The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single turnover enzyme that displays a stable product state after double-stranded DNA cleavage. Here, we present cryo-EM structures of pre-catalytic, post-catalytic, and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg2+. In the pre-catalytic state, Cas9 adopts the “checkpoint” conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the post-catalytic state involves a dramatic ~34 Å swing of the HNH domain and disorder of the REC2 recognition domain. The post-catalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the pre-catalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and nucleic acids provide new insights into the mechanism of genome editing by Cas9.

Selenocysteine is the only genetically encoded amino acid in humans whose biosynthesis occurs on its cognate transfer RNA (tRNA). O-Phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS) catalyzes the final step of selenocysteine formation by a poorly understood tRNAdependent mechanism. The crystal structure of human tRNA Sec in complex with SepSecS, phosphoserine, and thiophosphate, together with in vivo and in vitro enzyme assays, supports a pyridoxal phosphate-dependent mechanism of Sec-tRNA Sec formation. Two tRNA Sec molecules, with a fold distinct from other canonical tRNAs, bind to each SepSecS tetramer through their 13-base pair acceptor-TΨC arm (where Ψ indicates pseudouridine). The tRNA binding is likely to induce a conformational change in the enzyme's active site that allows a phosphoserine covalently attached to tRNA Sec , but not free phosphoserine, to be oriented properly for the reaction to occur.The 21st amino acid, selenocysteine (Sec), is distinct from other amino acids not only because it lacks its own tRNA synthetase, but also because it is the only one that is synthesized on the cognate tRNA in all domains of life [reviewed in (1-4)] in a process that is reminiscent of the tRNA-dependent synthesis of glutamine, asparagine, and cysteine in prokaryotes (4). The importance of Sec is illustrated by the embryonic lethal phenotype of the tRNA Sec knockout mouse (5) and by the presence of Sec in the active sites of enzymes involved in removing reactive oxidative species and in thyroid hormone activation (6,7). It is intriguing that the codon for Sec is UGA, which is normally a translational stop signal (1). During translation of selenoprotein mRNAs, UGA is recoded by the interaction of a

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Pigment epithelium-derived factor (PEDF), a noninhibitory member of the serpin superfamily, is the most potent inhibitor of angiogenesis in the mammalian ocular compartment. It also has neurotrophic activity, both in the retina and in the central nervous system, and is highly up-regulated in young versus senescent fibroblasts. To provide a structural basis for understanding its many biological roles, we have solved the crystal structure of glycosylated human PEDF to 2.85 Å. The structure revealed the organization of possible receptor and heparin-binding sites, and showed that, unlike any other previously characterized serpin, PEDF has a striking asymmetric charge distribution that might be of functional importance. These results provide a starting point for future detailed structure͞function analyses into possible mechanisms of PEDF action that could lead to development of therapeutics against uncontrolled angiogenesis.

CheY is the best characterized member of the response regulator superfamily, and as such it has become the principal model for understanding the initial molecular mechanisms of signaling in two-component systems. Normal signaling by response regulators requires phosphorylation, in combination with an activation mechanism whose conformational effects are not completely understood. CheY activation involves three events, phosphorylation, a conformational change in the ␤ 4 -␣ 4 loop, and a rotational restriction of the side chain of tyrosine 106. An outstanding question concerns the nature of an active conformation in the apoCheY population. The details of this 1.08-Å resolution crystal structure of wild-type apoCheY shows the ␤ 4 -␣ 4 loop in two distinctly different conformations that sterically correlate with the two rotameric positions of the tyrosine 106 side chain. One of these conformational states of CheY is the inactive form, and we propose that the other is a meta-active form, responsible for the active properties seen in apoCheY.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are related intestinal pathogens that harbor highly similar pathogenicity islands known as the locus of enterocyte effacement (LEE). Despite their genetic similarity, these two pathogens disrupt epithelial tight junction barrier function with distinct kinetics. EHEC-induced reduction in transepithelial electrical resistance (TER), a measure of barrier function disruption, is significantly slower and more modest in comparison to that induced by EPEC. The variation in bacterial adherence only partially accounted for these differences. The LEE-encoded effector protein EspF has been shown to be critical for EPEC-induced alterations in TER. EspF from both EPEC and EHEC is expressed and secreted upon growth in tissue culture medium. The mutation of EHEC cesF suggested that the optimal expression and secretion of EHEC EspF required its chaperone CesF, as has been shown for EPEC. In contrast to EPEC espF and cesF, mutation of the corresponding EHEC homologs did not dramatically alter the decrease in TER. These differences could possibly be explained by the presence of additional espF-like sequences (designated U-and M-espF, where the letter designations refer to the specific cryptic prophage sequences on the EHEC chromosome closest to the respective genes) in EHEC. Reverse transcription-PCR analyses revealed coordinate regulation of EHEC U-espF and the LEE-encoded espF, with enhanced expression in bacteria grown in Dulbecco-Vogt modified Eagle's medium compared to bacteria grown in Luria broth. Both EHEC espF and U-espF complemented an EPEC espF deletion strain for barrier function alteration. The overexpression of U-espF, but not espF, in wild-type EHEC potentiated the TER response. These studies reveal further similarities and differences in the pathogenesis of EPEC and EHEC.

Spectrin and ankyrin participate in membrane organization, stability, signal transduction, and protein targeting; their interaction is critical for erythrocyte stability. Repeats 14 and 15 of ␤I-spectrin are crucial for ankyrin recognition, yet the way spectrin binds ankyrin while preserving its repeat structure is unknown. We have solved the crystal structure of the ␤I-spectrin 14,15 di-repeat unit to 2.1 Å resolution and found 14 residues critical for ankyrin binding that map to the end of the helix C of repeat 14, the linker region, and the B-C loop of repeat 15. The tilt (64°) across the 14,15 linker is greater than in any published di-repeat structure, suggesting that the relative positioning of the two repeats is important for ankyrin binding. We propose that a lack of structural constraints on linker and inter-helix loops allows proteins containing spectrin-like di-repeats to evolve diverse but specific ligand-recognition sites without compromising the structure of the repeat unit. The linker regions between repeats are thus critical determinants of both spectrin's flexibility and polyfunctionality. The putative coupling of flexibility and ligand binding suggests a mechanism by which spectrin might participate in mechanosensory regulation. (Blood. 2009;113:5377-5384) IntroductionFirst discovered in the human erythrocyte and closely associated with a variety of familial hemolytic anemias, the spectrin-ankyrin cytoskeleton has emerged as the classical paradigm of a polyfunctional organizing membrane scaffold. Ubiquitous in higher eukaryotes, the spectrin-ankyrin skeleton contributes to membrane stability, the organization of membrane proteins and lipids, the recruitment to membranes of cytosolic proteins and signaling complexes, the tethering of organized protein mosaics to filamentous actin or to the motors effecting microtubule-directed transport, and the facilitated transport of membrane proteins through the secretory and endocytic pathways. 1 Reflecting these diverse but fundamental roles, hereditary or experimental disruption of spectrin or ankyrin leads to many pathologies, including hemolytic disease, 2 embryonic lethality, cancer and developmental defects, 3 pump and channel failures and endoplasmic reticulum (ER) retention disorders, 4 neuromuscular syndromes and sudden cardiac death. 5,6 The participation of spectrin and ankyrin in so many cellular processes reflects their ability to organize multiple membrane and cytosolic proteins and lipids into membrane microdomains, linking them to the filamentous skeleton. Polyfunctionality is a critical attribute of both proteins. Ankyrins derive this capacity largely by the juxtaposition of multiple 33-residue repeat units, each composed of 2 helices linked by a ␤-turn. 7 Selectivity is achieved by minor sequence variation within the ␤-turn of each ankyrin-repeat and by the juxtaposition of repeats with differing sequence. Less is known about how spectrin binds its ligands with high affinity and specificity. In humans, there are 7 spectrin genes encoding 5...

We have determined the X-ray crystal structure to 1.8 A resolution of the Ca(2+) complex of complement-like repeat 7 (CR7) from the low-density lipoprotein receptor-related protein (LRP) and characterized its calcium binding properties at pH 7.4 and 5. CR7 occurs in a region of the LRP that binds to the receptor-associated protein, RAP, and other protein ligands in a Ca(2+)-dependent manner. The calcium coordination is identical to that found in LB5 and consists of carboxyls from three conserved aspartates and one conserved glutamate, and the backbone carbonyls of a tryptophan and another aspartate. The overall fold of CR7 is similar to those of CR3 and CR8 from the LRP and LB5 from the LDL receptor, though the low degree of sequence homology of residues not involved in calcium coordination or in disulfide formation results in a distinct pattern of surface residues for each domain, including CR7. The thermodynamic parameters for Ca(2+) binding at both extracellular and endosomal pHs were determined by isothermal titration calorimetry for CR7 and for related complement-like repeats CR3, CR8, and LB5. Although the drop in pH resulted in a reduction in calcium affinity in each case, the changes were very variable in magnitude, being as low as a 2-fold reduction for CR3. This suggests that a pH-dependent change in calcium affinity alone cannot be responsible for the release of bound protein ligands from the LRP at the pH prevailing in the endosome, which in turn requires one or more other pH-dependent effects for regulating protein ligand release.