Mechanistic understanding of pre-mRNA splicing requires detailed structural information on various states of the spliceosome. Here we report the cryo electron microscopy (cryo-EM) structure of the human spliceosome just before exon ligation (the C complex) at an average resolution of 3.76 Å. The splicing factor Prp17 stabilizes the active site conformation. The step II factor Slu7 adopts an extended conformation, binds Prp8 and Cwc22, and is poised for selection of the 3'-splice site. Remarkably, the intron lariat traverses through a positively charged central channel of RBM22; this unusual organization suggests mechanisms of intron recruitment, confinement, and release. The protein PRKRIP1 forms a 100-Å α helix linking the distant U2 snRNP to the catalytic center. A 35-residue fragment of the ATPase/helicase Prp22 latches onto Prp8, and the quaternary exon junction complex (EJC) recognizes upstream 5'-exon sequences and associates with Cwc22 and the GTPase Snu114. These structural features reveal important mechanistic insights into exon ligation.

Netrin-1 is a guidance cue that can trigger either attraction or repulsion effects on migrating neurons, depending on the repertoire of receptors available on the growth cone. How a single chemotropic molecule can act in such contradictory ways has long been a puzzle at the molecular level. Here we present the crystal structure of netrin-1 in complex with the Deleted in Colorectal Cancer (DCC) receptor. We show that one netrin-1 molecule can simultaneously bind to two DCC molecules through a DCC-specific site and through a unique generic receptor binding site, where sulfate ions staple together positively charged patches on both DCC and netrin-1. Furthermore, we demonstrate that UNC5A can replace DCC on the generic receptor binding site to switch the response from attraction to repulsion. We propose that the modularity of binding allows for the association of other netrin receptors at the generic binding site, eliciting alternative turning responses.

Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted therapeutics. SF3B1, part of the U2 small nuclear RNP (snRNP), is targeted by splicing modulators, including E7107, the first to enter clinical trials, and, more recently, H3B-8800. Modulating splicing represents a first-in-class opportunity in drug discovery, and elucidating the structural basis for the mode of action opens up new possibilities for structure-based drug design. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the SF3b subcomplex (SF3B1, SF3B3, PHF5A, and SF3B5) bound to E7107 at 3.95 Å. This structure shows that E7107 binds in the branch point adenosine-binding pocket, forming close contacts with key residues that confer resistance upon mutation: SF3B1 R1074H and PHF5A Y36C . The structure suggests a model in which splicing modulators interfere with branch point adenosine recognition and supports a substrate competitive mechanism of action (MOA). Using several related chemical probes, we validate the pose of the compound and support their substrate competitive MOA by comparing their activity against both strong and weak pre-mRNA substrates. Finally, we present functional data and structure-activity relationship (SAR) on the PHF5A R38C mutation that sensitizes cells to some chemical probes but not others. Developing small molecule splicing modulators represents a promising therapeutic approach for a variety of diseases, and this work provides a significant step in enabling structure-based drug design for these elaborate natural products. Importantly, this work also demonstrates that the utilization of cryo-EM in drug discovery is coming of age.

Drugs that can protect against organ damage are urgently needed, especially for diseases such as sepsis and brain stroke. We have discovered that terazosin (TZ), a widely marketed alpha1-adrenergic receptor agonist, alleviated organ damage and improved survival in rodent models of stroke and sepsis. Through combined studies of enzymology and X-ray crystallography, we have discovered that TZ binds to a novel target, phosphoglycerate kinase 1 (Pgk1) and activates its enzymatic activity, probably through 1,3-diamino-6,7-dimethoxyisoquinoline's ability to promote ATP release from Pgk1. Mechanistically, the ATP generated from Pgk1 may enhance the chaperone activity of Hsp90, an ATPase known to associate with Pgk1. Upon activation, Hsp90 promotes multi-stress resistance. Our studies have demonstrated that TZ has a novel protein target, Pgk1, and has revealed its corresponding biological effect. As a clinical drug, TZ may be quickly translated into treatment of devastating diseases including stroke and sepsis.

The structure and biochemical function of the hot dog-fold thioesterase PaaI operative in the aerobic phenylacetate degradation pathway are examined. PaaI showed modest activity with phenylacetyl-coenzyme A, suggestive of a role in coenzyme A release from this pathway intermediate in the event of limiting downstream pathway enzymes. Minimal activity was observed with aliphatic acyl-coenzyme A thioesters, which ruled out PaaI function in the lower phenylacetate pathway. PaaI was most active with ring-hydroxylated phenylacetyl-coenzyme A thioesters. The x-ray crystal structure of the Escherichia coli thioesterase is reported and analyzed to define the structural basis of substrate recognition and catalysis. The contributions of catalytic and substrate binding residues, thus, identified were examined through steady-state kinetic analysis of site-directed mutant proteins.

The β-phosphoglucomutase (β-PGM) of the haloacid dehalogenase enzyme superfamily (HADSF) catalyzes the conversion of β-glucose 1-phosphate (βG1P) to glucose 6-phosphate (G6P) using Asp8 of the core domain active-site to mediate phosphoryl transfer from β-glucose 1,6-(bis)phosphate (βG1,6bisP) to βG1P. Herein we explore the mechanism by which hydrolysis of the β-PGM phosphoAsp8 is avoided during the time that the active site must remain open to solvent in order to allow the exchange of the bound product G6P with the substrate βG1P. Based on structural information, a model of catalysis is proposed in which the general acid/base (Asp10) side chain moves from a position where it forms a hydrogen bond to the Thr16-Ala17 of the domain-domain linker, to a functional position where it forms a hydrogen bond to the substrate leaving-group O and a His20-Lys76 pair of the cap domain. This repositioning of the general acid/base within the core domain active site is coordinated with substrate-induced closure of the cap domain over the core domain. The model predicts that Asp10 is required for general acid/base catalysis and for stabilization of the enzyme in the cap-closed conformation. It also predicts that hinge residue Thr16 plays a key role in productive domain-domain association, that hydrogen bond interaction with the Thr16 backbone amide NH is required to prevent phospho-Asp8 hydrolysis in the cap-open conformation, and that the His20-Lys76 pair plays an important role in substrate-induced cap closure. The model is examined via kinetic analyses of Asp10, Thr16, His20, and Lys76 site-directed mutants. Replacement of the Asp10 by Ala, Ser, Cys, Asn, or Glu resulted in no observable activity. The kinetic consequences of the replacement of linker residue Thr16 with Pro include a reduced rate of Asp8 phosphorylation by βG1,6bisP, a reduced rate of cycling of the phosphorylated enzyme to convert βG1P to G6P, and an enhanced rate of phosphoryl transfer from phospho-Asp8 to water. The X-ray structure of the T16P mutant at 2.7 Å resolution provides a snapshot of the enzyme in an unnatural cap-open conformation where the Asp10 side chain is located in the core-domain active site. The His20 and Lys76 site- * Address correspondence to Debra Dunaway-Mariano email: dd39@unm.edu, phone: 505-277-3383, fax: 505-277-2609 and Karen N. Allen, phone: 617-358-5544, fax: 617-358-5554, email: drkallen@bu.edu. c present address: Department of Chemistry, Boston University, Boston, MA 02215, USA 1 Abbreviations used are: α-PGM, α-phosphoglucomutase; α-PGM/PMM, dual specificity α-phosphoglucomutase/α-phosphomannomutase; β-PGM, β-phosphoglucomutase; E, β-PGM -Mg 2+ ; E-P, phospho-β-PGM-Mg 2+ ; βG1P, β-D-glucose 1-phosphate; βG1,6bisP, β-D-glucose 1,6-(bis)phosphate; αG1P, α-D-glucose 1-phosphate; αG1,6bisP, α-D-glucose 1,6-(bis)phosphate; PEP, phosphoenol pyruvate; SA, specific activity. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 August 6. Published in final edited form as:Biochemistry. Phosp...

DCC (Deleted in Colorectal Cancer) is a single-pass transmembrane protein that belongs to the immunoglobulin superfamily. It was originally identified as a prognostic tumor marker and then subsequently found to be a receptor for netrin-1. DCC plays a key role in axon guidance and also in a number of other important cellular processes. This review describes the current progress of the structural biology of DCC with an emphasis on how DCC is involved in the dual functionality of netrin-1 as a chemo-attractant as well as a repellent in axon guidance, referred to as bi-functionality. A perspective about other DCC ligands and the signaling mechanism of the cytoplasmic tail of DCC is also recapitulated.

SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in multiple KRAS cancer cell lines. SHOC2 forms a heterotrimeric complex with MRAS and PP1C that is essential for regulating RAF and MAPK-pathway activation by dephosphorylating a specific phosphoserine on RAF kinases. Here we present the high-resolution crystal structure of SHOC2-MRAS-PP1C (SMP) complex and apo-SHOC2. Our structures reveal that SHOC2, MRAS and PP1C form a stable ternary complex where all three proteins synergistically interact with each other. Our results show that dephosphorylation of RAF substrates by PP1C is enhanced upon interacting with SHOC2 and MRAS. The SMP complex only forms when MRAS is in an active state and is dependent on SHOC2 functioning as a scaffolding protein in the complex by bringing PP1C and MRAS together. Our results provide structural insights into the role of the SMP complex in RAF activation, how mutations found in Noonan syndrome enhance the complex formation and reveal new avenues for therapeutic interventions.C. elegans as a positive modulator of the MAPK pathway 12,13 . It is a ubiquitously expressed protein composed primarily of predicted leucine-rich repeats (LRRs). N-terminal to the LRR domains, SHOC2 contains a ~90-residue long sequence that is predicted to be intrinsically disordered and has been suggested to be necessary for complex formation with MRAS and PP1C 11,14 . Germline mutations in SHOC2 (S2G, M173I, and Q269H/H270Y) have been detected in NS 11,[15][16][17] . SHOC2 plays a vital role in transformation, metastasis, epithelial-tomesenchymal transition, and MAPK pathway inhibitor resistance [18][19][20][21] . Multiple genome-scale, single-gene CRISPR/Cas9 fitness screens in human cancer cells have suggested selective dependency of RAS mutant cells on SHOC2 20,[22][23][24] . SHOC2 was also identified as the strongest synthetic lethal target in the presence of MEK inhibitors in KRAS mutant lung and pancreatic cancer cell lines 19 . Thus, SHOC2 may provide a unique therapeutic opportunity within the RTK-RAS-MAPK pathway in oncogenic RAS cells.The SMP complex formation is initiated following MRAS activation as SHOC2 and PP1C bind only to MRAS-GTP 25 . The canonical RAS family members HRAS, KRAS, and NRAS, also bind SHOC2, although with considerably lower affinity than MRAS 26 . The nature of the selectivity for MRAS is not known. MRAS shares ~50% sequence identity with the canonical RAS proteins and contains an extra ten amino acids at the N-terminus. Activating mutations in MRAS are very rare in cancer; however, gain-of-function mutations (G23V, T68I, Q71R) in MRAS have been identified in NS patients 27,28 . In the SMP complex, PP1C provides the enzymatic activity for dephosphorylation. PP1C is a class of serine/threonine phosphatases with three highly conserved isoforms (PP1CA, PP1CB, and PP1CC with >90% sequence identity) that are ubiquitously expressed and catalyze the dephosphorylation of a substantial fraction of phosphoserine/threonine in all eukaryotic cells [29][30][31] . Mutation...