Superantigen architecture: Functional decoration on a conserved scaffold

A defining and consistent feature of the bacterial superantigens from Staphylococcus aureus and Streptococcus pyogenes is their strongly conserved three-dimensional structure. Structural studies to date show that the array of more than 280 amino acid sequences known for superantigens (SAgs) and staphylococcal superantigen-like (SSL) proteins all have the same fold-a structure in which the same three-dimensional arrangement of α-helices and β-sheets is traced by each amino acid sequence, with the same topology (for recent reviews, see references 29 and 43). A typical SAg structure comprises two domains-an N-terminal β -barrel domain called an OB-fold (4, 25) and a C-terminal β-grasp domain in which a long α-helix packs on to a mixed parallel and antiparallel β-sheet. These two domains are traversed by an α-helix that lies at the N terminus of the protein and packs against the β-grasp domain, thus linking the N- and C-terminal domains

Infinite stacking of alternating polyfluoroaryl rings and bromide anions

The crystal structure of 1-(4-bromo-2,3,5,6-tetrafluorophenyl)-3-benzylimidazolium bromide comprises columns of parallel bromotetrafluorophenyl rings with an interplanar distance of 6.936(6) Å separated by bromide anions

Expression and purification of an adenylation domain from a eukaryotic nonribosomal peptide synthetase: Using structural genomics tools for a challenging target

Nonribosomal peptide synthetases (NRPSs) are large multimodular and multidomain enzymes that are involved in synthesising an array of molecules that are important in human and animal health. NRPSs are found in both bacteria and fungi but most of the research to date has focused on the bacterial enzymes. This is largely due to the technical challenges in producing active fungal NRPSs, which stem from their large size and multidomain nature. In order to target fungal NRPS domains for biochemical and structural characterisation, we tackled this challenge by using the cloning and expression tools of structural genomics to screen the many variables that can influence the expression and purification of proteins. Using these tools we have screened 32 constructs containing 16 different fungal NRPS domains or domain combinations for expression and solubility. Two of these yielded soluble protein with one, the third adenylation domain of the SidN NRPS (SidNA3) from the grass endophyte Neotyphodium lolii, being tractable for purification using Ni-affinity resin. The initial purified protein exhibited poor solution behaviour but optimisation of the expression construct and the buffer conditions used for purification, resulted in stable recombinant protein suitable for biochemical characterisation, crystallisation and structure determination

Structural analysis of the GH43 enzyme Xsa43E from Butyrivibrio proteoclasticus

The rumen of dairy cattle can be thought of as a large, stable fermentation vat and as such it houses a large and diverse community of microorganisms. The bacterium Butyrivibrio proteoclasticus is a representative of a significant component of this microbial community. It is a xylan-degrading organism whose genome encodes a large number of open reading frames annotated as fibre-degrading enzymes. This suite of enzymes is essential for the organism to utilize the plant material within the rumen as a fuel source, facilitating its survival in this competitive environment. Xsa43E, a GH43 enzyme from B. proteoclasticus, has been structurally and functionally characterized. Here, the structure of selenomethionine-derived Xsa43E determined to 1.3 Å resolution using single-wavelength anomalous diffraction is reported. Xsa43E possesses the characteristic five-bladed β-propeller domain seen in all GH43 enzymes. GH43 enzymes can have a range of functions, and the functional characterization of Xsa43E shows it to be an arabinofuranosidase capable of cleaving arabinose side chains from short segments of xylan. Full functional and structural characterization of xylan-degrading enzymes will aid in creating an enzyme cocktail that can be used to completely degrade plant material into simple sugars. These molecules have a range of applications as starting materials for many industrial processes, including renewable alternatives to fossil fuels

Solving the structure of the mycobacterial chromosome condensing protein Lsr2 in complex with DNA

Lsr2 is a DNA binding protein that is highly conserved in mycobacteria and related actinomycetes and it is thought to be essential in Mycobacterium tuberculosis. Previous studies have shown that Lsr2 is involved in down-regulating a range of genes involved in cell wall synthesis and metabolic functions and it is proposed that it does this by organisation of bacterial chromatin. We solved the structure of the N-terminal dimerisation domain of Lsr2 using crystallographic ab initio approaches1 whereas the C-terminal DNA binding domain structure was solved by others using NMR2. Electron microscopy shows that Lsr2 organises DNA into large helical structures involving several strands1. Whilst DNA binding by individual domains has been modelled based on the NMR structure, the exact mechanism of DNA binding and chromosome organisation by the entire protein is unknown. Lsr2 contains a long flexible loop between the two domains which may lead to the protein having a large range of movement, allowing it to bind DNA in a dynamic way. The Lsr2 dimer is also capable of forming linear oligomers through the interaction of overlapping N terminal residues and evidence of this has been shown in our crystal structure and using TEM1. We wish to solve the structure of Lsr2 bound to DNA. For the purposes of crystallisation of Lsr2 we have engineered the removal of important N-terminal residues involved in oligomersiation to prevent this process occurring in solution. The truncated form of Lsr2 provides a sub-population of purified Lsr2 that is DNA-free and we have utilised this population for binding specific dsDNA oligonucleotides for crystallisation. To date, we have determined the optimal length of DNA required and have refined the exact combination of nucleotides for ideal protein binding. We have progressed through the crystallisation of Lsr2 bound to a range of oligonucleotides. One oligonucleotide has yielded the most success to date with crystals that have diffracted to 2.7 Å resolution. Attempts to solve the structure using molecular replacement have thus far been unsuccessful. Our current focus is to reproduce crystals for improving resolution and for heavy atom soaking to assist with solving the structure

Enzyme evolution: innovation is easy, optimization is complicated

Enzymes have been evolving to catalyze new chemical reactions for billions of years, and will continue to do so for billions more. Here, we review examples in which evolutionary biochemists have used big data and high-throughput experimental tools to shed new light on the enormous functional diversity of extant enzymes, and the evolutionary processes that gave rise to it. We discuss the role that gene loss has played in enzyme evolution, as well as the more familiar processes of gene duplication and divergence. We also review insightful studies that relate not only catalytic activity, but also a host of other biophysical and cellular parameters, to organismal fitness. Finally, we provide an updated perspective on protein engineering, based on our new-found appreciation that most enzymes are sloppy and mediocre

Reconstructed Ancestral Enzymes Impose a Fitness Cost upon Modern Bacteria Despite Exhibiting Favourable Biochemical Properties

Ancestral sequence reconstruction has been widely used to study historical enzyme evolution, both from biochemical and cellular perspectives. Two properties of reconstructed ancestral proteins/enzymes are commonly reported—high thermostability and high catalytic activity—compared with their contemporaries. Increased protein stability is associated with lower aggregation rates, higher soluble protein abundance and a greater capacity to evolve, and therefore, these proteins could be considered “superior” to their contemporary counterparts. In this study, we investigate the relationship between the favourable in vitro biochemical properties of reconstructed ancestral enzymes and the organismal fitness they confer in vivo. We have previously reconstructed several ancestors of the enzyme LeuB, which is essential for leucine biosynthesis. Our initial fitness experiments revealed that overexpression of ANC4, a reconstructed LeuB that exhibits high stability and activity, was only able to partially rescue the growth of a ΔleuB strain, and that a strain complemented with this enzyme was outcompeted by strains carrying one of its descendants. When we expanded our study to include five reconstructed LeuBs and one contemporary, we found that neither in vitro protein stability nor the catalytic rate was correlated with fitness. Instead, fitness showed a strong, negative correlation with estimated evolutionary age (based on phylogenetic relationships). Our findings suggest that, for reconstructed ancestral enzymes, superior in vitro properties do not translate into organismal fitness in vivo. The molecular basis of the relationship between fitness and the inferred age of ancestral LeuB enzymes is unknown, but may be related to the reconstruction process. We also hypothesise that the ancestral enzymes may be incompatible with the other, contemporary enzymes of the metabolic network.France. Agence nationale de la recherch

Editorial: Biotechnological Uses of Archaeal Proteins

Many industrial/biotechnological processes take place under extreme conditions of temperature, pH, salinity, or pressure which are not suitable for activities of proteins from model eukaryotic or common neutrophilic, mesophilic, and prokaryotic microorganisms. In contrast, Archaea offer a large panel of extremophile organisms that express proteins that are able to remain properly folded and functional under the harshest biophysical conditions

The Structure of the Oligomerization Domain of Lsr2 from Mycobacterium tuberculosis Reveals a Mechanism for Chromosome Organization and Protection

Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNA-binding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal cross-linking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 Å resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ß-ß-a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel β-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated