Materials and Methods Figs. S1 to S3 Table S1 References S1 SUPPORTING MATERIAL Materials and Methods Preparation of Re I (CO) 3 (dmp)(H 124)|(W 122)|AzCu I Mutant azurins were expressed and Re I (CO) 3 (dmp)(H 124)|(W 122)|AzCu I was prepared using previously published protocols (S1,S2). Crystal Structure of Re I (CO) 3 (dmp)(H 124)|(W 122)|AzCu II Crystals of Re(4,7-dimethyl-1,10-phenanthroline)(CO) 3 (H 124){T 124 H|K 122 W|H 83 Q}(Cu II)azurin (Re I (CO) 3 (dmp)(H 124)|(W 122)|AzCu II ; space group I222, cell dimensions 63.22 × 69.08 × 68.94 Å 3 ; α = β = γ = 90.00°, one molecule per asymmetric unit) grew from 4 μL drops made from equal volumes of 30 mg/mL Re I (CO) 3 (dmp)(H 124)|(W 122)|AzCu II in 25 mM HEPES pH 7.5 and reservoir by vapor diffusion. The drops were equilibrated against 500 μL of reservoir

GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.

Numerous oncogenic mutations occur within the BRAF kinase domain (BRAF(KD)). Here we show that stable BRAF-MEK1 complexes are enriched in BRAF(WT) and KRAS mutant (MT) cells but not in BRAF(MT) cells. The crystal structure of the BRAF(KD) in a complex with MEK1 reveals a face-to-face dimer sensitive to MEK1 phosphorylation but insensitive to BRAF dimerization. Structure-guided studies reveal that oncogenic BRAF mutations function by bypassing the requirement for BRAF dimerization for activity or weakening the interaction with MEK1. Finally, we show that conformation-specific BRAF inhibitors can sequester a dormant BRAF-MEK1 complex resulting in pathway inhibition. Taken together, these findings reveal a regulatory role for BRAF in the MAPK pathway independent of its kinase activity but dependent on interaction with MEK.

Nitric oxide synthases (NOSs) are multidomain metalloproteins first identified in mammals as being responsible for the synthesis of the wide-spread signaling and protective agent nitric oxide (NO). Over the past 10 years, prokaryotic proteins that are homologous to animal NOSs have been identified and characterized, both in terms of enzymology and biological function. Despite some interesting differences in cofactor utilization and redox partners, the bacterial enzymes are in many ways similar to their mammalian NOS (mNOS) counterparts and, as such, have provided insight into the structural and catalytic properties of the NOS family. In particular, spectroscopic studies of thermostable bacterial NOSs have revealed key oxyheme intermediates involved in the oxidation of substrate L-arginine (Arg) to product NO. The biological functions of some bacterial NOSs have only more recently come to light. These studies disclose new roles for NO in biology, such as taking part in toxin biosynthesis, protection against oxidative stress, and regulation of recovery from radiation damage.

Cryoreduction EPR/ENDOR/step-annealing measurements with substrate complexes of oxy-gsNOS (3; gsNOS is nitric oxide synthase from Geobacillus stearothermophilus) confirm that Compound I (6) is the reactive heme species that carries out the gsNOS-catalyzed (Stage I) oxidation of L-arginine to N-hydroxy-L-arginine (NOHA), whereas the active species in the (Stage II) oxidation of NOHA to citrulline and HNO/NO(-) is the hydroperoxy-ferric form (5). When 3 is reduced by tetrahydrobiopterin (BH4), instead of an externally supplied electron, the resulting BH4(+) radical oxidizes HNO/NO(-) to NO. In this report, radiolytic one-electron reduction of 3 and its complexes with Arg, Me-Arg, and NO(2)Arg was shown by EPR and (1)H and (14,15)N ENDOR spectroscopies to generate 5; in contrast, during cryoreduction of 3/NOHA, the peroxo-ferric-gsNOS intermediate (4/NOHA) was trapped. During annealing at 145 K, ENDOR shows that 5/Arg and 5/Me-Arg (but not 5/NO(2)Arg) generate a Stage I primary product species in which the OH group of the hydroxylated substrate is coordinated to Fe(III), characteristic of 6 as the active heme center. Analysis shows that hydroxylation of Arg and Me-Arg is quantitative. Annealing of 4/NOHA at 160 K converts it first to 5/NOHA and then to the Stage II primary enzymatic product. The latter contains Fe(III) coordinated by water, characteristic of 5 as the active heme center. It further contains quantitative amounts of citrulline and HNO/NO(-); the latter reacts with the ferriheme to form the NO-ferroheme upon further annealing. Stage I delivery of the first proton of catalysis to the (unobserved) 4 formed by cryoreduction of 3 involves a bound water that may convey a proton from L-Arg, while the second proton likely derives from the carboxyl side chain of Glu 248 or the heme carboxylates; the process also involves proton delivery by water(s). In the Stage II oxidation of NOHA, the proton that converts 4/NOHA to 5/NOHA likely is derived from NOHA itself, a conclusion supported by the pH invariance of the process. The present results illustrate how the substrate itself modulates the nature and reactivity of intermediates along the monooxygenase reaction pathway.

The ReI(CO)3(4,7-dimethyl-1,10-phenanthroline)(histidine-124)(tryptophan-122) complex, denoted ReI(dmp)(W122), of Pseudomonas aeruginosa azurin behaves as a single photoactive unit that triggers very fast electron transfer (ET) from a distant (2 nm) CuI center in the protein. Analysis of time-resolved (ps-μs) IR spectroscopic and kinetics data collected on ReI(dmp)(W122)AzM (M = ZnII CuII, CuI; Az = azurin) and position-122 tyrosine (Y), phenylalanine (F), and lysine (K) mutants together with excited-state DFT/TDDFT calculations and X-ray structural characterization reveal the character, energetics, and dynamics of the relevant electronic states of the ReI(dmp)(W122) unit and a cascade of photoinduced ET and relaxation steps in the corresponding Re-azurins. Optical population of ReI(imidazole-H124)(CO)3→dmp 1CT states is followed by ~110 fs intersystem crossing and ~600 ps structural relaxation to a 3CT state whose IR spectrum indicates a mixed ReI(CO)3,A→dmp/π→π*(dmp) character for aromatic amino acids A122 (A = W, Y, F) and ReI(CO)3→dmp MLCT for ReI(dmp)(K122)AzCuII. In a few ns, the 3CT state of ReI(dmp)(W122)AzM establishes an equilibrium with the ReI(dmp•−)(W122•+)AzM charge-separated state, 3CS, whereas the 3CT state of the other Y, F, and K122 proteins decays to the ground state. In addition to this main pathway, 3CS is populated by fs and ps W(indole)→ReII ET from 1CT and the initially “hot” 3CT states, respectively. The 3CS state undergoes a tens-of-ns dmp•−→W122•+ ET recombination leading to the ground state or, in the case of the CuI azurin, competitively fast (~30 ns over 1.12 nm) CuI→W•+ ET producing ReI(dmp•−)(W122)AzCuII. The overall photoinduced CuI→Re(dmp) ET through ReI(dmp)(W122)AzCuI occurs over a 2 nm distance in <50 ns after excitation, the intervening fast 3CT-3CS equilibrium being the principal accelerating factor. No reaction was observed for the three Y, F, and K122 analogues. Although the presence of Re(dmp)(W122)AzCuII oligomers in solution was documented by mass spectrometry and phosphorescence anisotropy, kinetics data do not indicate any significant interference from intermolecular ET steps. The ground-state dmp-indole ππ interaction together with well-matched W/W•+ and excited-state ReII(CO)3(dmp•−)/ReI(CO)3(dmp•−) potentials, that result in very rapid electron interchange and 3CT - 3CS energetic proximity, are the main factors responsible for the unique ET behavior of ReI(dmp)(W122)-containing azurins.

No abstract

No abstract