Mitiko Gō

Ancient divergence of long and short isoforms of adenylate kinase molecular evolution of the nucleoside monophosphate kinase family

Adenylate kinases (AK) from vertebrates are separated into three isoforms, AK1, AK2 and AK3, based on structure, subcellular localization and substrate specificity. AK1 is the short type with the amino acid sequence being 27 residues shorter than sequences of the long types, AK2 and AK3. A phylogenetic tree prepared for the AK isozymes and other members of the nucleoside monophosphate (NMP) kinase family shows that the divergence of long and short types occurred first and then differentiation in subcellular localization or substrate specificity took place. The first step involved a drastic change in the three‐dimensional structure of the LID domain. The second step was caused mainly by smaller changes in amino acid sequences.

INSECT PROTHORACICOTROPIC HORMONE : A NEW MEMBER OF THE VERTEBRATE GROWTH FACTOR SUPERFAMILY

Prothoracicotropic hormone (PTTH) is a brain neurosecretory protein that controls insect development. PTTH of the silkmoth Bombyx mori is a homodimeric protein, the subunit of which consists of 109 amino acids. Clear‐cut sequence similarity to any other proteins has not been observed. By disulfide‐bond pattern analysis and modeling of the PTTH structure based on the known three‐dimensional (3D) structures of growth factor family with cystine‐knot motif, we propose that the PTTH protomer adopts the fold unique to the structural superfamily of the growth factors, β‐nerve growth factor (β‐NGF), transforming growth factor‐β2 (TGF‐β2), and platelet‐derived growth factor‐BB (PDGF‐BB). The insect neurohormone PTTH appears to be a member of the growth factor superfamily, sharing a common ancestral gene with the three vertebrate growth factors, β‐NGF, TGF‐β2 and PDGF‐BB.

Evolutionary clustering and functional similarity of RNA-binding proteins

RNA‐binding proteins (RNPs) involved in splicing, processing and translation regulation contain one to four RNA‐binding domains. We constructed a phylogenetic tree for the RNA‐binding domains, including those of poly(A)‐binding protein (PABP), splicing factors, chloroplast RNPs, hnRNPs, snRNP U1‐70K, nucleolin and Drosophila sex determinants. Proteins with similar functions were found to have closely related RNA‐binding domains and common domain organizations. In light of these observation, one can assume the function of an RNA‐binding protein, based on the evolutionary relationship between its RNA‐binding domain(s) and domain organization, as compared with other RNPs.

Variable Subunit Contact and Cooperativity of Hemoglobins

Abstract. Tertiary structures of proteins are conserved better than their primary structures during evolution. Quaternary structures or subunit organizations, however, are not always conserved. A typical case is found in hemoglobin family. Although human, Scapharca, and Urechis have tetrameric hemoglobins, their subunit contacts are completely different from each other. We report here that only one or two amino acid replacements are enough to create a new contact between subunits. Such a small number of chance replacements is expected during the evolution of hemoglobins. This result explains why different modes of subunit interaction evolved in animal hemoglobins. In contrast, certain interactions between subunits are necessary for cooperative oxygen binding. Cooperative oxygen binding is observed often in dimeric and tetrameric hemoglobins. Conformational change of a subunit induced by the first oxygen binding to the heme group is transmitted through the subunit contacts and increases the affinity of the second oxygen. The tetrameric hemoglobins from humans and Scapharca have cooperativity in spite of their different modes of subunit contact, but the one from Urechis does not. The relationship between cooperativity and the mode of subunit contacts is not clear. We compared the atomic interactions at the subunit contact surface of cooperative and non-cooperative tetrameric hemoglobins. We show that heme-contact modules M3–M6 play a key role in the subunit contacts responsible for cooperativity. A module was defined as a contiguous peptide segment having compact conformation and its average length is about 15 amino acid residues. We show that the cooperative hemoglobins have interactins involving at least two pairs of modules among the four heme-contact modules at subunit contact.

Cloning and characterization of the gene encoding Halobacterium halobium adenylate kinase

The gene (AK) encoding adenylate kinase (AK) of Halobacterium halobium was cloned. AK consisted of 648 bp and coded for 216 amino acids (aa). S1 mapping and primer extension experiments indicated that the transcription start point (tsp) was located immediately upstream from the start codon. The TAT-like promoter sequence was found at a position 20-24 bp upstream from tsp. The most striking property of the enzyme was a putative Zn finger-like structure with four cysteines. It might contribute to the structural stability of the molecule in high-salt conditions. Phylogenetic analysis indicated two lineages of the AK family, the short and long types which diverged a long time ago, possibly before the separation of prokaryotes and eukaryotes. Although the H. halobium AK belongs to the long-type AK lineage, it is located in an intermediary position between the two lineages of the phylogenetic tree, indicating early divergence of the gene along the long-type lineage.

Conformational characterization of designed minibarnase.

We have designed a minibarnase by removing one module from barnase, a bacterial RNase from Bacillus amyloliquefaciens. Barnase, consisting of 110 amino acid residues, is decomposed into six modules, M1-M6. Module is defined as a peptide segment consisting of contiguous amino acid residues that makes a small compact conformation within a globular domain. To understand the role of module in protein architecture, we analyzed NMR and CD spectra of a minibarnase, which lacked 26 amino acid residues corresponding to module M2. We demonstrated the formation of hydrophobic cores in the minibarnase similar to those of barnase. Although its conformational stability against acids and heat was reduced in comparison with barnase, the minibarnase retained cooperative folding character (two-state folding). Therefore, the folding of the minibarnase consisting of modules M1 and M3-M6 is independent to some extent of module M2. This finding may be useful for future module-based protein design.

Adaptive amino acid replacements accompanied by domain fusion in reverse transcriptase

Two basic processes are involved in protein evolution: One is amino acid replacement and another is reorganization of structural or functional units of proteins. Multidomain or multifunctional proteins are thought to have evolved by fusion of smaller structural units such as modules or domains. Reverse transcriptase (RT) is one of such fused proteins. The N-terminal part forms of globular domain with polymerase activity and the C-terminal part forms another globular domain with ribonuclease H activity (RNase H domain). There are single-domain enzymes which are homologous with the RNase H domain. The group of enzymes is called type I ribonuclease H (RNase HI). It is most likely that the ancestors of RNase HI and the polymerase domain were fused and became contemporary RT. At fusion, amino acid replacements presumably occurred at the interface of the domains to reinforce the interdomain interactions. Such replaced amino acid residues are conserved during evolution of the fused enzyme. We analyzed the pattern of amino acid replacement at each residue site in the free form, RNase HI group, and the integrated form, RNase H domain group. Then we compared the patterns between the two forms. Drastic fitting replacements of amino acid residues occurred at four of 29 residue sites involved in interdomain contact. Hydrophilic amino acid residues of the free form were substituted with hydrophobic or ambivalent ones in the integrated form. These substitutions aid in stabilizing the fused conformation by hydrophobic interactions at the interface of the domains. These observations imply that domain fusion could have occurred with only a relatively small number of adaptive amino acid substitutions.

Module Organization in Proteins and Exon Shuffling

Molecular mechanisms leading to drastic evolutionary change is essentially a recombination of genetic information. Exon shuffling, is one such mechanism used in the creation of novel proteins. The remnants of the shuffling are observed in the split gene structures of eukaryotic cells. Since it was found that the introns in the genes of the globin family corresponded to module joints of the globin chains, such correspondence has been widely observed in various genes. Modules are defined as compact conformational units in the three-dimensional structures of globular proteins. This article focuses on the close correlation between intron positions and module boundaries in several genes and their products. The intron-module relationship shows that exon shuffling is module shuffling in protein language. However, no introns are found at some module joints. It is suggested that many introns were lost during evolution. Though most of the eukaryotic genes are split by introns, introns are located in only a few exceptional genes in the case of prokaryotes. Module organization in proteins is observed also in prokaryotes as well as in eukaryotes. It is highly possible that prokaryotic genes were split by introns and these introns were lost after prokaryote-eukaryote divergence. Imperfect conservation of introns in eukaryotes shows that the module organization, conserved in the three-dimensional structures of contemporary proteins, gives us useful information concerning the evolutionary history of exon shuffling