Deciphering the Catalytic Machinery in 30S Ribosome Assembly GTPase YqeH

YqeH, a circularly permuted GTPase (cpGTPase), which is conserved across bacteria and eukaryotes including humans is important for the maturation of small (30S) ribosomal subunit in Bacillus subtilis. Recently, we have shown that it binds 30S in a GTP/GDP dependent fashion. However, the catalytic machinery employed to hydrolyze GTP is not recognized for any of the cpGTPases, including YqeH. This is because they possess a hydrophobic substitution in place of a catalytic glutamine (present in Ras-like GTPases). Such GTPases were categorized as HAS-GTPases and were proposed to follow a catalytic mechanism, different from the Ras-like proteins.MnmE, another HAS-GTPase, but not circularly permuted, utilizes a potassium ion and water mediated interactions to drive GTP hydrolysis. Though the G-domain of MnmE and YqeH share only approximately 25% sequence identity, the conservation of characteristic sequence motifs between them prompted us to probe GTP hydrolysis machinery in YqeH, by employing homology modeling in conjunction with biochemical experiments. Here, we show that YqeH too, uses a potassium ion to drive GTP hydrolysis and stabilize the transition state. However, unlike MnmE, it does not dimerize in the transition state, suggesting alternative ways to stabilize switches I and II. Furthermore, we identify a potential catalytic residue in Asp-57, whose recognition, in the absence of structural information, was non-trivial due to the circular permutation in YqeH. Interestingly, when compared with MnmE, helix alpha2 that presents Asp-57 is relocated towards the N-terminus in YqeH. An analysis of the YqeH homology model, suggests that despite such relocation, Asp-57 may facilitate water mediated catalysis, similarly as the catalytic Glu-282 of MnmE. Indeed, an abolished catalysis by D57I mutant supports this inference.An uncommon means to achieve GTP hydrolysis utilizing a K(+) ion has so far been demonstrated only for MnmE. Here, we show that YqeH also utilizes a similar mechanism. While the catalytic machinery is similar in both, mechanistic differences may arise based on the way they are deployed. It appears that K(+) driven mechanism emerges as an alternative theme to stabilize the transition state and hydrolyze GTP in a subset of GTPases, such as the HAS-GTPases

RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state

Flaviviral RNA-dependent RNA polymerases (RdRps) initiate replication of the single-stranded RNA genome in the absence of a primer. The template sequence 5'-CU-3' at the 3'-end of the flaviviral genome is highly conserved. Surprisingly, flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. We show that GTP stimulates de novo RNA synthesis by RdRp from Japanese encephalitis virus (jRdRp) also. Crystal structures of jRdRp complexed with GTP and ATP provide a basis for specific recognition of GTP. Comparison of the jRdRp(GTP) structure with other viral RdRp-GTP structures shows that GTP binds jRdRp in a novel conformation. Apo-jRdRp structure suggests that the conserved motif F of jRdRp occupies multiple conformations in absence of GTP. Motif F becomes ordered on GTP binding and occludes the nucleotide triphosphate entry tunnel. Mutational analysis of key residues that interact with GTP evinces that the jRdRp(GTP) structure represents a novel pre-initiation state. Also, binding studies show that GTP binding reduces affinity of RdRp for RNA, but the presence of the catalytic Mn2+ ion abolishes this inhibition. Collectively, these observations suggest that the observed pre-initiation state may serve as a check-point to prevent erroneous template-independent RNA synthesis by jRdRp during initiation

Outcome of four pretreatment regimes on hemodynamics during electroconvulsive therapy: A double-blind randomized controlled crossover trial

Context: Electroconvulsive therapy (ECT) is associated with tachycardia and hypertension. Aims: The aim of this study was to compare two doses of dexmedetomidine, esmolol, and lignocaine with respect to hemodynamics, seizure duration, emergence agitation (EA), and recovery profile. Methodology: Thirty patients undergoing ECT were assigned to each of the following pretreatment regimes over the course of five ECT sessions in a randomized crossover design: Group D1 (dexmedetomidine 1 μg/kg), Group D0.5 (dexmedetomidine0.5 μg/kg), Group E (esmolol 1 mg/kg), Group L (lignocaine 1 mg/kg), and Group C (saline as placebo) before induction. Heart rate (HR), mean arterial pressure (MAP), seizure duration, EA, and time to discharge were evaluated. Results: Groups D1, D0.5, and esmolol had significantly reduced response of HR, MAP compared to lignocaine and control groups at 1, 3, 5 min after ECT (P 0.05). Time to eye opening and time to discharge were comparable in all groups (P > 0.05) except Group D1 (P = 0.001). EA score was least in Group D1 (P = 0.000). Conclusion: Dexmedetomidine 1 μg/kg, 0.5 μg/kg, and esmolol produced significant amelioration of cardiovascular response to ECT without affecting seizure duration, results being best with dexmedetomidine 1 μg/kg. However, the latter has the shortcoming of delayed recovery

Sequence and structural examination of SMO-1.

<p>(A) Multiple sequence alignment of SMO-1 with its homologues SUMO1 and SUMO2. Identical residues across the homologues are boxed in black while similar residues are boxed in grey. (B) ¹⁵N-HSQC of SMO-1 protein is shown with backbone amide peaks labelled by residue numbers. The side chain amides are connected by dashed lines. The folded peaks of arginine side chains are marked by an asterisk.</p

Structural and functional analysis of SMO-1, the SUMO homolog in <i>Caenorhabditis elegans</i>

<div><p>SUMO proteins are important post-translational modifiers involved in multiple cellular pathways in eukaryotes, especially during the different developmental stages in multicellular organisms. The nematode <i>C</i>. <i>elegans</i> is a well known model system for studying metazoan development and has a single SUMO homolog, SMO-1. Interestingly, SMO-1 modification is linked to embryogenesis and development in the nematode. However, high-resolution information about SMO-1 and the mechanism of its conjugation is lacking. In this work, we report the high-resolution three dimensional structure of SMO-1 solved by NMR spectroscopy. SMO-1 has flexible N-terminal and C-terminal tails on either side of a rigid beta-grasp folded core. While the sequence of SMO-1 is more similar to SUMO1, the electrostatic surface features of SMO-1 resemble more with SUMO2/3. SMO-1 can bind to typical SUMO Interacting Motifs (SIMs). SMO-1 can also conjugate to a typical SUMOylation consensus site as well as to its natural substrate HMR-1. Poly-SMO-1 chains were observed <i>in-vitro</i> even though SMO-1 lacks any consensus SUMOylation site. Typical deSUMOylation enzymes like Senp2 can cleave the poly-SMO-1 chains. Despite being a single gene, the SMO-1 structure allows it to function in a large repertoire of signaling pathways involving SUMO in <i>C</i>. <i>elegans</i>. Structural and functional features of SMO-1 studies described here will be useful to understand its role in development.</p></div

Comparison of structures and electrostatic surface potential between SMO-1 and its SUMO homologues.

<p>(A), (B) and (C) The top panel shows the cartoon representation of SUMO1, SMO-1 and SUMO2 (all in orange). The SIM interacting region of SUMO1 and SUMO2 is marked by an oval in broken line. The middle panel shows the APBS calculated (at 298 K) electrostatic surface potential of respective proteins in the orientation shown in top panel. The bottom panel shows the same electrostatic surface potential at an orientation rotated 180 degree about the vertical shown as shown. Color gradient scheme of the electrostatic surface is shown for each surface. Positively charged surfaces are colored blue, neutral surfaces are in white and negatively charged surfaces are in red. The unit of the color gradient shown is kcal/(mol.<i>e</i>).</p

SMO-1 forms chains in <i>in vitro</i> SUMOylation assays.

<p>(A) Sequence of FITC fluorophore labelled peptide used as substrate in <i>in vitro</i> SUMOylation assays. The lysines competent for SUMOylation are in bigger font and in bold face. All subsequent images were obtained by observing FITC fluorescence at 519 nm unless otherwise mentioned. (B) SUMOylation reaction products with peptide shown in (A) and SUMO1/SMO-1 resolved on SDS-PAGE gel. The negative control is lane labelled “no ATP”. Bands of free peptide and peptide conjugated with one, two or multiple (n) SMO-1 are marked. The molecular weight marker positions are indicated. (C) Time course of SUMOylation reaction between peptide shown in (A) and SMO-1. Different time points are indicated. O/N means overnight (~12 hours) incubation of reaction. Band identities and molecular weight marker positions are shown as in (B). (D) SUMOylation reaction using FITC-labelled SMO-1 and in absence of any other peptide substrate. The reaction products and the negative control (without ATP) are resolved and marked. (E) De-conjugation of SUMOylated peptides by SENP2 enzyme. The SUMOylation reaction products shown in (B) were run with or without treatment with SENP2. (F) De-conjugation reaction of SMO-1-YFP construct with SENP2. The SDS-PAGE gel is imaged by observing YFP fluorescence. (G) SUMOylation reaction between FITC-labelled SMO-1 and HMR-1A, a known protein substrate of SMO-1 in <i>C</i>. <i>elegans</i>. The reaction product band is marked. Negative controls–without ATP and without HMR-1A is also shown. (H) SUMOylation reaction described in (G) is shown with the negative control where all lysines of HMR-1A were mutated to arginines to create the SUMOylation incompetent HMR-1A-K2R construct.</p

NMR solution structure of SMO-1.

<p>(A) 20 lowest energy NMR solution structures of SMO-1calculated by CYANA. The SMO-1 chains (in orange) are depicted in cartoon representation. (B) The best model structure of SMO-1 protein is shown in orange. The chain termini and secondary elements are labelled.</p