Liisa Laakkonen

A Complete Neandertal Mitochondrial Genome Sequence Determined by High-Throughput Sequencing

A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000 year-old Neandertal individual with 8341 mtDNA sequences identified among 4.8 Gb of DNA generated from approximately 0.3 g of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs, and allows an estimate of the divergence date between the two mtDNA lineages of 660,000 +/- 140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared with other primate lineages, suggesting that the effective population size of Neandertals was small.

The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site.

Cell respiration is catalyzed by the heme-copper oxidase superfamily of enzymes, which comprises cytochrome c and ubiquinol oxidases. These membrane proteins utilize different electron donors through dissimilar access mechanisms. We report here the first structure of a ubiquinol oxidase, cytochrome bo3, from Escherichia coli. The overall structure of the enzyme is similar to those of cytochrome c oxidases; however, the membrane-spanning region of subunit I contains a cluster of polar residues exposed to the interior of the lipid bilayer that is not present in the cytochrome c oxidase. Mutagenesis studies on these residues strongly suggest that this region forms a quinone binding site. A sequence comparison of this region with known quinone binding sites in other membrane proteins shows remarkable similarities. In light of these findings we suggest specific roles for these polar residues in electron and proton transfer in ubiquinol oxidase.

Trends in inversion barriers. I. Group-15 hydrides

Inversion barriers for the group‐15 hydrides NH3, PH3, AsH3, SbH3 and BiH3 have been studied using ab initio self‐consistent‐field methods including electron correlation and relativistic effects. A modified symmetric inversion potential is introduced to describe the inversion from the minimum C3v arrangement through the D3h transition state. Tunneling rates and frequencies are calculated at the Hartree–Fock and Mo/ller–Plesset (MP2) level within the Wentzel–Kramers–Brillouin approximation. At the MP2 level the calculated 0+/0− ν2 frequency splitting of the vibronic ground state of NH3/ND3 (0.729 cm−1/0.041 cm−1) is in excellent agreement with the experimental values (0.794 cm−1/0.053 cm−1). The tunneling rate for PH3 suggests that previously published values are wrong by orders of magnitude. Correlation effects do not change the barriers significantly in accordance with Freed’s theorem. This has been studied in more detail for BiH3 at the quadratic configuration‐interaction (QCI) level. Relativistic effec...

Glutamine Synthetase and Glucose-6-Phosphate Isomerase Are Adhesive Moonlighting Proteins of Lactobacillus crispatus Released by Epithelial Cathelicidin LL-37

Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His(6)-GS, His(6)-GPI, His(6)-enolase, and His(6)-GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His(6)-GS and His(6)-GPI proteins bound to type I collagen, and His(6)-GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His(6)-GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells.

The Omptins of Yersinia pestis and Salmonella enterica Cleave the Reactive Center Loop of Plasminogen Activator Inhibitor 1

Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis.

A molecular model of the human UDP-glucuronosyltransferase 1A1, its membrane orientation, and the interactions between different parts of the enzyme.

The vertebrate UDP-glucuronosyltransferases (UGTs) are membrane-bound enzymes of the endoplasmic reticulum that process both endogenous and exogenous substrates. The human UGTs are well known biologically, but biophysical understanding is scarce, largely because of problems in purification. The one resolved crystal structure covers the C-terminal domain of the human UGT2B7. Here, we present a homology model of the complete monomeric human UGT1A1, the enzyme that catalyzes bilirubin glucuronidation. The enzyme can be seen as composed of four different domains: two large ones, the N- and C-terminal domains, and two small ones, the “envelope” helices and the transmembrane segment that includes the cytoplasmic tail. The hydrophobic core of the N-terminal domain and the two envelope helices that connect the large domains are shown to be structurally well conserved even among distant homologs and can thus be modeled with good certainty according to plant and bacterial structures. We consider alternative solutions for the highly variable N-terminal regions that probably contribute to substrate binding. The bilirubin binding site, known pathological mutations in UGT1A1, and other specific residues have been examined in the context of the model with regard to available experimental data. A putative orientation of the protein relative to the membrane has been derived from the location of predicted N-glycosylation sites. The model presents extensive interactions between the N- and C-terminal domains, the two envelope helices, and the membrane. Together, these interactions could allow for a concerted large-scale conformational change during catalysis.

Charge parameterization of the metal centers in cytochrome c oxidase

Reliable atomic point charges are of key importance for a correct description of the electrostatic interactions when performing classical, force field based simulations. Here, we present a systematic procedure for point charge derivation, based on quantum mechanical methodology suited for the systems at hand. A notable difference to previous procedures is to include an outer region around the actual system of interest. At the cost of increasing the system sizes, here up to 265 atoms, including the surroundings achieves near‐neutrality for the systems as well as structural stability, important factors for reliable charge distributions. In addition, the common problem of converting between CH bonds and CC bonds at the border vanishes. We apply the procedure to the four redox‐active metal centers of cytochrome c oxidase: CuA, haem a, haem a3, and CuB. Several relevant charge and ligand states are considered. Charges for two different force fields, CHARMM and AMBER, are presented.

Fibrinolytic and coagulative activities of Yersinia pestis

The outer membrane protease Pla belongs to the omptin protease family spread by horizontal gene transfer into Gram-negative bacteria that infect animals or plants. Pla has adapted to support the life style of the plague bacterium Yersinia pestis. Pla has a β-barrel fold with 10 membrane-spanning β strands and five surface loops, and the barrel surface contains bound lipopolysaccharide (LPS) that is critical for the conformation and the activity of Pla. The biological activity of Pla is influenced by the structure of the surface loops around the active site groove and by temperature-induced LPS modifications. Several of the putative virulence-related functions documented for Pla in vitro address control of the human hemostatic system, i.e., coagulation and fibrinolysis. Pla activates human plasminogen to the serine protease plasmin and activates the physiological plasminogen activator urokinase. Pla also inactivates the protease inhibitors alpha-2-antiplasmin and plasminogen activator inhibitor 1 (PAI-1) and prevents the activation of thrombin-activatable fibrinolysis inhibitor (TAFI). These functions enhance uncontrolled fibrinolysis which is thought to improve Y. pestis dissemination and survival in the mammalian host, and lowered fibrin(ogen) deposition has indeed been observed in mice infected with Pla-positive Y. pestis. However, Pla also inactivates an anticoagulant, the tissue factor (TF) pathway inhibitor, which should increase fibrin formation and clotting. Thus, Pla and Y. pestis have complex interactions with the hemostatic system. Y. pestis modifies its LPS upon transfer to the mammalian host and we hypothesize that the contrasting biological activities of Pla in coagulation and fibrinolysis are influenced by LPS changes during infection.

Probing the ability of the coat and vertex protein of the membrane-containing bacteriophage PRD1 to display a meningococcal epitope

Bacteriophage PRD1 is an icosahedral dsDNA virus with a diameter of 740 A and an outer protein shell composed of 720 copies of major coat protein P3. Spike complexes at the vertices are composed of a pentameric base (protein P31) and a spike structure (proteins P5 and P2) where the N-terminal region of the trimeric P5 is associated with the base and the C-terminal region of P5 is associated with receptor-binding protein P2. The functionality of proteins P3 and P5 was investigated using insertions and deletions. It was observed that P3 did not tolerate changes whereas P5 tolerated changes much more freely. These properties support the hypothesis that viruses have core structures and functions, which remain stable over time, as well as other elements, responsible for host interactions, which are evolutionally more fluid. The insertional probe used was the apex of exposed loop 4 of group B meningococcal outer membrane protein PorA, a medically important subunit vaccine candidate. It was demonstrated that the epitope could be displayed on the virus surface as part of spike protein P5.

Polyglutamine variation in a flowering time protein correlates with island age in a Hawaiian plant radiation

BackgroundA controversial topic in evolutionary developmental biology is whether morphological diversification in natural populations can be driven by expansions and contractions of amino acid repeats in proteins. To promote adaptation, selection on protein length variation must overcome deleterious effects of multiple correlated traits (pleiotropy). Thus far, systems that demonstrate this capacity include only ancient or artificial morphological diversifications. The Hawaiian Islands, with their linear geological sequence, present a unique environment to study recent, natural radiations. We have focused our research on the Hawaiian endemic mints (Lamiaceae), a large and diverse lineage with paradoxically low genetic variation, in order to test whether a direct relationship between coding-sequence repeat diversity and morphological change can be observed in an actively evolving system.ResultsHere we show that in the Hawaiian mints, extensive polyglutamine (CAG codon repeat) polymorphism within a homolog of the pleiotropic flowering time protein and abscisic acid receptor FCA tracks the natural environmental cline of the island chain, consequent with island age, across a period of 5 million years. CAG expansions, perhaps following their natural tendency to elongate, are more frequent in colonists of recently-formed, nutrient-rich islands than in their forebears on older, nutrient-poor islands. Values for several quantitative morphological variables related to reproductive investment, known from Arabidopsis fca mutant studies, weakly though positively correlate with increasing glutamine tract length. Together with protein modeling of FCA, which indicates that longer polyglutamine tracts could induce suboptimally mobile functional domains, we suggest that CAG expansions may form slightly deleterious alleles (with respect to protein function) that become fixed in founder populations.ConclusionIn the Hawaiian mint FCA system, we infer that contraction of slightly deleterious CAG repeats occurred because of competition for resources along the natural environmental cline of the island chain. The observed geographical structure of FCA variation and its correlation with morphologies expected from Arabidopsis mutant studies may indicate that developmental pleiotropy played a role in the diversification of the mints. This discovery is important in that it concurs with other suggestions that repetitive amino acid motifs might provide a mechanism for driving morphological evolution, and that variation at such motifs might permit rapid tuning to environmental change.