Summary Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop-1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the Trigger Loop (TL), allowing visualization of its open state. Overall, our observations suggest that “open/closed” conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation.

Na ؉ -H ؉ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodiumphosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser 290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1␣ (PP1␣), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser 290 . Mutating 257 VPF 259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1␣-mediated Ser 290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that ␤-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally . 4 The abbreviations used are: PTH, parathyroid hormone; PTHR, PTH receptor; NHERF1, Na ϩ /H ϩ -exchanger regulatory factor 1; GnTI Ϫ , N-acetylglucosaminyltransferase-deficient HEK-293S cells; TAP-NHERF1, tandem affinity purification-tagged NHERF1; FLIM, fluorescence lifetime imaging; HDX, hydrogen/deuterium exchange mass spectrometry; pSer 290 , phosphorylated Ser 290 ; NP40, Nonidet P-40; TAMRA, carboxytetramethylrhodamine; SILAC, stable isotope labeling of amino acids in cell culture; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; ND, nondeuterated; FD, fully deuterated; EBD, ezrin-binding domain; PDB, Protein Data Bank; 2Me-4OMe-TM, 7-hydroxy-5,5-dimethyl-10-(4-methoxy-2-methylphenyl)-dibenzo-[b,e]-silin-3(5H)-one; ANOVA, analysis of variance; ID, intrinsically disordered; TTN, tautomycetin; RPTEC, renal proximal tubule epithelial cell; pen/strep, penicillin and streptomycin; CFP, cyan fluorescent protein; FERM, Ezrin-Radixin-Moesin; MD, molecular dynamics.

Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) is a scaffolding protein containing 2 PDZ domains that coordinates the assembly and trafficking of transmembrane receptors and ion channels. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to the either PDZ domain, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A), uniquely bind only one PDZ domain. We sought to define the structural determinants responsible for the specificity of interaction between NHERF1 PDZ domains and NPT2A. By performing all-atom/explicit-solvent molecular dynamics (MD) simulations in combination with biological mutagenesis, fluorescent polarization (FP) binding assays, and isothermal titration calorimetry (ITC), we found that in addition to canonical interactions of residues at 0 and -2 positions, Arg at the -1 position of NPT2A plays a critical role in association with Glu43 and His27 of PDZ1 that are absent in PDZ2. Experimentally introduced mutation in PDZ1 (Glu43Asp and His27Asn) decreased binding to NPT2A. Conversely, introduction of Asp183Glu and Asn167His mutations in PDZ2 promoted the formation of favorable interactions yielding micromolar K Ds. The results describe novel determinants within both the PDZ domain and outside the canonical PDZ-recognition motif that are responsible for discrimination of NPT2A between two PDZ domains. The results challenge general paradigms for PDZ recognition and suggest new targets for drug development.

BackgroundThe scavenging ability of sufficient divalent metal ions is pivotal for pathogenic bacteria to survive in the host. ATP-binding cassette (ABC)-type metal transporters provide a considerable amount of different transition metals for bacterial growth. TroA is a substrate binding protein for uptake of multiple metal ions. However, the function and structure of the TroA homologue from the epidemic Streptococcus suis isolates (SsTroA) have not been characterized.Methodology/Principal FindingsHere we determined the crystal structure of SsTroA from a highly pathogenic streptococcal toxic shock syndrome (STSS)-causing Streptococcus suis in complex with zinc. Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed that apo-SsTroA binds Zn2+ and Mn2+. Both metals bind to SsTroA with nanomolar affinity and stabilize the protein against thermal unfolding. Zn2+ and Mn2+ induce distinct conformational changes in SsTroA compared with the apo form as confirmed by both circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra. NMR data also revealed that Zn2+/Mn2+ bind to SsTroA in either the same site or an adjacent region. Finally, we found that the folding of the metal-bound protein is more compact than the corresponding apoprotein.Conclusions/SignificanceOur findings reveal a mechanism for uptake of metal ions in S. suis and this mechanism provides a reasonable explanation as to how SsTroA operates in metal transport.