E1 enzymes activate ubiquitin-like proteins and transfer them to cognate E2 enzymes. Atg7, a noncanonical E1, activates two ubiquitin-like proteins, Atg8 and Atg12, and plays a crucial role in autophagy. Here, we report crystal structures of full-length Atg7 and its C-terminal domain bound to Atg8 and MgATP, as well as a solution structure of Atg8 bound to the extreme C-terminal domain (ECTD) of Atg7. The unique N-terminal domain (NTD) of Atg7 is responsible for Atg3 (E2) binding, whereas its C-terminal domain is comprised of a homodimeric adenylation domain (AD) and ECTD. The structural and biochemical data demonstrate that Atg8 is initially recognized by the C-terminal tail of ECTD and is then transferred to an AD, where the Atg8 C terminus is attacked by the catalytic cysteine to form a thioester bond. Atg8 is then transferred via a trans mechanism to the Atg3 bound to the NTD of the opposite protomer within a dimer.

CRKI (SH2-SH3) and CRKII (SH2-SH3-SH3) are splicing isoforms of the oncoprotein CRK that regulate transcription and cytoskeletal reorganization for cell growth and motility by linking tyrosine kinases to small G proteins. CRKI shows substantial transforming activity, whereas the activity of CRKII is low, and phosphorylated CRKII has no biological activity whatsoever. The molecular mechanisms underlying the distinct biological activities of the CRK proteins remain elusive. We determined the solution structures of CRKI, CRKII and phosphorylated CRKII by NMR and identified the molecular mechanism that gives rise to their activities. Results from mutational analysis using rodent 3Y1 fibroblasts were consistent with those from the structural studies. Together, these data suggest that the linker region modulates the binding of CRKII to its targets, thus regulating cell growth and motility.

Destrin is an isoprotein of cofilin that regulates actin cytoskeleton in various eukaryotes. We determined the tertiary structure of destrin by triple-resonance multidimensional nuclear magnetic resonance. In spite of there being no significant amino acid sequence homology, we found that the folding of destrin was strikingly similar to that of repeated segments in the gelsolin family, which resulted in a new protein fold group. Sequential dissimilarity of the actin-binding helix of destrin to that of gelsolin explains the Ca2+-independent actin-binding of destrin. Possible mechanisms of phosphorylation-sensitive phosphoinositide-competitive actin binding, of pH-dependent filament severing, and of nuclear translocation with actin in response to stresses, are discussed on the basis of the tertiary structure.

The hippocampus is often a difficult structure to visualize with magnetic resonance imaging (MRI) and functional MRI (fMRI) due to its convoluted nature and susceptibility to signal dropout. Improving our ability to pinpoint changes in neural activity using fMRI in this structure remains an important challenge. Current fMRI/MRI methods typically do not permit visualization of the hippocampus and surrounding cortex at a resolution less than one mm. We present here improvements to our previous methods for obtaining structural MR images of the hippocampus, which provided an in-plane resolution of 0.4 mm2 mm and two-dimensional “flat” maps of the hippocampus with an interpolated isotropic resolution of 0.4mm3 (Engel et al., 1997; Zeineh et al., 2000). We present changes to existing structural imaging sequences that now augment the resolution of previous scans, permitting visualization of the anterior portion of CA1, parts of the dentate gyrus, and CA23. These imaging improvements are of relevance generally to the field of imaging because they permit higher overall resolution imaging of the hippocampus than previously possible (at 3 Tesla). We also introduce a novel application of a computational interpolation method that improves our ability to capture the convoluted three-dimensional shape of the hippocampus. Furthermore, we have developed a quantitative method for obtaining group activation patterns based on producing averaged flat maps using vector field warping techniques, allowing localization of activations to specific hippocampal subregions across groups of subjects. Together, these methods provide a means to improve imaging of neural activity in the human hippocampus and surrounding cortex during cognitive tasks.

Src-homology 3 (SH3) domains mediate signal transduction by binding to proline-rich motifs in target proteins. We have determined the high-resolution NMR structure of the complex between the amino-terminal SH3 domain of GRB2 and a ten amino acid peptide derived from the guanine nucleotide releasing factor Sos. The NMR data show that the peptide adopts the conformation of a left-handed polyproline type II helix and interacts with three major sites on the SH3 domain. The orientation of the bound peptide is opposite to that of proline-rich peptides bound to the SH3 domains of Abl, Fyn and p85.

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We have developed an improved isotope-filtered pulse scheme in combination with a double-tuned filter, a hyperbolic secant inversion pulse, and a z-filter with a pulsed field gradient. These filtering pulse schemes have been incorporated into several one-, two-, and three-dimensional experiments, which were applied to the (13)C/(15)N uniformly labeled N-terminal SH3 domain of Grb2 complexed with the unlabeled Sos-derived peptide. The proton resonances of the Sos-derived peptide were unambiguously assigned using isotope-filtered DQF-COSY, TOCSY and NOESY spectra. Furthermore, in the isotope-filtered, isotope-edited 3D NOESY spectrum, intermolecular NOEs between the labeled protein and the unlabeled peptide could be identified. Through these applications, we demonstrate the high filtering efficiency of the presented pulse scheme.

The phagocyte NADPH oxidase plays a crucial role in host defense against microbial infections by generating reactive oxygen species. It is a multisubunit enzyme composed of membrane-bound flavocytochrome b 558 as well as cytosolic components, including p47 phox , which is essential for assembly of the complex. When phagocytes are activated, the cytosolic components of the NADPH oxidase translocate to flavocytochrome b 558 due to binding of the tandem Src homology 3 (SH3) domains of p47 phox to a proline-rich region in p22 phox , a subunit of flavocytochrome b 558 . Using NMR titration, we first identified the proline-rich region of p22 phox that is essential for binding to the tandem SH3 domains of p47 phox . We subsequently determined the solution structure of the p47 phox tandem SH3 domains complexed with the proline-rich peptide of p22 phox using NMR spectroscopy. In contrast to the intertwined dimer reported for the crystal state, the solution structure is a monomer. The central region of the p22 phox peptide forms a polyproline type II helix that is sandwiched by the N-and C-terminal SH3 domains, as was observed in the crystal structure, whereas the C-terminal region of the peptide takes on a short ␣-helical conformation that provides an additional binding site with the N-terminal SH3 domain. Thus, the C-terminal ␣-helical region of the p22 phox peptide increases the binding affinity for the tandem SH3 domains of p47 phox more than 10-fold.NADPH oxidase plays a crucial role in the killing of invading microbes by neutrophils and other phagocytic cells by reducing oxygen to superoxide anion using NADPH as an electron donor. NADPH oxidase is composed of the membrane-bound flavocytochrome b 558 , a heterodimer of gp91 phox and p22 phox , and four cytosolic regulatory components, p47phox , p67 phox , p40 phox , and Rac (1-5). Recently, p47 phox , p67 phox , and p40 phox were reported to form a ternary complex in the resting state (6). Upon activation of the cell, the p47 phox -p67 phox -p40 phox complex is relocated from the cytosol to the plasma membrane where it associates with flavocytochrome b 558 , forming an active NADPH oxidase complex. The interaction between p47 phox and p22 phox plays a critical role in the formation of this complex (7-10).The p47 phox protein contains in order from the N-to the C termini, a Phox homology (PX) 2 domain, tandem SH3 domains, a polybasic or autoinhibitory region (PBR/AIR), and a proline-rich region (PRR). The membrane-bound protein p22phox also contains a PRR in its C-terminal cytosolic region. In the resting state, the tandem SH3 domains of p47 phox are masked by an intramolecular interaction with the PBR/AIR, which maintains the enzyme in an inactive form (10 -12). Upon cell stimulation, some serine residues in the PBR/AIR domain of p47 phox are phosphorylated by a specific kinase, which induces a conformational change. This leads to changes in intramolecular interactions (13-16) and exposure of the tandem SH3 domains, permitting their binding with the PRR of p22 phox (...