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The canonical function of glutamyl‐tRNA synthetase (GluRS) is to glutamylate tRNAGlu. Yet not all bacterial GluRSs glutamylate tRNAGlu; many glutamylate both tRNAGlu and tRNAGln, while some glutamylate only tRNAGln and not the cognate substrate tRNAGlu. Understanding the basis of the unique specificity of tRNAGlx is important. Mutational studies have hinted at hotspot residues, both on tRNAGlx and GluRS, which play crucial roles in tRNAGlx‐specificity. However, its underlying structural basis remains unexplored. The majority of biochemical studies related to tRNAGlx‐specificity have been performed on GluRS from Escherichia coli and other proteobacterial species. However, since the early crystal structures of GluRS and tRNAGlu‐bound GluRS were from non‐proteobacterial species (Thermus thermophilus), proteobacterial biochemical data have often been interpreted in the context of non‐proteobacterial GluRS structures. Marked differences between proteobacterial and non‐proteobacterial GluRSs have been demonstrated; therefore, it is important to understand tRNAGlx‐specificity vis‐a‐vis proteobacterial GluRS structures. To this end, we solved the crystal structure of a double mutant GluRS from E. coli. Using the solved structure and several other currently available proteo‐ and non‐proteobacterial GluRS crystal structures, we probed the structural basis of the tRNAGlx‐specificity of bacterial GluRSs. Specifically, our analyses suggest a unique role played by the tRNAGlx D‐helix contacting loop of GluRS in the modulation of tRNAGln‐specificity. While earlier studies have identified functional hotspots on tRNAGlx that control the tRNAGlx‐specificity of GluRS, this is the first report of complementary signatures of tRNAGlx‐specificity in GluRS. [ABSTRACT FROM AUTHOR]

Electron bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate-potential electron donor are split so that one is sent along a high-potential pathway to a high-potential acceptor and the other is sent along a low-potential pathway to a low-potential acceptor. This process allows endergonic reactions to be driven by exergonic ones and is an alternative, less recognized, mechanism of energy coupling to the well-known chemiosmotic principle. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron bifurcation in HydABC remains enigmatic in spite of intense research efforts over the last few years. Structural information may provide the basis for a better understanding of spectroscopic and functional information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure shows a heterododecamer composed of two independent 'halves' each made of two strongly interacting HydABC heterotrimers connected via a [4Fe-4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and for proton reduction. We identified two conformations of a flexible iron-sulfur cluster domain: a 'closed bridge' and an 'open bridge' conformation, where a Zn2+ site may act as a 'hinge' allowing domain movement. Based on these structural revelations, we propose a possible mechanism of electron bifurcation in HydABC where the flavin mononucleotide serves a dual role as both the electron bifurcation center and as the NAD+ reduction/NADH oxidation site. [ABSTRACT FROM AUTHOR]

Sensory type [FeFe] hydrogenases are predicted to play a role in transcriptional regulation by detecting the H2 level of the cellular environment. These hydrogenases contain the hydrogenase domain with distinct modifications in the active site pocket, followed by a Per-Arnt-Sim (PAS) domain. As yet, neither the physiological function nor the biochemical or spectroscopic properties of these enzymes have been explored. Here, we present the characterization of an artificially maturated, putative sensory [FeFe] hydrogenase from Thermotoga maritima (HydS). This enzyme shows lower hydrogen conversion activity than prototypical [FeFe] hydrogenases and a reduced inhibition by CO. Using FTIR spectroelectrochemistry and EPR spectroscopy, three redox states of the active site were identified. The spectroscopic signatures of the most oxidized state closely resemble those of the Hox state from the prototypical [FeFe] hydrogenases, while the FTIR spectra of both singly and doubly reduced states show large differences. The FTIR bands of both the reduced states are strongly red-shifted relative to the Hox state, indicating reduction at the diiron site, but with retention of the bridging CO ligand. The unique functional and spectroscopic features of HydS are discussed with regard to the possible role of altered amino acid residues influencing the electronic properties of the H-cluster. [ABSTRACT FROM AUTHOR]

The putative zinc-binding domain (pZBD) in Escherichia coli glutamyl-tRNA synthetase (GluRS) is known to correctly position the tRNA acceptor arm and modulate the amino acid-binding site. However, its functional role in other bacterial species is not clear since many bacterial GluRSs lack a zinc-binding motif in the pZBD. From experimental studies on pZBD-swapped E. coli GluRS, with Thermosynechoccus elongatus GluRS, Burkholderia thailandensis GluRS and E. coli glutamyl-queuosine-tRNAAsp synthetase (Glu-Q-RS), we show that E. coli GluRS, containing the zinc-free pZBD of B. thailandensis, is as functional as the zinc-bound wild-type E. coli GluRS, whereas the other constructs, all zinc-bound, show impaired function. A pZBD-tinkered version of E. coli GluRS that still retained Zn-binding capacity, also showed reduced activity. This suggests that zinc is not essential for the pZBD to be functional. From extensive structural and sequence analyses from whole genome database of bacterial GluRS, we further show that in addition to many bacterial GluRS lacking a zinc-binding motif, the pZBD is actually deleted in some bacteria, all containing either glutaminyl-tRNA synthetase (GlnRS) or a second copy of GluRS (GluRS2). Correlation between the absence of pZBD and the occurrence of glutamine amidotransferase CAB (GatCAB) in the genome suggests that the primordial role of the pZBD was to facilitate transamidation of misacylated Glu-tRNAGln via interaction with GatCAB, whereas its role in tRNAGlu interaction may be a consequence of the presence of pZBD. [ABSTRACT FROM AUTHOR]

The nature of interaction between glutamyl-tRNA synthetase (GluRS) and its tRNA substrate is unique in bacteria in that many bacterial GluRS are capable of recognizing two tRNA substrates: tRNAGlu and tRNAGln. To properly understand this distinctive GluRS-tRNA interaction it is important to pursue detailed structure-function studies; however, because of the fact that tRNA-GluRS interaction in bacteria is also associated with phylum-specific idiosyncrasies, the structure-function correlation studies must also be phylum-specific. GluRS from Thermus thermophilus and Escherichia coli, which belong to evolutionarily distant phyla, are the biochemically best characterized. Of these, only the structure of T. thermophilus GluRS is available. To fully unravel the subtleties of tRNAGlu-GluRS interaction in E. coli, a model bacterium that can also be pathogenic, determination of the E. coli GluRS structure is essential. However, previous attempts have failed to crystallize E. coli GluRS. By mapping crystal contacts of a homologous GluRS onto the E. coli GluRS sequence, two surface residues were identified that might have been hindering crystallization attempts. Accordingly, these two residues were mutated and crystallization of the double mutant was attempted. Here, the design, expression, purification and crystallization of an engineered E. coli GluRS in which two surface residues were mutated to optimize crystal contacts are reported. [ABSTRACT FROM AUTHOR]

Integrase Interactor 1 (INI1/hSNF5) is a component of the hSWI/SNF chromatin remodeling complex. The INI1 gene is either deleted or mutated in rhabdoid cancers like ATRT (Atypical terratoid and rhabdoid tumor). INI1 is also a host factor for HIV-1 replication. INI1 binds DNA non-specifically. However, the mechanism of DNA binding and its biological role are unknown. From agarose gel retardation assay (AGRA), Ni-NTA pull-down and atomic force microscopy (AFM) studies we show that amino acids 105–183 of INI1 comprise the minimal DNA binding domain (DBD). The INI1 DBD is absent in plants and in yeast SNF5. It is present in Caenorhabditis elegans SNF5, Drosophila melanogaster homologue SNR1 and is a highly conserved domain in vertebrates. The DNA binding property of this domain in SNR1, that is only 58% identical to INI1/hSNF5, is conserved. Analytical ultracentrifugation studies of INI1 DBD and INI1 DBD:DNA complexes at different concentrations show that the DBD exists as a monomer at low protein concentration and two molecules of monomer binds one molecule of DNA. At high protein concentration, it exists as a dimer and binds two DNA molecules. Furthermore, isothermal calorimetry (ITC) experiments demonstrate that the DBD monomer binds DNA with a stoichiometry (N) of ∼0.5 and Kd  = 0.94 µM whereas the DBD dimer binds two DNA molecules sequentially with K’d1 = 222 µM and K’d2 = 1.16 µM. Monomeric DBD binding to DNA is enthalpy driven (ΔH = –29.9 KJ/mole). Dimeric DBD binding to DNA is sequential with the first binding event driven by positive entropy (ΔH’1 = 115.7 KJ/mole, TΔS’1 = 136.8 KJ/mole) and the second binding event driven by negative enthalpy (ΔH’2 = –106.3 KJ/mole, TΔS’2 = –75.7 KJ/mole). Our model for INI1 DBD binding to DNA provides new insights into the mechanism of DNA binding by INI1. [ABSTRACT FROM AUTHOR]

The canonical function of glutamyl-tRNA synthetase (GluRS) is to glutamylate tRNA Glu . Yet not all bacterial GluRSs glutamylate tRNA Glu ; many glutamylate both tRNA Glu and tRNA Gln , while some glutamylate only tRNA Gln and not the cognate substrate tRNA Glu . Understanding the basis of the unique specificity of tRNA Glx is important. Mutational studies have hinted at hotspot residues, both on tRNA Glx and GluRS, which play crucial roles in tRNA Glx -specificity. However, its underlying structural basis remains unexplored. The majority of biochemical studies related to tRNA Glx -specificity have been performed on GluRS from Escherichia coli and other proteobacterial species. However, since the early crystal structures of GluRS and tRNA Glu -bound GluRS were from non-proteobacterial species (Thermus thermophilus), proteobacterial biochemical data have often been interpreted in the context of non-proteobacterial GluRS structures. Marked differences between proteobacterial and non-proteobacterial GluRSs have been demonstrated; therefore, it is important to understand tRNA Glx -specificity vis-a-vis proteobacterial GluRS structures. To this end, we solved the crystal structure of a double mutant GluRS from E. coli. Using the solved structure and several other currently available proteo- and non-proteobacterial GluRS crystal structures, we probed the structural basis of the tRNA Glx -specificity of bacterial GluRSs. Specifically, our analyses suggest a unique role played by the tRNA Glx D-helix contacting loop of GluRS in the modulation of tRNA Gln -specificity. While earlier studies have identified functional hotspots on tRNA Glx that control the tRNA Glx -specificity of GluRS, this is the first report of complementary signatures of tRNA Glx -specificity in GluRS., (© 2023 Wiley Periodicals LLC.)