▪ Abstract Most prokaryotic signal-transduction systems and a few eukaryotic pathways use phosphotransfer schemes involving two conserved components, a histidine protein kinase and a response regul...

Two-component signal transduction

The large ribosomal subunit catalyzes peptide bond formation and binds initiation, termination, and elongation factors. We have determined the crystal structure of the large ribosomal subunit from Haloarcula marismortui at 2.4 angstrom resolution, and it includes 2833 of the subunit's 3045 nucleotides and 27 of its 31 proteins. The domains of its RNAs all have irregular shapes and fit together in the ribosome like the pieces of a three-dimensional jigsaw puzzle to form a large, monolithic structure. Proteins are abundant everywhere on its surface except in the active site where peptide bond formation occurs and where it contacts the small subunit. Most of the proteins stabilize the structure by interacting with several RNA domains, often using idiosyncratically folded extensions that reach into the subunit's interior.

https://science.sciencemag.org/content/sci/289/5481/905.full.pdf

The complete atomic structure of the large ribosomal subunit at 2.4 A resolution.

Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein–RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.

Structure of the 30S ribosomal subunit.

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Calcium ATPase is a member of the P-type ATPases that transport ions across the membrane against a concentration gradient. Here we have solved the crystal structure of the calcium ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a) at 2.6 A resolution with two calcium ions bound in the transmembrane domain, which comprises ten alpha-helices. The two calcium ions are located side by side and are surrounded by four transmembrane helices, two of which are unwound for efficient coordination geometry. The cytoplasmic region consists of three well separated domains, with the phosphorylation site in the central catalytic domain and the adenosine-binding site on another domain. The phosphorylation domain has the same fold as haloacid dehalogenase. Comparison with a low-resolution electron density map of the enzyme in the absence of calcium and with biochemical data suggests that large domain movements take place during active transport.

http://www.iam.u-tokyo.ac.jp/StrBiol/resource/nature2000.pdf

Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 Å resolution

The tertiary structure of the macrophage migration inhibitory factor (MIF) from rat liver (12,300 Mr) is presented at 2.2 A resolution. Each monomer consists of two β/α/β motifs aligned in quasi two-fold symmetry, comprising a domain consisting of a four-stranded mixed β-sheet and two antiparallel α-helices. The protein exists as a trimer in the crystal. An extra β-strand that is almost perpendicular to the other β-strands joins to the β-sheet of the neighbouring monomer in the trimer. Unexpected similarities were detected between MIF and two kinds of isomerase.

Crystal structure of the macrophage migration inhibitory factor from rat liver.

Structures of nitric oxide reductase (NOR) in the ferric resting and the ferrous CO states have been solved at 2.0 A resolution. These structures provide significant new insights into how NO is reduced in biological systems. The haem distal pocket is open to solvent, implicating this region as a possible NADH binding site. In combination with mutagenesis results, a hydrogen-bonding network from the water molecule adjacent to the iron ligand to the protein surface of the distal pocket through the hydroxyl group of Ser 286 and the carboxyl group of Asp 393 can be assigned to a pathway for proton delivery during the NO reduction reaction.

Crystal structure of nitric oxide reductase from denitrifying fungus Fusarium oxysporum.

The prokaryotic protein HU, integration host factor (IHF) from Escherichia coli, and transcription factor 1 (TF1) from bacteriophage SPO1 are closely related molecules. Biochemical results suggest that the role of these proteins is to bind and bend DNA. From the high-resolution structure of HU, we propose a model for this interaction with DNA. Crucial amino acid differences between the proteins can be rationalized in terms of their different specific functions.

A protein structural motif that bends DNA.

The crystal structure of human MRP14 (S100A9), a Ca(2+)-dependent regulator protein in inflammatory process.

The C-terminal DNA-binding domain of OmpR, a positive regulator involved in osmoregulation expression of the ompF and ompC genes in Escherichia coli, has a helix-turn-helix variant motif. The 'turn' region, consisting of 11 residues, forms an RNA polymerase contact site.

Isao Tanaka

Papers

Crystal structure of the macrophage migration inhibitory factor from rat liver.

Crystal structure of nitric oxide reductase from denitrifying fungus Fusarium oxysporum.

A protein structural motif that bends DNA.

The crystal structure of human MRP14 (S100A9), a Ca(2+)-dependent regulator protein in inflammatory process.

Escherichia coli positive regulator OmpR has a large loop structure at the putative RNA polymerase interaction site.