A PTIP-PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex.

Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP-PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities.

Molecular Basis of the Mechanisms Controlling MASTL.

The human MASTL (Microtubule-associated serine/threonine kinase-like) gene encodes an essential protein in the cell cycle. MASTL is a key factor preventing early dephosphorylation of M-phase targets of Cdk1/CycB. Little is known about the mechanism of MASTL activation and regulation. MASTL contains a non-conserved insertion of 550 residues within its activation loop, splitting the kinase domain, and making it unique. Here, we show that this non-conserved middle region (NCMR) of the protein is crucial for target specificity and activity. We performed a phosphoproteomic assay with different MASTL constructs identifying key phosphorylation sites for its activation and determining whether they arise from autophosphorylation or exogenous kinases, thus generating an activation model. Hydrogen/deuterium exchange data complements this analysis revealing that the C-lobe in full-length MASTL forms a stable structure, whereas the N-lobe is dynamic and the NCMR and C-tail contain few localized regions with higher-order structure. Our results indicate that truncated versions of MASTL conserving a cryptic C-Lobe in the NCMR, display catalytic activity and different targets, thus establishing a possible link with truncated mutations observed in cancer-related databases.

Large-Scale Biophysical Evaluation of Protein PEGylation Effects: In Vitro Properties of 61 Protein Entities.

PEGylation is the most widely used method to chemically modify protein biopharmaceuticals, but surprisingly limited public data is available on the biophysical effects of protein PEGylation. Here we report the first large-scale study, with site-specific mono-PEGylation of 15 different proteins and characterization of 61 entities in total using a common set of analytical methods. Predictions of molecular size were typically accurate in comparison with actual size determined by size-exclusion chromatography (SEC) or dynamic light scattering (DLS). In contrast, there was no universal trend regarding the effect of PEGylation on the thermal stability of a protein based on data generated by circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorimetry (DSF). In addition, DSF was validated as a fast and inexpensive screening method for thermal unfolding studies of PEGylated proteins. Multivariate data analysis revealed clear trends in biophysical properties upon PEGylation for a subset of proteins, although no universal trends were found. Taken together, these findings are important in the consideration of biophysical methods and evaluation of second-generation biopharmaceutical drug candidates.

Functional and structural properties of a novel protein and virulence factor (Protein sHIP) in Streptococcus pyogenes.

Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.

Non-linear effects of macromolecular crowding on enzymatic activity of multi-copper oxidase.

Enzymes catalyze biochemical reactions in highly crowded environments where the amount of macromolecules may occupy up to 40% of the volume. Here we report how cell-like conditions tune catalytic parameters for the monomeric multi-copper oxidase, Saccharomyces cerevisiae Fet3p, in vitro. At low amounts of crowding agent, we detect increases in both of K(M) (weaker substrate binding) and k(cat) (improved catalytic efficiency), whereas at higher crowding levels, both parameters were reduced. Presence of crowding agents does not affect Fet3p structural content but increases thermal resistance. The observations are compatible with ordering of a non-optimal substrate-binding site and restricted internal dynamics as a result of excluded volume effects making the protein less structurally 'strained'.

Structure of levitated Si-Ge melts studied by high-energy x-ray diffraction in combination with reverse Monte Carlo simulations.

The short-range order in liquid Si, Ge and binary Six-Ge1-xalloys (x= 0.25, 0.50, 0.75) was studied by x-ray diffraction and reverse Monte Carlo simulations. Experiments were performed in the normal and supercooled liquid states by using the containerless technique of aerodynamic levitation with CO2laser heating, enabling deeper supercooling of liquid Si and Si-Ge alloys than previously reported. The local atomic structure of liquid Si and Ge resembles theß-tin structure. The first coordination numbers of about 6 for all compositions are found to be independent of temperature indicating the supercooled liquids studied retain this high-density liquid (HDL) structure. However, there is evidence of developing local tetrahedral ordering, as manifested by a shoulder on the right side of the first peak inS(Q) which becomes more prominent with increasing supercooling. This result is potentially indicative of a continuous transition from the stable HDLß-tin (high pressure) phase, towards a metastable low-density liquid phase, reminiscent of the diamond (ambient pressure) structure.

Liquid-liquid phase transition in supercooled yttria-alumina.

The structure and thermal characteristics of aerodynamically levitated samples of yttria-alumina in the liquid, supercooled liquid and solid phases were explored in an extensive series of high energy x-ray diffraction, small angle neutron scattering, and pyrometric cooling measurements. Particular focus was placed on the compound (Y2O3)(x)(Al2O3)(1-x) with x = 0.2 for which a liquid-liquid phase transition at a temperature of 1788 K has recently been reported. No structural or thermal signature in support of this metastable phase transition could be found.

Fragile X mental retardation syndrome: structure of the KH1-KH2 domains of fragile X mental retardation protein.

Fragile X syndrome is the most common form of inherited mental retardation in humans, with an estimated prevalence of about 1 in 4000 males. Although several observations indicate that the absence of functional Fragile X Mental Retardation Protein (FMRP) is the underlying basis of Fragile X syndrome, the structure and function of FMRP are currently unknown. Here, we present an X-ray crystal structure of the tandem KH domains of human FMRP, which reveals the relative orientation of the KH1 and KH2 domains and the location of residue Ile304, whose mutation to Asn is associated with a particularly severe incidence of Fragile X syndrome. We show that the Ile304Asn mutation both perturbs the structure and destabilizes the protein.

Structure of Csx1-cOA4 complex reveals the basis of RNA decay in Type III-B CRISPR-Cas.

Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA4 complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA4 binding site and a ssRNA catalytic pocket. cOA4 undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA4 and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5'-C-C-3'. The degradation of cOA4 by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications.

Chemical short-range order in liquid Al-Ni alloys.

The chemical short-range structure was studied in liquid Al-Ni alloys by x-ray absorption spectroscopy as a function of temperature and composition. A containerless technique, combining aerodynamic levitation and inductive heating, was used to position and melt the samples. The fluorescence yield x-ray absorption at the Ni K edge was measured by a multichannel solid-state Ge detector. The number of heteroatomic pairs around the scatterer is higher than for a homogeneous alloy.

Molecular basis of Tousled-Like Kinase 2 activation.

Tousled-like kinases (TLKs) are required for genome stability and normal development in numerous organisms and have been implicated in breast cancer and intellectual disability. In humans, the similar TLK1 and TLK2 interact with each other and TLK activity enhances ASF1 histone binding and is inhibited by the DNA damage response, although the molecular mechanisms of TLK regulation remain unclear. Here we describe the crystal structure of the TLK2 kinase domain. We show that the coiled-coil domains mediate dimerization and are essential for activation through ordered autophosphorylation that promotes higher order oligomers that locally increase TLK2 activity. We show that TLK2 mutations involved in intellectual disability impair kinase activity, and the docking of several small-molecule inhibitors of TLK activity suggest that the crystal structure will be useful for guiding the rationale design of new inhibition strategies. Together our results provide insights into the structure and molecular regulation of the TLKs.

Approaching the speed limit for Greek Key beta-barrel formation: transition-state movement tunes folding rate of zinc-substituted azurin.

Azurin is a blue-copper protein with a beta-barrel structure of Greek Key topology. In vitro, copper can be substituted with zinc without change in protein structure. We here analyze the kinetic folding behavior of zinc-substituted Pseudomonas aeruginosa azurin. Our findings can be summarized in three key conclusions: first, zinc remains strongly bound to the polypeptide upon unfolding, suggesting that the cofactor may bind to the protein before polypeptide folding in vivo. Second, the semi-logarithmic plot of folding and unfolding rates for zinc-substituted azurin as a function of denaturant concentration exhibits curvature due to a changing transition-state structure. Third, the extrapolated folding speed in water for zinc-substituted azurin is similar to that of other proteins with the same topology, implying that there is a speed limit that can be modulated by stability-driven transition-state movement for formation of beta-barrel structures with Greek Key topology.

New insights into Fragile X syndrome. Relating genotype to phenotype at the molecular level.

Lack of functional Fragile X mental retardation protein (FMRP) is the primary cause of the Fragile-mental retardation syndrome in humans. In most cases, the disease results from transcriptional silencing of fragile mental retardation gene 1, fmr1, which encodes FMRP. However, a single missense mutation (I304N) in the second KH domain of FMRP gives rise to a particularly severe case of Fragile X syndrome. A Drosophila homolog of FMRP has been identified, Drosophila Fragile X related protein (dFXRP). The corresponding missense mutation in dFXRP, the I307N, has pronounced effects on the in vivo activity of the protein. The effect of the point mutation on the structure and function of FMRP is unclear, and published data are contradictory. No in vitro structural or stability studies have been performed on dFXRP. Here we show that a construct that contains only the tandem KH1-KH2 domains is a stable, well-folded unit suitable for detailed structural and functional characterization. Using this KH1-KH2 construct we explicitly test a hypothesis that has been proposed to explain the effect of the Ile-->Asn mutation: that it causes complete unfolding of the protein. Here we show that the I307N point mutation does not completely unfold the KH domain. The KH1-KH2 construct bearing I307N substitution is stable in isolation and adopts a native-like fold. Thus our data favor alternative explanations for the in vivo observed loss of dFXRP activity associated with I307N mutation: (a) the point mutation might affect intra and/or inter-molecular interactions of dFXRP; or (b) it might impair dFXRP's interactions with its RNA target(s).

Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose).

Intrinsically disordered proteins can phase separate from the soluble intracellular space, and tend to aggregate under pathological conditions. The physiological functions and molecular triggers of liquid demixing by phase separation are not well understood. Here we show in vitro and in vivo that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing. PAR levels are markedly induced at sites of DNA damage, and we provide evidence that PAR-seeded liquid demixing results in rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites. Demixing, which relies on electrostatic interactions between positively charged RGG repeats and negatively charged PAR, is amplified by aggregation-prone prion-like domains, and orchestrates the earliest cellular responses to DNA breakage. We propose that PAR-seeded liquid demixing is a general mechanism to dynamically reorganize the soluble nuclear space with implications for pathological protein aggregation caused by derailed phase separation.

If space is provided, bulky modification on the rim of azurin's beta-barrel results in folded protein.

Pseudomonas aeruginosa azurin is a blue-copper protein with a beta-barrel fold. Here we report that, at conditions where thermal unfolding of apo-azurin is reversible, the reaction occurs in a single step with a transition midpoint (T(m)) of 69 degrees C (pH 7). The active-site mutation His117Gly creates a cavity in the beta-barrel near the surface but does not perturb the overall fold (T(m) of 64 degrees C, pH 7). Oxidation of the active-site cysteine (Cysteine-112) in wild-type azurin, which occurs readily at higher temperatures, results in a modified protein that cannot adopt a native-like structure. In sharp contrast, Cysteine-112 oxidation in His117Gly azurin yields a modified apo-azurin that appears folded and displays cooperative, reversible unfolding (T(m) approximately 55 degrees C, pH 7). We conclude that azurin's beta-barrel is a rigid structural element that constrains the structure of its surface; a bulky modification can only be accommodated if complementary space is provided.

The impact of structural integrity and route of administration on the antibody specificity against three cow's milk allergens - a study in Brown Norway rats.

BACKGROUND: Characterisation of the specific antibody response, including the epitope binding pattern, is an essential task for understanding the molecular mechanisms of food allergy. Examination of antibody formation in a controlled environment requires animal models. The purpose of this study was to examine the amount and types of antibodies raised against three cow's milk allergens; ß-lactoglobulin (BLG), α-lactalbumin (ALA) and ß-casein upon oral or intraperitoneal (i.p.) administration. A special focus was given to the relative amount of antibodies raised against linear versus conformational epitopes. METHODS: Specific antibodies were raised in Brown Norway (BN) rats. BN rats were dosed either (1) i.p. with the purified native cow's milk allergens or (2) orally with skimmed milk powder (SMP) alone or together with gluten, without the use of adjuvants. The allergens were denatured by reduction and alkylation, resulting in unfolding of the primary structure and a consequential loss of conformational epitopes. The specific IgG1 and IgE responses were analysed against both the native and denatured form of the three cow's milk allergens, thus allowing examination of the relative amount of linear versus conformational epitopes. RESULTS: The inherent capacity to induce specific IgG1 and IgE antibodies were rather similar upon i.p. administration for the three cow's milk allergens, with BLG = ALA > ß-casein. Larger differences were found between the allergens upon oral administration, with BLG > ALA > ß-casein. Co-administration of SMP and gluten had a great impact on the specific antibody response, resulting in a significant reduced amount of antibodies. Together results indicated that most antibodies were raised against conformational epitopes irrespectively of the administration route, though the relative proportions between linear and conformational epitopes differed remarkably between the allergens. CONCLUSIONS: This study showed that the three-dimensional (3D) structure has a significant impact on the antibodies raised for both systemic and orally administered allergens. A remarkable difference in the antibody binding patterns against linear and conformational epitope was seen between the allergens, indicating that the structural characteristics of proteins may heavily affect the induced antibody response.

Identification of differentially expressed proteins in ovarian cancer using high-density protein microarrays.

Ovarian cancer is a leading cause of deaths, yet many aspects of the biology of the disease and a routine means of its detection are lacking. We have used protein microarrays and autoantibodies from cancer patients to identify proteins that are aberrantly expressed in ovarian tissue. Sera from 30 cancer patients and 30 healthy individuals were used to probe microarrays containing 5,005 human proteins. Ninety-four antigens were identified that exhibited enhanced reactivity from sera in cancer patients relative to control sera. The differential reactivity of four antigens was tested by using immunoblot analysis and tissue microarrays. Lamin A/C, SSRP1, and RALBP1 were found to exhibit increased expression in the cancer tissue relative to controls. The combined signals from multiple antigens proved to be a robust test to identify cancerous ovarian tissue. These antigens were also reactive with tissue from other types of cancer and thus are not specific to ovarian cancer. Overall our studies identified candidate tissue marker proteins for ovarian cancer and demonstrate that protein microarrays provide a powerful approach to identify proteins aberrantly expressed in disease states.

Studies of Pseudomonas aeruginosa azurin mutants: cavities in beta-barrel do not affect refolding speed.

Pseudomonas aeruginosa azurin is a blue-copper protein with a Greek-key fold. Removal of copper produces an apoprotein with the same structure as holoazurin. To address the effects on thermodynamic stability and folding dynamics caused by small cavities in a beta-barrel, we have studied the behavior of the apo-forms of wild-type and two mutant (His-46-Gly and His-117-Gly) azurins. The equilibrium- and kinetic-folding and unfolding reactions appear as two-state processes for all three proteins. The thermodynamic stability of the two mutants is significantly decreased as compared with the stability of wild-type azurin, in accord with cavities in or near the hydrophobic interior having an overall destabilizing effect. Large differences are also found in the unfolding rates: the mutants unfold much faster than wild-type azurin. In contrast, the folding-rate constants are almost identical for the three proteins and closely match the rate-constant predicted from the native-state topology of azurin. We conclude that the topology is more important than equilibrium stability in determining the folding speed of azurin.

Methionine-121 coordination determines metal specificity in unfolded Pseudomonas aeruginosa azurin.

Pseudomonas aeruginosa azurin binds copper so tightly that it remains bound even upon polypeptide unfolding. Copper can be substituted with zinc without change in protein structure, and also in this complex the metal remains bound upon protein unfolding. Previous work has shown that native-state copper ligands Cys112 and His117 are two of at least three metal ligands in the unfolded state. In this study we use isothermal titration calorimetry and spectroscopic methods to test if the native-state ligand Met121 remains a metal ligand upon unfolding. From studies on a point-mutated version of azurin (Met121Ala) and a set of model peptides spanning the copper-binding C-terminal part (including Cys112, His117 and Met121), we conclude that Met121 is a metal ligand in unfolded copper-azurin but not in the case of unfolded zinc-azurin. Combination of unfolding and metal-titration data allow for determination of copper (Cu(II) and Cu(I)) and zinc affinities for folded and unfolded azurin polypeptides, respectively.