Structural basis for mobility in the 1.1 A crystal structure of the NG domain of Thermus aquaticus Ffh

The NG domain of the prokaryotic signal recognition protein Ffh is a two-domain GTPase that comprises part of the prokaryotic signal recognition particle (SRP) that functions in co-translational targeting of proteins to the membrane. The interface between the N and G domains includes two highly conserved sequence motifs and is adjacent in sequence and structure to one of the conserved GTPase signature motifs. Previous structural studies have shown that the relative orientation of the two domains is dynamic. The N domain of Ffh has been proposed to function in regulating the nucleotide-binding interactions of the G domain. However, biochemical studies suggest a more complex role for the domain in integrating communication between signal sequence recognition and interaction with receptor. Here, we report the structure of the apo NG GTPase of Ffh from Thermus aquaticus refined at 1.10 A resolution. Although the G domain is very well ordered in this structure, the N domain is less well ordered, reflecting the dynamic relationship between the two domains previously inferred. We demonstrate that the anisotropic displacement parameters directly visualize the underlying mobility between the two domains, and present a detailed structural analysis of the packing of the residues, including the critical alpha4 helix, that comprise the interface. Our data allows us to propose a structural explanation for the functional significance of sequence elements conserved at the N/G interface.     

Purification of RanGDP, RanGTP, and RanGMPPNP by ion exchange chromatography

Ran is a small GTPase that cycles between a guanosine diphosphate (GDP)-bound form (RanGDP) and a guanosine triphosphate (GTP)-bound form (RanGTP) and plays important roles in nuclear transport and mitosis. For studies of Ran function and its interactions with partner proteins, pure RanGDP and RanGTP complexes are critical. Ran complexed with the nonhydrolyzable GTP analog, GMPPNP (RanGMPPNP), is used instead of RanGTP when inhibition of hydrolysis is required. In this study, we demonstrate that the binding of Ran to a UNO Q ion exchange column is remarkably sensitive to small shifts in MgCl(2) concentration, and we use this property to purify recombinant RanGTP, RanGMPPNP, and RanGDP complexes. At 10 mM MgCl(2), Ran was found predominantly in the flow-through and, thus, was separated from the vast majority of bacterial proteins. After reducing the concentration of MgCl(2) to 5 mM, further purification of RanGTP, RanGMPPNP, and RanGDP was achieved by loading onto ion exchange columns and elution with an NaCl gradient. Purity of the resulting preparations was confirmed by releasing the bound nucleotide and checking it against a known nucleotide by high-performance liquid chromatography (HPLC). To further confirm the purity and function of the Ran preparations, appropriate protein-binding, enzymatic, and nuclear import assays were carried out. These methods should facilitate studies of cellular processes involving Ran by providing pure functional Ran-nucleotide complexes.     

Treatment of Na?ve Patients with Chronic Hepatitis C Genotypes 2 and 3 with Pegylated Interferon Alpha and Ribavirin in a Real World Setting: Relevance for the New Era of DAA

Evidence based clinical guidelines are implemented to treat patients efficiently that include efficacy, tolerability but also health economic considerations. This is of particular relevance to the new direct acting antiviral agents that have revolutionized treatment of chronic hepatitis C. For hepatitis C genotypes 2/3 interferon free treatment is already available with sofosbuvir plus ribavirin. However, treatment with sofosbuvir-based regimens is 10–20 times more expensive compared to pegylated interferon alfa and ribavirin (PegIFN/RBV). It has to be discussed if PegIFN/RBV is still an option for easy to treat patients. We assessed the treatment of patients with chronic hepatitis C genotypes 2/3 with PegIFN/RBV in a real world setting according to the latest German guidelines. Overall, 1006 patients were recruited into a prospective patient registry with 959 having started treatment. The intention-to-treat analysis showed poor SVR (GT2 61%, GT3 47%) while patients with adherence had excellent SVR in the per protocol analysis (GT2 96%, GT3 90%). According to guidelines, 283 patients were candidates for shorter treatment duration, namely a treatment of 16 weeks (baseline HCV-RNA <800.000 IU/mL, no cirrhosis and RVR). However, 65% of these easy to treat patients have been treated longer than recommended that resulted in higher costs but not higher SVR rates. In conclusion, treatment with PegIFN/RBV in a real world setting can be highly effective yet similar effective than PegIFN± sofosbuvir/RBV in well-selected naïve G2/3 patients. Full adherence to guidelines could be further improved, because it would be important in the new era with DAA, especially to safe resources.     

Degradation of nanoRNA is performed by multiple redundant RNases in Bacillus subtilis

Escherichia coli possesses only one essential oligoribonuclease (Orn), an enzyme that can degrade oligoribonucleotides of five residues and shorter in length (nanoRNA). Firmicutes including Bacillus subtilis do not have an Orn homolog. We had previously identified YtqI (NrnA) as functional analog of Orn in B. subtilis. Screening a genomic library from B. subtilis for genes that can complement a conditional orn mutant, we identify here YngD (NrnB) as a second nanoRNase in B. subtilis. Like NrnA, NrnB is a member of the DHH/DHHA1 protein family of phosphoesterases. NrnB degrades nanoRNA 5-mers in vitro similarily to Orn. Low expression levels of NrnB are sufficient for orn complementation. YhaM, a known RNase present in B. subtilis, degrades nanoRNA efficiently in vitro but requires high levels of expression for only partial complementation of the orn^– strain. A triple mutant (nrnA^–, nrnB^–, yhaM^–) in B. subtilis is viable and shows almost no impairment in growth. Lastly, RNase J1 seems also to have some 5′-to-3′ exoribonuclease activity on nanoRNA and thus can potentially finish degradation of RNA. We conclude that, unlike in E. coli, degradation of nanoRNA is performed in a redundant fashion in B. subtilis.     

Interaction of the MARCKS peptide with PIP2 in phospholipid monolayers

In this present work we have studied the effect of MARCKS (151–175) peptide on a mixed DPPC/PIP₂ monolayer. By means of film balance, fluorescence microscopy, x-ray reflection/diffraction and neutron reflection measurements we detected changes in the lateral organization of the monolayer and changes in the perpendicular orientation of the PIP₂ molecules depending on the presence of MARCKS (151–175) peptide in the subphase. In the mixed monolayer, the PIP₂ molecules are distributed uniformly in the disordered phase of the monolayer, whereas the PI(4,5) groups elongate up to 10 Å below the phosphodiester groups. This elongation forms the precondition for the electrostatic interaction of the MARCKS peptide with the PIP₂ molecules. Due to the enrichment of PIP₂ in the disordered phase, the interaction with the peptide occurs primarily in this phase, causing the PI(4,5) groups to tilt toward the monolayer interface.