Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca 2؉ /calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electronsupplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg 1400 , and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.

Flap EndoNuclease-1 (FEN-1) and the processivity factor proliferating cell nuclear antigen (PCNA) are central to DNA replication and repair. To clarify the molecular basis of FEN-1 specificity and PCNA activation, we report here structures of FEN-1:DNA and PCNA:FEN-1-peptide complexes, along with fluorescence resonance energy transfer (FRET) and mutational results. FEN-1 binds the unpaired 3' DNA end (3' flap), opens and kinks the DNA, and promotes conformational closing of a flexible helical clamp to facilitate 5' cleavage specificity. Ordering of unstructured C-terminal regions in FEN-1 and PCNA creates an intermolecular beta sheet interface that directly links adjacent PCNA and DNA binding regions of FEN-1 and suggests how PCNA stimulates FEN-1 activity. The DNA and protein conformational changes, composite complex structures, FRET, and mutational results support enzyme-PCNA alignments and a kinked DNA pivot point that appear suitable to coordinate rotary handoffs of kinked DNA intermediates among enzymes localized by the three PCNA binding sites.

Farnesyl pyrophosphate synthetase (FPPS) synthesizes farnesyl pyrophosphate through successive condensations of isopentyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. Nitrogen-containing bisphosphonate drugs used to treat osteoclast-mediated bone resorption and tumor-induced hypercalcemia are potent inhibitors of the enzyme. Here we present crystal structures of substrate and bisphosphonate complexes of FPPS. The structures reveal how enzyme conformational changes organize conserved active site residues to exploit metal-induced ionization and substrate positioning for catalysis. The structures further demonstrate how nitrogen-containing bisphosphonates mimic a carbocation intermediate to inhibit the enzyme. Together, these FPPS complexes provide a structural template for the design of novel inhibitors that may prove useful for the treatment of osteoporosis and other clinical indications including cancer.Post-translational modification of C-terminal CAAX sequences by covalent attachment of isoprenyl chains is crucial for intracellular localization and proper function of small GTPases such as Ras, Rac, Rho, and CDC42 (1, 2). The substrates for these modifications are the 15-carbon isoprenoid farnesyl pyrophosphate (FPP) 1 or the 20-carbon isoprenoid geranyl-geranyl pyrophosphate synthesized by enzymes of the mevalonate pathway (3) (Fig. 1A). A key branch point enzyme of the mevalonate pathway is farnesyl pyrophosphate synthetase (FPPS), a ϳ30-kDa Mg 2ϩ -dependent homodimeric enzyme that synthesizes (E,E)-FPP from isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) (4, 5) (Fig. 1B). Interest in understanding FPPS activity stems from the recent discovery that FPPS is the molecular target of nitrogencontaining bisphosphonates (6,7,31,32). Bisphophonates are non-cleavable pyrophosphate (P-O-P) analogues in which the central oxygen is replaced by a carbon (P-C-P) with various side chains (Fig. 1C). Against parasitic organisms (8, 9) these agents have been shown in vitro to disrupt cell growth through FPPS inhibition. In people, bisphosphonates are targeted to bone tissue (10) where FPPS inhibition in bone-resorbing osteoclasts is a current therapeutic approach for treating postmenopausal osteoporosis (11,12). Because of their bone-targeting properties, bisphosphonates have also found use as agents to treat tumor-induced hypercalcemia (13), Paget's disease (14), and osteolytic metastases (15).Although structures of apo-and ligand-bound avian FPPS have been solved (16,17), the active sites are unassembled and do not provide substantial information concerning catalysis. Thus, to resolve the molecular basis of catalysis, and also to understand the structural features governing bisphosphonate recognition, we determined the structures of unliganded Staphylococcus aureus FPPS (FPPS-Sa), as well as two Escherichia coli FPPS (FPPS-Ec) ternary complexes. These ternary complexes include a 2.4-Å "substrate-bound" structure containing IPP and the noncleavable DMAPP analogue dimethyla...

The second-generation antiandrogen enzalutamide was recently approved for patients with castration-resistant prostate cancer. Despite its success, the duration of response is often limited. For previous antiandrogens, one mechanism of resistance is mutation of the androgen receptor (AR). To prospectively identify AR mutations that might confer resistance to enzalutamide, we performed a reporter-based mutagenesis screen and identified a novel mutation, F876L, which converted enzalutamide into an AR agonist. Ectopic expression of AR F876L rescued the growth inhibition of enzalutamide treatment. Molecular dynamics simulations performed on antiandrogen–AR complexes suggested a mechanism by which the F876L substitution alleviates antagonism through repositioning of the coactivator recruiting helix 12. This model then provided the rationale for a focused chemical screen which, based on existing antiandrogen scaffolds, identified three novel compounds that effectively antagonized AR F876L (and AR WT) to suppress the growth of prostate cancer cells resistant to enzalutamide.DOI: http://dx.doi.org/10.7554/eLife.00499.001

Flap endonuclease (FEN-1) removes 5' overhanging flaps in DNA repair and processes the 5' ends of Okazaki fragments in lagging strand DNA synthesis. The crystal structure of Pyrococcus furiosus FEN-1, active-site metal ions, and mutational information indicate interactions for the single- and double-stranded portions of the flap DNA substrate and identify an unusual DNA-binding motif. The enzyme's active-site structure suggests that DNA binding induces FEN-1 to clamp onto the cleavage junction to form the productive complex. The conserved FEN-1 C terminus binds proliferating cell nuclear antigen (PCNA) and positions FEN-1 to act primarily as an exonuclease in DNA replication, in contrast to its endonuclease activity in DNA repair. FEN-1 mutations altering PCNA binding should reduce activity during replication, likely causing DNA repeat expansions as seen in some cancers and genetic diseases.

Background: DNA apurinic/apyrimidinic (AP) sites are toxic and mutagenic if unrepaired by AP endonucleases. Results: Structural, mutational, and computational analyses of prototypic AP endonucleases APE1 and Nfo identify surprising similarities. Conclusion: APE1 and Nfo reveal functional equivalences illuminating their catalytic reaction. Significance: A conserved catalytic geometry is specific to AP site removal despite different enzyme structures and metal ions.

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Endonuclease IV is the archetype for a conserved apurinic/apyrimidinic (AP) endonuclease family that primes DNA repair synthesis by cleaving the DNA backbone 5' of AP sites. The crystal structures of Endonuclease IV and its AP-DNA complex at 1.02 and 1.55 A resolution reveal how an alpha8beta8 TIM barrel fold can bind dsDNA. Enzyme loops intercalate side chains at the abasic site, compress the DNA backbone, bend the DNA approximately 90 degrees, and promote double-nucleotide flipping to sequester the extrahelical AP site in an enzyme pocket that excludes undamaged nucleotides. These structures suggest three Zn2+ ions directly participate in phosphodiester bond cleavage and prompt hypotheses that double-nucleotide flipping and sharp bending by AP endonucleases provide exquisite damage specificity while aiding subsequent base excision repair pathway progression.