Identifying protein construct variants with increased crystallization propensity--a case study

This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity.     

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.     

The refined 2.0 A X-ray crystal structure of the complex formed between bovine beta-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes

The stoichiometric complex formed between bovine beta-trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI-I) was crystallized and its X-ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0-2.0 A). CMTI-I is of ellipsoidal shape; it lacks helices or beta-sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS-3I-20I, Cys-10I-22I and Cys-16I-28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val-2I (P4)-Glu-9I (P4') which contains the reactive site bond Arg-5I-Ile-6I and is in a conformation observed also for other serine proteinase inhibitors.     

Proteomics of light-harvesting proteins in different plant species. Analysis and comparison by liquid chromatography-electrospray ionization mass spectrometry. Photosystem I

The light-harvesting proteins (Lhca) of photosystem I (PSI) from four monocot and five dicot species were extracted from plant material, separated by reversed-phase high-performance liquid chromatography (HPLC) and subsequently identified on the basis of their intact molecular masses upon on-line hyphenation with electrospray ionization mass spectrometry. Although their migration behavior in gel electrophoresis was very similar, the elution times among the four antenna types in reversed-phase-HPLC differed significantly, even more than those observed for the light-harvesting proteins of photosystem II. Identification of proteins is based on the good agreement between the measured intact molecular masses and the values calculated on the basis of their nucleotide-derived amino acid sequences, which makes the intact molecular masses applicable as intact mass tags. These values match excellently for Arabidopsis, most probably because of the availability of high-quality DNA sequence data. In all species examined, the four antennae eluted in the same order, namely Lhca1 > Lhca3 > Lhca4 > Lhca2. These characteristic patterns enabled an unequivocal assignment of the proteins in preparations from different species. Interestingly, in all species examined, Lhca1 and Lhca2 were present in two or three isoforms. A fifth antenna protein, corresponding to the Lhca6 gene, was found in tomato (Lycopersicon esculentum). However PSI showed a lower heterogeneity than photosystem II. In most plant species, Lhca2 and Lhca4 proteins are the most abundant PSI antenna proteins. The HPLC method used in this study was found to be highly reproducible, and the chromatograms may serve as a highly confident fingerprint for comparison within a single and among different species for future studies of the PSI antenna.     

Scaling of feeding biomechanics in the horn shark Heterodontus francisci: ontogenetic constraints on durophagy

Organismal performance changes over ontogeny as the musculoskeletal systems underlying animal behavior grow in relative size and shape. As performance is a determinant of feeding ecology, ontogenetic changes in the former can influence the latter. The horn shark Heterodontus francisci consumes hard-shelled benthic invertebrates, which may be problematic for younger animals with lower performance capacities. Scaling of feeding biomechanics was investigated in H. francisci (n=16, 19–59 cm standard length (SL)) to determine the biomechanical basis of allometric changes in feeding performance and whether this performance capacity constrains hard-prey consumption over ontogeny. Positive allometry of anterior (8–163 N) and posterior (15–382 N) theoretical bite force was attributed to positive allometry of cross-sectional area in two jaw adducting muscles and mechanical advantage at the posterior bite point (0.79–1.26). Mechanical advantage for anterior biting scaled isometrically (0.52). Fracture forces for purple sea urchins Strongylocentrotus purpuratus consumed by H. francisci ranged from 24 to 430 N. Comparison of these fracture forces to the bite force of H. francisci suggests that H. francisci is unable to consume hard prey early in its life history, but can consume the majority of S. purpuratus by the time it reaches maximum size. Despite this constraint, positive allometry of biting performance appears to facilitate an earlier entry into the durophagous niche than would an isometric ontogenetic trajectory. The posterior gape of H. francisci is significantly smaller than the urchins capable of being crushed by its posterior bite force. Thus, the high posterior bite forces of H. francisci cannot be fully utilized while consuming prey of similar toughness and size to S. purpuratus, and its potential trophic niche is primarily determined by anterior biting capacity.     

Characterization of the Pivotal Carbon Metabolism of Streptococcus suis Serotype 2 under ex Vivo and Chemically Defined in Vitro Conditions by Isotopologue Profiling

Streptococcus suis is a neglected zoonotic pathogen that has to adapt to the nutritional requirements in the different host niches encountered during infection and establishment of invasive diseases. To dissect the central metabolic activity of S. suis under different conditions of nutrient availability, we performed labeling experiments starting from [¹³C]glucose specimens and analyzed the resulting isotopologue patterns in amino acids of S. suis grown under in vitro and ex vivo conditions. In combination with classical growth experiments, we found that S. suis is auxotrophic for Arg, Gln/Glu, His, Leu, and Trp in chemically defined medium. De novo biosynthesis was shown for Ala, Asp, Ser, and Thr at high rates and for Gly, Lys, Phe, Tyr, and Val at moderate or low rates, respectively. Glucose degradation occurred mainly by glycolysis and to a minor extent by the pentose phosphate pathway. Furthermore, the exclusive formation of oxaloacetate by phosphoenolpyruvate (PEP) carboxylation became evident from the patterns in de novo synthesized amino acids. Labeling experiments with S. suis grown ex vivo in blood or cerebrospinal fluid reflected the metabolic adaptation to these host niches with different nutrient availability; however, similar key metabolic activities were identified under these conditions. This points at the robustness of the core metabolic pathways in S. suis during the infection process. The crucial role of PEP carboxylation for growth of S. suis in the host was supported by experiments with a PEP carboxylase-deficient mutant strain in blood and cerebrospinal fluid.     

Ethylene Glycol Metabolism by Pseudomonas putida

In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.     

The whereabouts of an ancient wanderer: global phylogeography of the solitary ascidian Styela plicata

Genetic tools have greatly aided in tracing the sources and colonization history of introduced species. However, recurrent introductions and repeated shuffling of populations may have blurred some of the genetic signals left by ancient introductions. Styela plicata is a solitary ascidian distributed worldwide. Although its origin remains unclear, this species is believed to have spread worldwide by travelling on ship's hulls. The goals of this study were to infer the genetic structure and global phylogeography of S. plicata and to look for present-day and historical genetic patterns. Two genetic markers were used: a fragment of the mitochondrial gene Cytochrome Oxidase subunit I (COI) and a fragment of the nuclear gene Adenine Nucleotide Transporter/ADP-ATP Translocase (ANT). A total of 368 individuals for COI and 315 for ANT were sequenced from 17 locations worldwide. The levels of gene diversity were moderate for COI to high for ANT. The Mediterranean populations showed the least diversity and allelic richness for both markers, while the Indian, Atlantic and Pacific Oceans had the highest gene and nucleotide diversities. Network and phylogenetic analyses with COI and ANT revealed two groups of alleles separated by 15 and 4 mutational steps, respectively. The existence of different lineages suggested an ancient population split. However, the geographic distributions of these groups did not show any consistent pattern, indicating different phylogeographic histories for each gene. Genetic divergence was significant for many population-pairs irrespective of the geographic distance among them. Stochastic introduction events are reflected in the uneven distribution of COI and ANT allele frequencies and groups among many populations. Our results confirmed that S. plicata has been present in all studied oceans for a long time, and that recurrent colonization events and occasional shuffling among populations have determined the actual genetic structure of this species.     

Differential crosstalk between P2X7 and arachidonic acid in activation of mitogen-activated protein kinases

Accumulating evidence indicates that astroglial syncytium plays key role in normal and pathological brain functions. Astrocytes both in vitro and in situ respond to extracellular adenine-based nucleotides via the activation of P2 receptors. Massive release of ATP from neurons and glial cells occurs as a result of pathological conditions of the brain leading to neuroinflammation and involving P2X7 receptors. In this study, we investigated whether P2X7 stimulation on cultured cortical astrocytes promoted a differential activation of mitogen-activated protein kinases (MAPKs), and whether the second messenger arachidonic acid (AA), which is also a key modulator of neuroinflammation, affected the P2X7-mediated MAPK phosphorylation. The results show that the synthetic P2X7 receptor agonist 2′,3′-O-(4-benzoyl)benzoyl-ATP (BzATP), induced a concentration-dependent phosphorylation of MAPK ERK1/2, JNK and p38. Stimulation of ERK1/2, JNK and p38 phosphorylation was also obtained by pathophysiological levels of extracellularly applied AA. Interestingly, a robust potentiation of ERK1/2 phosphorylation was elicited by co-application of BzATP and AA, whereas no differences were observed in JNK or p38 phosphosignals. The kinases activation showed a differential dependence on the presence of extracellular Ca²⁺. The potentiation of BzATP-mediated ERK1/2 phosphorylation was also observed in human embryonic kidney cells (HEK293) stably transfected with rat P2X7, but not in HEK cells expressing truncated P2X7 receptor lacking the full cytoplasmic carboxy-terminal or in those carrying the structurally related rat P2X2. AA and BzATP synergism in ERK1/2 activation was abolished by cyclo-oxygenase and lipoxygenase pathway inhibitors.The result that ERK1/2-mediated transduction pathway is synergistically modulated by ATP and AA signalling depicts possible novel pharmacological targets for interfering with pathological activation of astroglial cells.     

Incorporation of beta-selenolo[3,2-b]pyrrolyl-alanine into proteins for phase determination in protein X-ray crystallography

beta-Selenolo[3,2-b]pyrrolyl-L-alanine that mimics tryptophan with the benzene ring of the indole moiety replaced by selenophene, was incorporated into human annexin V and barstar. This was achieved by fermentation and expression in a Trp-auxotrophic Escherichia coli host strain using the selective pressure incorporation method. The seleno- proteins were obtained in yields comparable to those of the wild-type proteins and exhibit full crystallographic isomorphism to the parent proteins, but expectedly show altered absorbance profiles and quenched tryptophan fluorescence. Since the occurrence of tryptophan residues in proteins is rare, incorporation of the electron-rich selenium-containing tryptophan surrogate into proteins represents a useful supplementation and even a promising novel alternative to selenomethionine for solving the phase problem in protein X-ray crystallography.