ZBP1 (zipcode-binding protein 1) was originally discovered as a trans-acting factor for the ''zipcode'' in the 39 untranslated region (UTR) of the b-actin mRNA that is important for its localization and translational regulation. Subsequently, ZBP1 has been found to be a multifunctional regulator of RNA metabolism that controls aspects of localization, stability, and translation for many mRNAs. To reveal how ZBP1 recognizes its RNA targets, we biochemically characterized the interaction between ZBP1 and the b-actin zipcode. The third and fourth KH (hnRNP K homology) domains of ZBP1 specifically recognize a bipartite RNA element located within the first 28 nucleotides of the zipcode. The spacing between the RNA sequences is consistent with the structure of IMP1 KH34, the human ortholog of ZBP1, that we solved by X-ray crystallography. The tandem KH domains are arranged in an intramolecular anti-parallel pseudodimer conformation with the canonical RNA-binding surfaces at opposite ends of the molecule. This orientation of the KH domains requires that the RNA backbone must undergo an~180°change in direction in order for both KH domains to contact the RNA simultaneously. The RNA looping induced by ZBP1 binding provides a mechanism for specific recognition and may facilitate the assembly of posttranscriptional regulatory complexes by remodeling the bound transcript.[Keywords: ZBP1; RNA-binding protein; KH domain; RNA localization] Supplemental material is available at http://www.genesdev.org.

A major driver of the pathophysiology of sickle cell disease (SCD) is polymerization of deoxygenated haemoglobin S (HbS), which leads to sickling and destruction of red blood cells (RBCs) and end-organ damage. Pharmacologically increasing the proportion of oxygenated HbS in RBCs may inhibit polymerization, prevent sickling and provide long term disease modification. We report that GBT440, a small molecule which binds to the N-terminal a chain of Hb, increases HbS affinity for oxygen, delays in vitro HbS polymerization and prevents sickling of RBCs. Moreover, in a murine model of SCD, GBT440 extends the half-life of RBCs, reduces reticulocyte counts and prevents ex vivo RBC sickling. Importantly, oral dosing of GBT440 in animals demonstrates suitability for once daily dosing in humans and a highly selective partitioning into RBCs, which is a key therapeutic safety attribute. Thus, GBT440 has the potential for clinical use as a disease-modifying agent in sickle cell patients.

The cocrystal structure of the PP7 bacteriophage coat protein in complex with its translational operator identifies a distinct mode of sequence-specific RNA recognition when compared to the well-characterized MS2 coat protein-RNA complex. The structure reveals the molecular basis of the PP7 coat protein's ability to selectively bind its cognate RNA, and it demonstrates that the conserved β-sheet surface is a flexible architecture that can evolve to recognize diverse RNA hairpins.The inherent sequence diversity generated by RNA replication, which allows RNA viruses to evolve, must be tempered by the need for conservation of functional elements within the viral genome. The coat proteins of single-stranded (ss) RNA bacteriophages have both structural and regulatory roles in the viral life cycle, as they assemble into the mature capsid and regulate translation of the phage replicase through binding and sequestration of an RNA hairpin that contains the initiation site 1 . The coat protein and translational operator form a pair that must coevolve, so that mutation in one must be accompanied by compensatory mutations in the other. The Pseudomonas aeruginosa bacteriophage PP7 is a model system for exploring coevolution because of the extensive sequence divergence of both the PP7 coat protein and translational operator from other ssRNA bacteriophages 2 . The PP7 and MS2 coat proteins share only 15% sequence identity, and their cognate RNA hairpins differ in the position of the bulged adenosine as well as in the size and nucleotide composition of the loop (Fig. 1a). Biochemical experiments have shown that both coat proteins bind their own RNA hairpins with high affinity (K d ~ 1 nM) and are able to discriminately bind in favor of their own RNA by ~1,000-fold 3,4 . In contrast to the PP7 coat protein, the Qβ coat protein also shares low sequence identity (21%) with the MS2 coat protein, yet it uses an RNAbinding mode similar to that of the MS2 coat protein [5][6][7] Fig. 1 and Supplementary Methods online). PP7ΔFG was generated by removing residues 67-75 and, notably, this truncation binds its translational operator with affinity similar to that of the fulllength protein ( Supplementary Fig. 2 online). The PP7ΔFG monomer adopts the topology characteristic of ssRNA bacteriophage coat proteins, with an N-terminal β hairpin, a fivestranded antiparallel β sheet and two C-terminal α helices ( Supplementary Fig. 3a online) [8][9][10][11] . Antiparallel association of protomers in the dimer positions the last β strands adjacent to one another, resulting in a ten-stranded β sheet that comprises the RNA-binding surface. The interwoven packing of the α helices between each other and the N-terminal β hairpins further stabilizes the dimer, combining with the β sheet interface to bury ~3,000 Å 2 of solvent-accessible surface area. The PP7ΔFG dimer has almost identical free and bound structures (r.m.s. deviation < 0.9 Å for all Cα atoms) and is similar to that of the MS2 coat protein (r.m.s. deviation of < 2.3 Å for all Cα atoms...

We report the discovery of a new potent allosteric effector of sickle cell hemoglobin, GBT440 (), that increases the affinity of hemoglobin for oxygen and consequently inhibits its polymerization when subjected to hypoxic conditions. Unlike earlier allosteric activators that bind covalently to hemoglobin in a 2:1 stoichiometry, binds with a 1:1 stoichiometry. Compound is orally bioavailable and partitions highly and favorably into the red blood cell with a RBC/plasma ratio of ∼150. This partitioning onto the target protein is anticipated to allow therapeutic concentrations to be achieved in the red blood cell at low plasma concentrations. GBT440 () is in Phase 3 clinical trials for the treatment of sickle cell disease (NCT03036813).

The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptional regulation, regulation of major signaling pathways, neural development, and type 1 diabetes. In conjunction with the contributions of other international structural genomics consortia, these efforts promise to provide an unprecedented database and materials repository for structureguided experimental and computational discovery of inhibitors for all classes of protein phosphatases.