Hamiltonian path analysis of viral genomes

Cryo-electron microscopy (EM) is undergoing a revolution, enabling the study of viral pathogens in unprecedented detail. The asymmetric EM reconstruction of bacteriophage MS2 at medium resolution (8.7 Å) by Koning et al.1, and the subsequent reconstruction at even higher resolution (3.6 Å) by Dai et al.2 revealed the structures of both the protein shell and the asym- metric genomic RNA and the unique maturation protein (A). It is the start of a wave of such structural data for viruses, and calls for the development of new analytical tools to describe the results. One approach is Hamiltonian path analysis (HPA) that we introduced to describe repeated, sequence-specific contacts between the MS2 genome and its protein shell3. Here, we describe how HPA is consistent with the new structures and, in turn, how it extends our understanding beyond the structural data alone

Structural and functional analyses of Rubisco from arctic diatom species reveal unusual posttranslational modifications

The catalytic performance of the major CO2-assimilating enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), restricts photosynthetic productivity. Natural diversity in the catalytic properties of Rubisco indicates possibilities for improvement. Oceanic phytoplankton contain some of the most efficient Rubisco enzymes, and diatoms in particular are responsible for a significant proportion of total marine primary production as well as being a major source of CO2 sequestration in polar cold waters. Until now, the biochemical properties and three-dimensional structures of Rubisco from diatoms were unknown. Here, diatoms from Arctic waters were collected, cultivated and analyzed for their CO2 fixing capability. We characterized the kinetic properties of five, and determined the crystal structures of four Rubiscos selected for their high CO2-fixing efficiency. The DNA sequences of the rbcL and rbcS genes of the selected diatoms were similar, reflecting their close phylogenetic relationship. The Vmax and KM for the oxygenase and carboxylase activities at 25°C and the specificity factors (Sc /o) at 15, 25 and 35°C, were determined. The Sc/o values were high, approaching those of mono- and dicot plants, thus exhibiting good selectivity for CO2 relative to O2. Structurally, diatom Rubiscos belong to Form I C/D, containing small subunits characterised by a short βA-βB loop and a carboxy-terminal extension that forms a β- hairpin structure (βE-βF loop). Of note, the diatom Rubiscos featured a number of posttranslational modifications of the large subunit, including 4-hydroxy-proline, betahydroxyleucine, hydroxylated, and nitrosylated cysteine, mono-, and di-hydroxylated lysine, and tri-methylated lysine. Our studies suggest adaptation toward achieving efficient CO2-fixation in Arctic diatom Rubiscos

Structural and functional analyses of Rubisco from arctic diatom species reveal unusual posttranslational modifications

The catalytic performance of the major CO2-assimilating enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), restricts photosynthetic productivity. Natural diversity in the catalytic properties of Rubisco indicates possibilities for improvement. Oceanic phytoplankton contain some of the most efficient Rubisco enzymes, and diatoms in particular are responsible for a significant proportion of total marine primary production as well as being a major source of CO2 sequestration in polar cold waters. Until now, the biochemical properties and three-dimensional structures of Rubisco from diatoms were unknown. Here, diatoms from Arctic waters were collected, cultivated and analyzed for their CO2 fixing capability. We characterized the kinetic properties of five, and determined the crystal structures of four Rubiscos selected for their high CO2-fixing efficiency. The DNA sequences of the rbcL and rbcS genes of the selected diatoms were similar, reflecting their close phylogenetic relationship. The Vmax and KM for the oxygenase and carboxylase activities at 25°C and the specificity factors (Sc/o) at 15, 25 and 35°C, were determined. The Sc/o values were high, approaching those of mono- and dicot plants, thus exhibiting good selectivity for CO2 relative to O2 Structurally, diatom Rubiscos belong to Form I C/D, containing small subunits characterised by a short βA-βB loop and a carboxy-terminal extension that forms a β-hairpin structure (βE-βF loop). Of note, the diatom Rubiscos featured a number of posttranslational modifications of the large subunit, including 4-hydroxy-proline, betahydroxyleucine, hydroxylated, and nitrosylated cysteine, mono-, and di-hydroxylated lysine, and tri-methylated lysine. Our studies suggest adaptation toward achieving efficient CO2-fixation in Arctic diatom Rubiscos

Asymmetric Genome Organization in an RNA Virus Revealed via Graph-Theoretical Analysis of Tomographic Data

Cryo-electron microscopy permits 3-D structures of viral pathogens to be determined in remarkable detail. In particular, the protein containers encapsulating viral genomes have been determined to high resolution using symmetry averaging techniques that exploit the icosahedral architecture seen in many viruses. By contrast, structure determination of asymmetric components remains a challenge, and novel analysis methods are required to reveal such features and characterize their functional roles during infection. Motivated by the important, cooperative roles of viral genomes in the assembly of single-stranded RNA viruses, we have developed a new analysis method that reveals the asymmetric structural organization of viral genomes in proximity to the capsid in such viruses. The method uses geometric constraints on genome organization, formulated based on knowledge of icosahedrally-averaged reconstructions and the roles of the RNA-capsid protein contacts, to analyse cryo-electron tomographic data. We apply this method to the low-resolution tomographic data of a model virus and infer the unique asymmetric organization of its genome in contact with the protein shell of the capsid. This opens unprecedented opportunities to analyse viral genomes, revealing conserved structural features and mechanisms that can be targeted in antiviral drug desig

Mechanical and Assembly Units of Viral Capsids Identified via Quasi-Rigid Domain Decomposition

Key steps in a viral life-cycle, such as self-assembly of a protective protein container or in some cases also subsequent maturation events, are governed by the interplay of physico-chemical mechanisms involving various spatial and temporal scales. These salient aspects of a viral life cycle are hence well described and rationalised from a mesoscopic perspective. Accordingly, various experimental and computational efforts have been directed towards identifying the fundamental building blocks that are instrumental for the mechanical response, or constitute the assembly units, of a few specific viral shells. Motivated by these earlier studies we introduce and apply a general and efficient computational scheme for identifying the stable domains of a given viral capsid. The method is based on elastic network models and quasi-rigid domain decomposition. It is first applied to a heterogeneous set of well-characterized viruses (CCMV, MS2, STNV, STMV) for which the known mechanical or assembly domains are correctly identified. The validated method is next applied to other viral particles such as L-A, Pariacoto and polyoma viruses, whose fundamental functional domains are still unknown or debated and for which we formulate verifiable predictions. The numerical code implementing the domain decomposition strategy is made freely available

Towards new β-lactam antibiotics

Antibiotics have had a profound impact on human health and belong to one of the largest-selling classes of drugs worldwide. Introduced into industrial production only some half century ago, these miracle drugs have been the main contributors to the recent increase in human life expectancy. However, the accelerated emergence of bacteria that are resistant to multiple antibiotic types now appears as the most serious threat to continuing success in the treatment of infectious diseases. Recent advances in our knowledge of the structures and mechanisms of enzymes in the biosynthetic pathways of penicillins and cephalosporins, amongst the most important antibiotics in current use, have identified a common structural core together with common iron- and cosubstrate-binding motifs. The diversity in the catalytic specificities of these oxygenases using very similar structural platforms suggests that altering the substrate and product specificities of these enzymes should be possible in the laboratory. This opens up new avenues for industrial production and medical utilisation.

Crystal structures of an oligopeptide-binding protein from the biosynthetic pathway of the beta-lactamase inhibitor clavulanic acid.

Clavulanic acid (CA) is a clinically important beta-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 A resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis

Probing sequence-specific RNA recognition by the bacteriophage MS2 coat protein.

We present the results of in vitro binding studies aimed at defining the key recognition elements on the MS2 RNA translational operator (TR) essential for complex formation with coat protein. We have used chemically synthesized operators carrying modified functional groups at defined nucleotide positions, which are essential for recognition by the phage coat protein. These experiments have been complemented with modification-binding interference assays. The results confirm that the complexes which form between TR and RNA-free phage capsids, the X-ray structure of which has recently been reported at 3.0 A, are identical to those which form in solution between TR and a single coat protein dimer. There are also effects on operator affinity which cannot be explained simply by the alteration of direct RNA-protein contacts and may reflect changes in the conformational equilibrium of the unliganded operator. The results also provide support for the approach of using modified oligoribonucleotides to investigate the details of RNA-ligand interactions