet for 16 weeks showed a 2-fold reduction in aortic root atherosclerosis. 7 The male apoE°RAG1°mice also exhibited modest reductions in plasma cholesterol levels. On the other hand, the extent of atherosclerosis in apoE°mice with either RAG1 or RAG2 deficiency fed a Western diet was similar to that found in immune-competent apoE°mice. 7,8 The role of B cells in the development of atherosclerosis is less clear. Antibodies to oxidized lipoproteins 9 and to heat shock protein 65 10 have been detected in the plasma of subjects with atherosclerotic cardiovascular disease. High titers of circulating autoantibodies to oxidized lipoproteins have also been detected in apoE°mice. 11 Recently, Zhou and Hansson 12 demonstrated the presence of CD22ϩ B cells and IgM in the aortic root lesions...

SUMMARY Ionotropic glutamate receptors (iGluRs) transduce the chemical signal of neurotransmitter release into membrane depolarization at excitatory synapses in the brain. The opening of the transmembrane ion channel of these ligand-gated receptors is driven by conformational transitions that are induced by the association of glutamate molecules to the ligand-binding domains (LBDs). Here, we describe the crystal structure of a GluA2 LBD tetramer in a configuration that involves an ~30° rotation of the LBD dimers relative to the crystal structure of the full-length receptor. The configuration is stabilized by an engineered disulfide crosslink. Biochemical and electrophysiological studies on full-length receptors incorporating either this crosslink or an engineered metal bridge show that this LBD configuration corresponds to an intermediate state of receptor activation. GluA2 activation therefore involves a combination of both intra-LBD (cleft closure) and inter-LBD dimer conformational transitions. Overall, these results provide a comprehensive structural characterization of an iGluR intermediate state.

Objective-We sought to examine whether there is a site-specific effect on atherosclerosis of the absence of mature T and B cells caused by a recombination activating-gene deficiency in LDL receptor-deficient mice and whether this effect is influence by the extent of backcrossing to C57BL/6 mice. Methods and Results-Male mice were fed atherogenic diets for 3 months. In strain 1 mice, in which Ϸ93% of the genes were from C57BL/6 mice, the absence of mature T and B cells led to a significant reduction in atherosclerosis in both the aortic sinus and the innominate artery. In strain 2 mice, in which Ϸ99ϩ% of the genes were from C57BL/6 mice, immune system deficiency led to a site-specific effect on atherosclerosis, with a reduction in atherosclerosis in the aortic sinus but not in the innominate artery, similar to previous results obtained with apolipoprotein EϪ/Ϫ mice. All of the immune system-incompetent mice had lower plasma total and VLDL cholesterol levels regardless of strain or diet, indicating that differences in lipid levels were unlikely to be responsible for these site-specific effects of immune system deficiency. Conclusions-These results suggest that immune system deficiency has a site-specific effect on atherosclerosis that is sensitive to the genetic background of the mice. A therosclerosis is a chronic, inflammatory response to hyperlipidemia. 1 As with other inflammation, components of both innate and adaptive immunity influence the progression of this inflammation. The cells that have received the most attention are the innate responders-monocytes/ macrophages-and the T and B cells of the adaptive immunity system. These cells participate either directly through their primary immune functions or indirectly, when mediated by the cytokines that they produce. The involvement of the adaptive immune system has been implicated by 2 kinds of studies in mice. First, the use of immune system-deficient models involving the absence of mature T and B cells (ie, recombination activating-gene deficiency [RAGϪ/Ϫ]), coupled with either apolipoprotein E deficiency (apoEϪ/Ϫ) or LDL receptor deficiency (LDLRϪ/Ϫ), results in a reduction of aortic sinus atherosclerosis, 2-5 implying that the net effect of the immune system through the course of atherogenesis is to promote vascular lesions. Consistent with this conclusion are the results from studies involving deficiency or overexpression of some cytokines secreted by T cells. 6 -9 In addition, recent data have strongly suggested that B cells are protective against the development of atherosclerosis. 10,11 Second, and related to the latter finding, is the observation that immunization of LDLRϪ/Ϫ mice with oxidized LDL and even with native LDL reduces atherosclerosis. 12 The effect of the immune system on atherosclerosis can be complex. In a previous study, we observed a site-specific effect of immune system deficiency on atherosclerosis. In that study, male apoEϪ/Ϫ RAG2Ϫ/Ϫ mice had markedly reduced aortic sinus atherosclerosis but not brachiocephalic (innominate) artery ...

No abstract