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Our 3.05 Å crystal structure of the E. coli Cascade complex exhibits a sea-horse-shaped architecture, similar to previous cryo-electron microscopy structures (10). Instead of forming a simple, compact structure, the [...], Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (Cas) proteins form the CRISPR/ Cas system to defend against foreign nucleic acids of bacterial and archaeal origin (1-9). In the I-E subtype CRISPR/Cas system, eleven subunits from five Cas proteins (Cas[A.sub.1][B.sub.2][C.sub.6][D.sub.1][E.sub.1]) assemble along a CRISPR RNA (crRNA) to form the Cascade complex[10.sub.-13]. Here we report on the 3.05 Å crystal structure of the 405-kilodalton Escherichia coli Cascade complex that provides molecular details beyond those available from earlier lower-resolution cryo-electron microscopy structures. The bound 61-nucleotide crRNA spans the entire 11-protein subunit-containing complex, where it interacts with all six CasC subunits (named CasC1-6), with its 5' and 3' terminal repeats anchored by CasD and CasE, respectively. The crRNA spacer region is positioned along a continuous groove on the concave surface generated by the aligned CasC1-6 subunits. The five long β-hairpins that project from individual CasC2-6 subunits extend across the crRNA, with each β-hairpin inserting into the gap between the last stacked base and its adjacent splayed counterpart, and positioned within the groove of the preceding CasC subunit. Therefore, instead of continuously stacking, the crRNA spacer region is divided into five equal fragments, with each fragment containing five stacked bases flanked by one flipped-out base. Each of those crRNA spacer fragments interacts with CasC in a similar fashion. Furthermore, our structure explains why the seed sequence, with its outward-directed bases, has a critical role in target DNA recognition. In conclusion, our structure of the Cascade complex provides novel molecular details of protein-protein and protein-RNA alignments and interactions required for generation of a complex mediating RNA-guided immune surveillance.

Carbon catabolite repression (CCR) is common in bacteria and higher organisms: up to 5-10% of all bacterial genes are subject to CCR regulation (1). Different organisms use different pathways to [...], Efficient carbon utilization is critical to the survival of microorganisms in competitive environments. To optimize energy usage, bacteria have developed an integrated control system to preferentially uptake carbohydrates that support rapid growth. The availability of a preferred carbon source, such as glucose, represses the synthesis and activities of proteins necessary for the transport and metabolism of secondary carbon sources. This regulatory phenomenon is defined as carbon catabolite repression1. In enteric bacteria, the key player of carbon catabolite repression is a component of the glucose-specific phosphotransferase system, enzyme IIA ([EIIA.sup.Glc]) (1,2). It is known that unphosphorylated [EIIA.sup.Glc] binds to and inhibits a variety of transporters when glucose is available (1,2). However, understanding the underlying molecular mechanism has been hindered by the complete absence of structures for any [EIIA.sup.Glc]--transporter complexes. Here we present the 3.9 Å crystal structure of Escherichia coli [EIIA.sup.Glc] in complex with the maltose transporter, an ATP-binding cassette (ABC) transporter. The structure shows that two [EIIA.sup.Glc] molecules bind to the cytoplasmic ATPase subunits, stabilizing the transporter in an inward-facing conformation and preventing the structural rearrangements necessary for ATP hydrolysis. We also show that the half-maximal inhibitory concentrations of the full-length [EIIA.sup.Glc] and an amino-terminal truncation mutant differ by 60-fold, consistent with the hypothesis that the amino-terminal region, disordered in the crystal structure, functions as a membrane anchor to increase the effective [EIIA.sup.Glc] concentration at the membrane (3,4). Together these data suggest a model of how the central regulatory protein [EIIA.sup.Glc] allosterically inhibits maltose uptake in E. coli.

The active site of the ribosome, the peptidyl transferase centre, catalyses two reactions, namely, peptide bond formation between peptidyl-tRNA and aminoacyl-tRNA as well as the release-factor-dependent hydrolysis of peptidyl-tRNA. Unlike [...]

L-fucose is a major constituent of N-linked glycans on the cell surfaces of microbes, plants and animals (7). It can serve as the sole carbon source for some bacteria. In [...], The major facilitator superfamily (MFS) transporters are an ancient and widespread family of secondary active transporters (1). In Escherichia coli, the uptake of L-fucose, a source of carbon for microorganisms, is mediated by an MFS proton symporter, FucP (2,3). Despite intensive study of the MFS transporters, atomic structure information is only available on three proteins and the outward-open conformation has yet to be captured (4-6). Here we report the crystal structure of FucP at 3.1 Å resolution, which shows that it contains an outward-open, amphipathic cavity. The similarly folded amino and carboxyl domains of FucP have contrasting surface features along the transport path, with negative electrostatic potential on the N domain and hydrophobic surface on the C domain. FucP only contains two acidic residues along the transport path, Asp 46 and Glu 135, which can undergo cycles of protonation and deprotonation. Their essential role in active transport is supported by both invivo and in vitro experiments. Structure-based biochemical analyses provide insights into energy coupling, substrate recognition and the transport mechanism of FucP.

In Gram-negative bacteria, efflux systems of the RND superfamily have significant functions in the intrinsic and acquired tolerance of antimicrobial agents, including silver and copper ions (1,2). The Gram-negative bacterium [...], Gram-negative bacteria, such as Escherichia coli, frequently use tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel various toxic compounds from the cell (1,2). The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions (3,4). No previous structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner-membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide new structural information about the HME subfamily of RND efflux pumps. The structures suggest that the metal-binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. This cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal-binding site, four methionine pairs--three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, using these methionine pairs or clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites.

ICDH (Enzyme Commission number (EC) 1.1.1.42) is located at a critical junction between the Krebs cycle and the glyoxylate bypass, an anaplerotic pathway that is essential for growth on substrates [...], The Escherichia coli isocitrate dehydrogenase kinase/phosphatase (AceK) is a unique bifunctional enzyme that phosphorylates or dephosphorylates isocitrate dehydrogenase (ICDH) in response to environmental changes, resulting in the inactivation or, respectively, activation of ICDH (1). ICDH inactivation short-circuits the Krebs cycle by enabling the glyoxlate bypass (2,3). It was the discovery of AceK and ICDH that established the existence of protein phosphorylation regulation in prokaryotes (1,4). As a 65-kDa protein, AceK is significantly larger than typical eukaryotic protein kinases. Apart from the ATP-binding motif, AceK does not share sequence homology with any eukaryotic protein kinase or phosphatase (5,6). Most intriguingly, AceK possesses the two opposing activities of protein kinase and phosphatase within one protein, and specifically recognizes only intact ICDH (7,8). Additionally, AceK has strong ATPase activity (9). It has been shown that AceK kinase, phosphatase and ATPase activities reside at the same site (6,10), although the molecular basis of such multifunctionality and its regulation remains completely unknown. Here we report the structures of AceK and its complex with ICDH. The AceK structure reveals a eukaryotic protein-kinase-like domain containing ATP and a regulatory domain with a novel fold. As an AceK phosphatase activator and kinase inhibitor, AMP is found to bind in an allosteric site between the two AceK domains. An AMP-mediated conformational change exposes and shields ATP, acting as a switch between AceK kinase and phosphatase activities, and ICDH-binding induces further conformational change for AceK activation. The substrate recognition loop of AceK binds to the ICDH dimer, allowing higher-order substrate recognition and interaction, and inducing critical conformational change at the phosphorylation site of ICDH.

Author(s): Alline R. Pacheco [1, 2]; Meredith M. Curtis [1, 2]; Jennifer M. Ritchie [3]; Diana Munera [3]; Matthew K. Waldor [3]; Cristiano G. Moreira [1, 2]; Vanessa Sperandio (corresponding [...]