Domain flexibility in retroviral proteases: structural implications for drug resistant mutations

Rigid body rotation of five domains and movements within their interfacial joints provide a rational context for understanding why HIV protease mutations that arise in drug resistant strains are often spatially removed from the drug or substrate binding sites. Domain motions associated with substrate binding in the retroviral HIV-1 and SIV proteases are identified and characterized. These motions are in addition to closure of the flaps and result from rotations of approximately 6-7 degrees at primarily hydrophobic interfaces. A crystal structure of unliganded SIV protease (incorporating the point mutation Ser 4 His to stabilize the protease against autolysis) was determined to 2.0 A resolution in a new space group, P3221. The structure is in the most "open" conformation of any retroviral protease so far examined, with six residues of the flaps disordered. Comparison of this and unliganded HIV structures, with their respective liganded structures by difference distance matrixes identifies five domains of the protease dimer that move as rigid bodies against one another: one terminal domain encompassing the N- and C-terminal beta sheet of the dimer, two core domains containing the catalytic aspartic acids, and two flap domains. The two core domains rotate toward each other on substrate binding, reshaping the binding pocket. We therefore show that, for enzymes, mutations at interdomain interfaces that favor the unliganded form of the target active site will increase the off-rate of the inhibitor, allowing the substrate greater access for catalysis. This offers a mechanism of resistance to competitive inhibitors, especially when the forward enzymatic reaction rate exceeds the rate of substrate dissociation.     

Endogenous retroviruses and the evolution of resistance to retroviral infection

The current AIDS epidemic has rekindled interest in the evolution of retroviruses and the development of resistance to infection. Retroviruses and their vertebrate hosts have coexisted for millions of years, during which time a variety of host defence mechanisms has evolved. One repeated strategy is to use endogenous retroviruses to combat infection by their exogenous relatives.     

DQ-haplotype analysis in rhesus macaques: implications for the evolution of these genes

The DQA1 and DQB1 alleles of 258 rhesus monkeys (Macaca mulatta) of different origin were typed by PCR-RFLP. Five novel MamuDQA1 and five novel -DQB1 alleles were detected and 15 Mamu-DQA1-DQB1 haplotypes were identified. Haplotype analysis confirmed the conservation of the DQA1*01-DQB1 *06 haplotypes in evolution. The most conspicuous finding was the tight linkage between the Mamu-DQA1 and -DQB1 alleles. Almost in every case the Mamu-DQA1 allele was linked to only one particular Mamu-DQB1 allele. Although there also are constraints in the formation of DQ haplotypes in humans, such tight linkages are not observed. These findings support the hypothesis of some kind of co-evolution between DQA1 and DQB1 alleles and may reflect a stronger force of natural selection in macaques than in humans.     

Gain-of-function mutations in FcgammaRI of NOD mice: implications for the evolution of the Ig superfamily

It has been postulated that, during evolution of the Ig superfamily, modifications of the function of individual receptors might occur by acquisition of exons and their subsequent modification, though evidence of this is lacking. Here we have analysed the interaction of mouse IgG subclasses with high-affinity FcgammaRI (CD64) which contains three Ig-like domains and is important in innate and adaptive immunity. This analysis has identified a mechanism by which the postulated modification of newly acquired exons provides gains in function. Thus, the most widely distributed FcgammaRI allele in mice (e.g. BALB/c), bound only a single IgG subclass, IgG2a, with high affinity. However, non-obese diabetic (NOD) mice expressed a unique allele that exhibits broader specificity and, in addition to binding IgG2a, FcgammaRI-NOD bound monomeric IgG3 and bound IgG2b with high affinity, an IgG subclass not bound by FcgammaRI of other mouse strains, either as monomer or multivalent immune complexes. Analysis of mutants of FcgammaRI wherein segments of the interdomain junctions were exchanged between FcgammaRI-BALB and FcgammaRI-NOD identified these regions as having major influence in 'gain-of-function' by the NOD form of FcgammaRI. Nucleotide sequence analysis of intron/exon boundaries encoding the interdomain junctions of the FcgammaRI alleles showed these to have arisen by mutation to alter existing or create new mRNA splice donor/acceptor sites, resulting in generation of modified junctions.     

A gonococcal porA pseudogene: implications for understanding the evolution and pathogenicity of Neisseria gonorrhoeae

There is an unanswered question from previous studies of 1H/2H-exchange of amide protons of barnase. Under certain conditions, there is a relatively abrupt change from EX2 towards EX1 kinetics as the temperature is slightly increased. The change in kinetics for different mutants is not directly related to their changes in stability. We have measured the stability of the folding intermediate of barnase (I) in 2H2O under a variety of conditions and calculated its population at different temperatures. The change in kinetics correlates with the change in the population of the folding intermediate. At higher temperatures and pH, the free energy of I becomes higher than that of the denatured state, D, and the kinetics becomes EX1. The data fit a simple kinetic scheme. Such changes in kinetics may be used to detect the presence of intermediates in the folding reaction at equilibrium in native conditions, but cannot distinguish whether they are on or off-pathway.     

Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: implications for retroviral assembly mechanisms

We have used electron cryo-microscopy and image analysis to examine the native structure of immature, protease-deficient (PR-) and mature, wild-type (WT) Moloney murine leukemia virus (MuLV). Maturational cleavage of the Gag polyprotein by the viral protease is associated with striking morphological changes. The PR- MuLV particles exhibit a rounded central core, which has a characteristic track-like shell on its surface, whereas the WT MuLV cores display a polygonal surface with loss of the track-like feature. The pleomorphic shape and inability to refine unique orientation angles suggest that neither the PR- nor the WT MuLV adheres to strict icosahedral symmetry. Nevertheless, the PR- MuLV particles do exhibit paracrystalline order with a spacing between Gag molecules of approximately 45 A and a length of approximately 200 A. Because of the pleomorphic shape and paracrystalline packing of the Gag-RNA complexes, we raise the possibility that assembly of MuLV is driven by protein-RNA, as well as protein-protein, interactions. The maturation process involves a dramatic reorganization of the packing arrangements within the ribonucleoprotein core with disordering and loosening of the individual protein components.     

Novel homologs of replication protein A in archaea: implications for the evolution of ssDNA-binding proteins

In Bacteria and Eukarya, ssDNA-binding proteins are central to most aspects of DNA metabolism. Until recently, however, no counterpart of an ssDNA-binding protein had been identified in the third domain of life, Archaea. Here, we report the discovery of a novel type of ssDNA-binding protein in the genomes of several archaeons. These proteins, in contrast to all known members of this protein family, possess four conserved DNA-binding sites within a single polypeptide or, in one case, two polypeptides. This peculiar structural organization allows us to propose a model for the evolution of this class of proteins.     

Ribosomal DNA and phylogeny of the Ascaridoidea (Nemata: Secernentea): implications for morphological evolution and classification

Nematodes of the superfamily Ascaridoidea are parasites of the alimentary tract of vertebrates and include species that are of medical and economic importance. Existing evolutionary hypotheses for these organisms have frequently been based on interpretation of one or few "key" structural or life history features. We used nuclear-encoded small (1764 characters) and large subunit (757 characters) ribosomal DNA sequences to estimate the phylogeny of representative taxa from this superfamily. Trees inferred by maximum parsimony and maximum likelihood methods strongly support clades that are primarily consistent with one recent classification of the group. In contrast, most previously proposed phylogenetic hypotheses were significantly worse when compared to the maximum likelihood tree by a statistical method. Hypotheses for the evolution of morphological and life history characters were explored by parsimony mapping these features on several tree topologies, including optimal molecular trees and alternative topologies reflecting traditional expectations deemed not worse in statistical tests. The results identify some consistent putative shared-derived morphological features, but also strongly suggest that some key features emphasized by previous workers represent ancestral states or highly homoplastic characters.     

There is no highly conserved embryonic stage in the vertebrates: implications for current theories of evolution and development

The design of Ru(II) and Os(II) complexes which are photoreactive with deoxyribonucleic acid (DNA) represents one of the main targets for the development of novel molecular tools for the study of DNA and, in the future, for the production of new, metal-based, anti-tumor drugs. In this review, we explain how it is possible to make a complex photoreactive with nucleobases and nucleic acids. According to the photophysical behaviour of the Ru(II) compounds, two types of photochemistry are expected: (1) photosubstitution of a ligand by a nucleobase and another monodentate ligand, which takes place from the triplet, metal-centred (3MC) state; this state is populated thermally from the lowest lying triplet metal to ligand charge transfer (3MLCT) state; (2) photoreaction from the 3MLCT state, corresponding to photoredox processes with DNA bases. The two photoreactivities are in competition. By modulating appropriately the redox properties of the 3MLCT state, an electron transfer process from the base to the excited complex takes place, and is directly correlated with DNA cleavage or the formation of an adduct of the complex to DNA. In this adduct, guanine is linked by N2 to the alpha-position of a non-chelating nitrogen of the polyazaaromatic ligand without destruction of the complex. Different strategies are explained which increase the affinity of the complexes for DNA and direct the complex photoreactivity to sites of special DNA topology or targeted sequences of bases. Moreover, the replacement of the Ru(II) ion by the Os(II) ion in the photoreactive complexes leads to an increased specificity of photoreaction. Indeed, only one type of photoreactivity (from the 3MLCT state) is present for the Os(II) complexes because the 3MC state is too high in energy to be populated at room temperature.     

Mitochondrial phylogeny of the European cyprinids: implications for their systematics, reticulate evolution, and colonization time

Two different mitochondrial genes, the cytochrome b and the 16S rDNA, support the same European cyprinid molecular phylogeny: the most basal subfamily is the paraphyletic Rasborinae, the Cyprininae are monophyletic, the Tincinae and Gobioninae are close to the Cyprininae or more basal lineages but not close to Leuciscinae or Alburninae, and the Leuciscinae are paraphyletic but can become monophyletic if we include the biphyletic alburninae and exclude the Phoxinini. The relationship of the Acheilognathinae remains obscure. Natural intergeneric and interspecific hybridizations are clearly demonstrated within the Leuciscinae, both from high bootstrap proportions and intermediate morphological features: Chondrostoma toxostoma and Rutilus rutilus, Scardinius erythrophthalmus and R. rutilus, and Leuciscus multicellus and Leuciscus soufia. Finally, the use of the nonsaturated and clockwise 16S mtDNA sequences have been used to infer from nonintrogressive taxa the time of the first European cyprinid cladogeneses. The estimation confirms the hypothesis of Alma?a and Banarescu that European cyprinid subfamilies started to diversify 35 mya and confirms the hypothesis of Bianco on the diversification of European leuciscines in the Mediterranean area during the late Messinian (6.5 to 5.3 mya).     

A P-loop-like motif in a widespread ATP pyrophosphatase domain: implications for the evolution of sequence motifs and enzyme activity

A conserved amino acid sequence motif was identified in four distinct groups of enzymes that catalyze the hydrolysis of the alpha-beta phosphate bond of ATP, namely GMP synthetases, argininosuccinate synthetases, asparagine synthetases, and ATP sulfurylases. The motif is also present in Rhodobacter capsulata AdgA, Escherichia coli NtrL, and Bacillus subtilis OutB, for which no enzymatic activities are currently known. The observed pattern of amino acid residue conservation and predicted secondary structures suggest that this motif may be a modified version of the P-loop of nucleotide binding domains, and that it is likely to be involved in phosphate binding. We call it PP-motif, since it appears to be a part of a previously uncharacterized ATP pyrophophatase domain. ATP sulfurylases, NtrL, and OutB consist of this domain alone. In other proteins, the pyrophosphatase domain is associated with amidotransferase domains (type I or type II), a putative citrulline-aspartate ligase domain or a nitrilase/amidase domain. Unexpectedly, statistically significant overall sequence similarity was found between ATP sulfurylase and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase, another protein of the sulfate activation pathway. The PP-motif is strongly modified in PAPS reductases, but they share with ATP sulfurylases another conserved motif which might be involved in sulfate binding. We propose that PAPS reductases may have evolved from ATP sulfurylases; the evolution of the new enzymatic function appears to be accompanied by a switch of the strongest functional constraint from the PP-motif to the putative sulfate-binding motif.     

Tobamovirus evolution: gene overlaps, recombination, and taxonomic implications

Tobamoviruses, mostly isolated from solanaceous plants, may represent ancient virus lineages that have codiverged with their hosts. Recently completed nucleotide sequences of six nonsolanaceous tobamoviruses allowed assessment of the codivergence hypothesis and support a third subgroup within tobamoviruses. The genomic sequences of 12 tobamoviruses and the partial sequences of 11 others have been analyzed. Comparisons of the predicted protein sequences revealed three clusters of tobamoviruses, corresponding to those infecting solanaceous species (subgroup 1), those infecting cucurbits and legumes (subgroup 2), and those infecting crucifers. The orchid-infecting odontoglossum ringspot tobamovirus was associated with subgroup 1 genomes by its coat and movement protein sequences, but with the crucifer-pathogenic tobamoviruses by the remainder of its genome, suggesting that it is the progeny of a recombinant. For four of five genomic regions, subgroup 1 and 3 genomes were equidistant from a subgroup 2 genome chosen for comparison, suggesting uniform rates of evolution. A phylogenetic tree of plant families based on the tobamoviruses they harbor was congruent with that based on rubisco sequences but had a different root, suggesting that codivergence was tempered by rare events of viruses of one family colonizing another family. The proposed subgroup 3 viruses probably have an origin of virion assembly in the movement protein gene, a large (25-codon) overlap of movement and coat protein open reading frames, and a comparably shorter genome. Codon-position-dependent base compositions and codon prevalences suggested that the coat protein frame of the overlap region was ancestral. Bootstrapped parsimony analysis of the nucleotides in the overlap region and of the sequences translated from the -1 frame (the subgroup 3 movement protein frame) of this region produced trees inconsistent with those deduced from other regions. The results are consistent with a model in which a no or short overlap organization was ancestral. Despite encoding of subgroup 2 and 3 movement protein C-termini by nonhomologous nucleotides, weak similarities between their amino acid sequences suggested convergent sequence evolution.     

Extraordinary ribosomal spacer length heterogeneity in a neotyphodium endophyte hybrid: implications for concerted evolution

An extraordinary level of length heterogeneity was found in the ribosomal DNA (rDNA) of an asexual hybrid Neotyphodium grass endophyte, isolate Lp1. This hybrid Neotyphodium endophyte is an interspecific hybrid between two grass endophytes, Neotyphodium lolii, and a sexual form, Epichl?e typhina, and the length heterogeneity was not found in either of these progenitor species. The length heterogeneity in the hybrid is localized to the intergenic spacer (IGS) and is the result of copy-number variation of a tandemly repeated subrepeat class within the IGS, the 111-/119-bp subrepeats. Copy number variation of this subrepeat class appears to be a consequence of mitotic unequal crossing over that occurs between these subrepeats. This implies that unequal crossing over plays a role in the concerted evolution of the whole rDNA. Changes in the pattern of IGS length variants occurred in just two rounds of single-spore purification. Analysis of the IGS length heterogeneity revealed features that are unexpected in a simple model of unequal crossing over. Potential refinements of the molecular details of unequal crossing over are presented, and we also discuss evidence for a combination of homogenization mechanisms that drive the concerted evolution of the Lp1 rDNA.     

A gene fusion at a homeobox locus: alterations in leaf shape and implications for morphological evolution

Compound leaves are seen in many angiosperm genera and are thought to be either fundamentally different from simple leaves or elaborations of simple leaves. The knotted1-like homeobox (knox) genes are known to regulate plant development. When overexpressed in homologous or heterologous species, this family of genes can cause changes in leaf morphology, including excessive leaf compounding in tomato. We describe here an instance of a spontaneously arisen fusion between a gene encoding a metabolic enzyme and a homeodomain protein. We show that the fusion results in overexpression of the homeodomain protein and a change in morphology that approximates the changes caused by overexpression of the same gene under the control of the cauliflower mosaic virus 35S promoter in transgenic plants. Exon-shuffling events can account for the modularity of proteins. If the shuffled exons are associated with altered promoters, changes in gene expression patterns can result. Our results show that gene fusions of this nature can cause changes in expression patterns that lead to altered morphology. We suggest that such phenomena may have played a role in the evolution of form.     

Antibiotic activity of polyketide products derived from combinatorial biosynthesis: implications for directed evolution

Aspirin and carotid endarterectomy represent the current method of prophylaxis against stroke in patients with symptomatic high grade carotid stenoses. Whilst balloon dilatation and stent placement is an accepted form of recannalization, elsewhere in the arterial circulation the technique is in its infancy within the supra-aortic vessels. Carotid endarterectomy is expensive, requires a general anaesthetic and is associated with an acceptable complication rate as demonstrated by the ECST and NASCET studies. The author's experience in 55 patients with symptomatic high grade atherosclerotic disease is described with a disabling stroke rate at 30 days similar to the surgical series (5%). Long-term follow-up is awaited. Metallic intra-arterial stents provide the opportunity to reduce the immediate postintervention residual stenosis and possibly reduce the long-term restenosis rate. Modifications of the technique are in development and may reduce the cerebral embolization that occurs during the procedure.