In the presence of excess nickel, Escherichia coli NikR regulates cellular nickel uptake by suppressing the transcription of the nik operon, which encodes the nickel uptake transporter, NikABCDE. Previously published in vitro studies have shown that NikR is capable of binding a range of divalent transition metal ions in addition to Ni2+, including Co2+, Cu2+, Zn2+, and Cd2+. To understand how the high-affinity nickel binding site of NikR is able to accommodate these other metal ions, and to improve our understanding of NikR's mechanism of binding to DNA, we have determined structures of the metal-binding domain (MBD) of NikR in the apo form and in complex with Cu2+ and Zn2+ ions and compared them with the previously published structures with Ni2+. We observe that Cu2+ ions bind in a manner very similar to that of Ni2+, with a square planar geometry but with longer bond lengths. Crystals grown in the presence of Zn2+ reveal a protein structure similar to that of apo MBD with a disordered alpha3 helix, but with two electron density peaks near the Ni2+ binding site corresponding to two Zn2+ ions. These structural findings along with biochemical data on NikR support a hypothesis that ordering of the alpha3 helix is important for repressor activation.

Proper ovarian development requires the cell type-specific transcription factor TAF4b, a subunit of the core promoter recognition complex TFIID. We present the 35 A structure of a cell type-specific core promoter recognition complex containing TAF4b and TAF4 (4b/4-IID), which is responsible for directing transcriptional synergy between c-Jun and Sp1 at a TAF4b target promoter. As a first step toward correlating potential structure/function relationships of the prototypic TFIID versus 4b/4-IID, we have compared their 3D structures by electron microscopy and single-particle reconstruction. These studies reveal that TAF4b incorporation into TFIID induces an open conformation at the lobe involved in TFIIA and putative activator interactions. Importantly, this open conformation correlates with differential activator-dependent transcription and promoter recognition by 4b/4-IID. By combining functional and structural analysis, we find that distinct localized structural changes in a megadalton macromolecular assembly can significantly alter its activity and lead to a TAF4b-induced reprogramming of promoter specificity.

The molecular structure of amyloid fibrils and the mechanism of their formation are of substantial medical and biological importance, but present an ongoing experimental and computational challenge. An early high-resolution view of amyloid-like structure was obtained on amyloid-like crystals of a small fragment of the yeast prion protein Sup35p: the peptide GNNQQNY. As GNNQQNY also forms amyloid-like fibrils under similar conditions, it has been theorized that the crystal’s structural features are shared by the fibrils. Here we apply magic-angle-spinning (MAS) NMR to examine the structure and dynamics of these fibrils. Previously multiple NMR signals were observed for such samples, seemingly consistent with the presence of polymorphic fibrils. Here we demonstrate that peptides with these three distinct conformations instead assemble together into composite protofilaments. Electron microscopy (EM) of the ribbon-like fibrils indicates that these protofilaments combine in differing ways to form striations of variable widths, presenting another level of structural complexity. Structural and dynamic NMR data reveal the presence of highly restricted side-chain conformations involved in interfaces between differently structured peptides, likely comprising interdigitated steric zippers. We outline molecular interfaces that are consistent with the observed EM and NMR data. The rigid and uniform structure of the GNNQQNY crystals is found to contrast distinctly with the more complex structural and dynamic nature of these "composite" amyloid fibrils. These results provide insight into the fibril-crystal distinction and also indicate a necessary caution with respect to the extrapolation of crystal structures to the study of fibril structure and formation.

The endoplasmic reticulum (ER) is a continuous, highly dynamic membrane compartment that is crucial for numerous basic cellular functions. The ER stretches from the nuclear envelope to the outer periphery of all living eukaryotic cells. This ubiquitous organelle shows remarkable structural complexity, adopting a range of shapes, curvatures, and length scales. Canonically, the ER is thought to be composed of two simple membrane elements: sheets and tubules. However, recent advances in superresolution light microscopy and three-dimensional electron microscopy have revealed an astounding diversity of nanoscale ER structures, greatly expanding our view of ER organization. In this review, we describe these diverse ER structures, focusing on what is known of their regulation and associated functions in mammalian cells.

The endoplasmic reticulum (ER) is a continuous, highly dynamic membrane compartment that is crucial for numerous basic cellular functions. The ER stretches from the nuclear envelope to the outer periphery of all living eukaryotic cells. This ubiquitous organelle shows remarkable structural complexity, adopting a range of shapes, curvatures, and length scales. Canonically, the ER is thought to be composed of two simple membrane elements: sheets and tubules. However, recent advances in superresolution light microscopy and three-dimensional electron microscopy have revealed an astounding diversity of nanoscale ER structures, greatly expanding our view of ER organization. In this review, we describe these diverse ER structures, focusing on what is known of their regulation and associated functions in mammalian cells.

Magnetic resonance imaging enables the noninvasive mapping of both anatomical white matter connectivity and dynamic patterns of neural activity in the human brain. We examine the relationship between the structural properties of white matter streamlines (structural connectivity) and the functional properties of correlations in neural activity (functional connectivity) within 84 healthy human subjects both at rest and during the performance of attention- and memory-demanding tasks. We show that structural properties, including the length, number, and spatial location of white matter streamlines, are indicative of and can be inferred from the strength of resting-state and task-based functional correlations between brain regions. These results, which are both representative of the entire set of subjects and consistently observed within individual subjects, uncover robust links between structural and functional connectivity in the human brain.

The Lrp/AsnC family of transcriptional regulatory proteins is found in both archaea and bacteria. Members of the family influence cellular metabolism in both a global (Lrp) and specific (AsnC) manner, often in response to exogenous amino acid effectors. In the present study we have determined both the first bacterial and the highest resolution structures for members of the family. Escherichia coli AsnC is a specific gene regulator whose activity is triggered by asparagine binding. Bacillus subtilis LrpC is a global regulator involved in chromosome condensation. Our AsnC-asparagine structure is the first for a regulator-effector complex and is revealed as an octameric disc. Key ligand recognition residues are identified together with a route for ligand access. The LrpC structure reveals a stable octamer supportive of a topological role in dynamic DNA packaging. The structures yield significant clues to the functionality of Lrp/AsnC-type regulators with respect to ligand binding and oligomerization states as well as to their role in specific and global DNA regulation.

The archaeon Sulfolobus solfataricus converts d-arabinose to 2-oxoglutarate by an enzyme set consisting of two dehydrogenases and two dehydratases. The third step of the pathway is catalyzed by a novel 2-keto-3-deoxy-D-arabinonate dehydratase (KdaD). In this study, the crystal structure of the enzyme has been solved to 2.1 A resolution. The enzyme forms an oval-shaped ring of four subunits, each consisting of an N-terminal domain with a four-stranded beta-sheet flanked by two alpha-helices, and a C-terminal catalytic domain with a fumarylacetoacetate hydrolase (FAH) fold. Crystal structures of complexes of the enzyme with magnesium or calcium ions and either a substrate analog 2-oxobutyrate, or the aldehyde enzyme product 2,5-dioxopentanoate revealed that the divalent metal ion in the active site is coordinated octahedrally by three conserved carboxylate residues, a water molecule, and both the carboxylate and the oxo groups of the substrate molecule. An enzymatic mechanism for base-catalyzed dehydration is proposed on the basis of the binding mode of the substrate to the metal ion, which suggests that the enzyme enhances the acidity of the protons alpha to the carbonyl group, facilitating their abstraction by glutamate 114. A comprehensive structural comparison of members of the FAH superfamily is presented and their evolution is discussed, providing a basis for functional investigations of this largely unexplored protein superfamily.

is a major human pathogen that has acquired alarming broad-spectrum antibiotic resistance. One group of secreted toxins with key roles during infection is the phenol-soluble modulins (PSMs). PSMs are amphipathic, membrane-destructive cytolytic peptides that are exported to the host-cell environment by a designated adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, the PSM transporter (PmtABCD). Here, we demonstrate that the minimal Pmt unit necessary for PSM export is PmtCD and provide its first atomic characterization by single-particle cryo-EM and x-ray crystallography. We have captured the transporter in the ATP-bound state at near atomic resolution, revealing a type II ABC exporter fold, with an additional cytosolic domain. Comparison to a lower-resolution nucleotide-free map displaying an "open" conformation and putative hydrophobic inner chamber of a size able to accommodate the binding of two PSM peptides provides mechanistic insight and sets the foundation for therapeutic design.

Alternative gene splicing gives rise to N-methyl-D-aspartate (NMDA) receptor ion channels with defined functional properties and unique contributions to calcium signaling in a given chemical environment in the mammalian brain. Splice variants possessing the exon-5-encoded motif at the amino-terminal domain (ATD) of the GluN1 subunit are known to display robustly altered deactivation rates and pH sensitivity, but the underlying mechanism for this functional modification is largely unknown. Here, we show through cryoelectron microscopy (cryo-EM) that the presence of the exon 5 motif in GluN1 alters the local architecture of heterotetrameric GluN1-GluN2 NMDA receptors and creates contacts with the ligand-binding domains (LBDs) of the GluN1 and GluN2 subunits, which are absent in NMDA receptors lacking the exon 5 motif. The unique interactions established by the exon 5 motif are essential to the stability of the ATD/LBD and LBD/LBD interfaces that are critically involved in controlling proton sensitivity and deactivation.