Diamond Blackfan anemia is mediated by hyperactive Nemo-like kinase.

Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome associated with ribosomal gene mutations that lead to ribosomal insufficiency. DBA is characterized by anemia, congenital anomalies, and cancer predisposition. Treatment for DBA is associated with significant morbidity. Here, we report the identification of Nemo-like kinase (NLK) as a potential target for DBA therapy. To identify new DBA targets, we screen for small molecules that increase erythroid expansion in mouse models of DBA. This screen identified a compound that inhibits NLK. Chemical and genetic inhibition of NLK increases erythroid expansion in mouse and human progenitors, including bone marrow cells from DBA patients. In DBA models and patient samples, aberrant NLK activation is initiated at the Megakaryocyte/Erythroid Progenitor (MEP) stage of differentiation and is not observed in non-erythroid hematopoietic lineages or healthy erythroblasts. We propose that NLK mediates aberrant erythropoiesis in DBA and is a potential target for therapy.

Sequence-specific DNA binding by the glucocorticoid receptor DNA-binding domain is linked to a salt-dependent histidine protonation.

We used isothermal titration calorimetry in the temperature range 21-25 degrees C to investigate the effect of pH on the calorimetric enthalpy (delta H(cal)) for sequence specific DNA-binding of the glucocorticoid receptor DNA-binding domain (GR DBD). Titrations were carried out in solutions containing 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol by volume, and 20 mM Tris, Hepes, Mops, or sodium phosphate buffers at pH 7.5. A strong dependence of delta H(cal) on the buffer ionization enthalpy is observed, demonstrating that the DNA binding of the GR DBD is linked to proton uptake at these conditions. The apparent increase in the pK(a) for an amino acid side chain upon DNA binding is supported by the results of complementary titrations, where delta H(cal) shows a characteristic dependence on the solution pH. delta H(cal) is also a function of the NaCl concentration, with opposite dependencies in Tris and Hepes buffers, respectively, such that a similar delta H(cal) value is approached at 300 mM NaCl. This behavior shows that the DNA-binding induced protonation is inhibited by increased concentrations of NaCl. A comparison with structural data suggests that the protonation involves a histidine (His451) in the GR DBD, because in the complex this residue is located close to a DNA phosphate at an orientation that is consistent with a charged-charged hydrogen bond in the protonated state. NMR spectra show that His451 is not protonated in the unbound protein at pH 7.5. The pH dependence in delta H(cal) can be quantitatively described by a shift of the pK(a) of His451 from approximately 6 in the unbound state to close to 8 when bound to DNA at low salt concentration conditions. A simple model involving a binding competition between a proton and a Na(+) counterion to the GR DBD-DNA complex reproduces the qualitative features of the salt dependence.

Characterization of sequence-specific DNA binding by the transcription factor Oct-1.

The DNA-binding domain of the Oct-1 transcription factor, POU, recognizes a defined DNA sequence known as the octamer element to regulate the expression of both general and cell-type-specific genes. The two-part DNA-binding domain partially encircles the DNA to recognize the eight base pairs of the octamer element. We have characterized the binding of Oct-1/POU to an octamer element using isothermal titration calorimetry. As found for other cognate protein/DNA complexes, the formation of the Oct-1 POU/DNA complex is associated with a large negative heat capacity change, DeltaC(p)()(, obs). However, the observed change is much greater than expected by empirical relationships with buried surface area. Supported by data from proteolysis studies on the free and DNA-bound protein, we propose that the discrepancy in heat capacity arises principally from the partial folding of the Oct-1 POU protein upon complex formation. Formation of the Oct-1 POU/DNA complex is strongly dependent on ionic strength, and the detailed quantification of this relationship suggests that six charged contacts are made between the protein and the phosphate groups of the DNA. This agrees with observations from the crystal structure of an Oct-1 POU/DNA complex.

Oligomerization of the chromatin-structuring protein H-NS.

H-NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H-NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H-NS protein from Salmonella typhimurium. The N-terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an alpha-helix, likely to be in coiled-coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full-length protein no single, defined oligomeric state is adopted. The C-terminal 48 residues do not participate in oligomerization and form a monomeric, DNA-binding domain. These N- and C-terminal domains are joined via a flexible linker which enables them to function independently within the context of the full-length protein. This novel mode of oligomerization may account for the unusual binding properties of H-NS.

Oct-1 POU and octamer DNA co-operate to recognise the Bob-1 transcription co-activator via induced folding.

The expression of immunoglobulin genes is controlled in part by the DNA-binding protein Oct-1 and the B cell-specific transcription co-activator, Bob1 (also known as OCA-B or OBF-1) that together form a complex on the Igkappa promoter. We have characterised the assembly of the ternary complex using biophysical methods. Bob1 binds specifically as a monomer to the complex of the Oct-1 DNA-binding domain (Oct-1 POU) and the Igkappa promoter, but binds weakly to either Oct-1 POU or the Igkappa promoter alone, indicating that both are required to make an avid complex. Ternary complex formation requires a defined DNA sequence, as the stability of the complex can be strongly affected by a single base-pair change or by removing 5-methyl groups from selected thymine bases.In isolation, Bob1 appears to have little secondary structure, but may become partially structured upon recruitment into the ternary complex as demonstrated by circular dichroism spectra and calorimetry. These and other findings suggest that ternary complex formation requires a defined geometry of the POU/DNA complex, and that the co-activator makes stereo-specific contacts to both the POU protein and the major groove of the DNA that induces its fold.

Thermodynamic characterization of non-sequence-specific DNA-binding by the Sso7d protein from Sulfolobus solfataricus.

We used isothermal titration calorimetry and fluorescence spectroscopy to investigate the thermodynamics of non-sequence-specific DNA-binding by the Sso7d protein from the archaeon Sulfolobus solfataricus. We report the Sso7d-poly(dGdC) binding thermodynamics as a function of buffer composition (Tris-HCl or phosphate), temperature (15 to 45 degrees C), pH (7.1 to 8.0), osmotic stress and solvent (H2O/2H2O), and compare it to poly (dAdT) binding; and we have previously also reported the salt concentration dependence. Binding isotherms can be represented by the McGhee-von Hippel model for non-cooperative binding, with a binding site size of four to five DNA base-pairs and binding free energies in the range DeltaG degrees approximately -7 to DeltaG degrees approximately -10 kcal mol-1, depending on experimental conditions. The non-specific nature of the binding is reflected in similar thermodynamics for binding to poly(dAdT) and poly(dGdC). The native lysine methylation of Sso7d has only minor effects on the binding thermodynamics. Sso7d binding to poly(dGdC) is endothermic at 25 degrees C with a binding enthalpy DeltaH degrees approximately 10 kcal mol-1 in both phosphate and Tris-HCl buffers at pH 7.6, indicating that DeltaH degrees does not include large contributions from coupled buffer ionization equilibria at this pH. The binding enthalpy is temperature dependent with a measured heat capacity change DeltaCp degrees=-0.25(+/-0.01) kcal mol-1 K-1 and extrapolations of thermodynamic data indicate that the complex is heat stable with exothermic binding close to the growth temperature (75 to 80 degreesC) of S. solfataricus. Addition of neutral solutes (osmotic stress) has minor effects on DeltaG degrees and the exchange of H2O for 2H2O has only a small effect on DeltaH degrees, consistent with the inference that complex formation is not accompanied by net changes in surface hydration. Thus, other mechanisms for the heat capacity change must be found. The observed thermodynamics is discussed in relation to the nature of non-sequence-specific DNA-binding by proteins.

Structure and dynamics of the glucocorticoid receptor DNA-binding domain: comparison of wild type and a mutant with altered specificity.

Nuclear magnetic resonance was used to compare parameters reflecting solution structure and dynamics of the glucocorticoid receptor DNA-binding domain (GRDBD), which binds specifically to a GRE binding site on DNA, and a triple mutant (GRDBDEGA), which binds to an ERE site. The studies were prompted by an earlier observation that the cooperativity for dimeric DNA-binding is 10 times higher for the GRDBDEGA-ERE association than for the GRDBD-GRE association (Lundbäck et al., 1994). The higher binding cooperativity of the mutant was unexpected since the triple mutation (G458E, S459G, and V462A) is made in the recognition helix and distant from the dimerization surface which is formed by residues in the fragment A477-N491. Sequential and long-range NOE connectivities and measured 3JHNHalpha coupling constants indicate that the overall structures of the two proteins are very similar, possibly with a less well-defined structure of the fragment K486-N491 in GRDBDEGA. However, chemical shift changes, line broadening, and increased amide proton exchange rates are observed for several residues at, or close to, the dimerization surface of the mutant. These observations are interpreted as a lower stability and/or several slowly interconverting folded conformations of this region of GRDBDEGA. The effects are likely to be due to the loss of a hydrogen bond which links S459 to the dimerization region in GRDBD. Different mechanisms for the increased binding cooperativity of the mutant are discussed, and it is noted that the properties of the GRDBDEGA dimerization region are reminiscent of those reported for the estrogen receptor DBD, which also binds to an ERE site.

Thermodynamics of sequence-specific protein-DNA interactions.

The molecular recognition processes in sequence-specific protein-DNA interactions are complex. The only feature common to all sequence-specific protein-DNA structures is a large interaction interface, which displays a high degree of complementarity in terms of shape, polarity and electrostatics. Many molecular mechanisms act in concert to form the specific interface. These include conformational changes in DNA and protein, dehydration of surfaces, reorganization of ion atmospheres, and changes in dynamics. Here we review the current understanding of how different mechanisms contribute to the thermodynamics of the binding equilibrium and the stabilizing effect of the different types of noncovalent interactions found in protein-DNA complexes. The relation to the thermodynamics of small molecule-DNA binding and protein folding is also briefly discussed.

Sequence-specific DNA-binding dominated by dehydration.

Fluorescence spectroscopy and isothermal titration calorimetry were used to study the thermodynamics of binding of the glucocorticoid receptor DNA-binding domain to four different, but similar, DNA-binding sites. The binding sites are two naturally occurring sites that differ in the composition of one base pair, i.e., an A-T to G-C mutation, and two sites containing chemical intermediates of these base pairs. The calorimetrically determined heat capacity change (Delta C(p)o(obs)) for glucocorticoid receptor DNA-binding domain binding agrees with that calculated for dehydration of solvent-accessible surface areas. A dominating effect of dehydration or solvent reorganization on the thermodynamics is also consistent with an observed linear relationship between observed enthalpy change (Delta Ho(obs)) and observed entropy change (Delta So(obs)) with a slope close to the experimental temperature. Comparisons with structural data allow us to rationalize individual differences between Delta Ho(obs) (and Delta So(obs)) for the four complexes. For instance, we find that the removal of a methyl group at the DNA-protein interface is enthalpically favorable but entropically unfavorable, which is consistent with a replacement by an ordered water molecule.

Solution structure and DNA-binding properties of a thermostable protein from the archaeon Sulfolobus solfataricus.

The archaeon Sulfolobus solfataricus expresses large amounts of a small basic protein, Sso7d, which was previously identified as a DNA-binding protein possibly involved in compaction of DNA. We have determined the solution structure of Sso7d. The protein consists of a triple-stranded anti-parallel beta-sheet onto which an orthogonal double-stranded beta-sheet is packed. This topology is very similar to that found in eukaryotic Src homology-3 (SH3) domains. Sso7d binds strongly (Kd < 10 microM) to double-stranded DNA and protects it from thermal denaturation. In addition, we note that epsilon-mono-methylation of lysine side chains of Sso7d is governed by cell growth temperatures, suggesting that methylation is related to the heat-shock response.

Thermodynamics of sequence-specific glucocorticoid receptor-DNA interactions.

The thermodynamics of sequence-specific DNA-protein interactions provide a complement to structural studies when trying to understand the molecular basis for sequence specificity. We have used fluorescence spectroscopy to study the chemical equilibrium between the wild-type and a triple mutant glucocorticoid receptor DNA-binding domain (GR DBD wt and GR DBDEGA, respectively) and four related DNA-binding sites (response elements). NMR spectroscopy was used to confirm that the structure of the two proteins is very similar in the uncomplexed state. Binding to DNA oligomers containing single half-sites and palindromic binding sites was studied to obtain separate determinations of association constants and cooperativity parameters involved in the dimeric DNA binding. Equilibrium parameters were determined at 10-35 degrees C in 85 mM NaCl, 100 mM KCl, 2 mM MgCl2, and 20 mM Tris-HCl at pH 7.4 (20 degrees C) and at low concentrations of an antioxidant and a nonionic detergent. GR DBDwt binds preferentially to a palindromic consensus glucocorticoid response element (GRE) with an association constant of (7.6 +/- 0.9) x 10(5) M-1 and a cooperativity parameter of 10 +/- 1 at 20 degrees C. GR DBDEGA has the highest affinity for an estrogen response element (ERE) with an association constant of (2.2 +/- 0.3) x 10(5) M-1 and a cooperativity parameter of 121 +/- 17 at 20 degrees C. The difference in cooperativity in the two binding processes, which indicates significant differences in binding modes, was confirmed using gel mobility assays. van't Hoff analysis shows that DNA binding in all cases in entropy driven within the investigated temperature range. We find that delta H0obs and delta S0obs for the formation of a GR DBDwt-GRE versus GR DBDEGA-ERE complex are significantly different despite very similar delta G0obs values. A comparison of GR DBDwt binding to two similar GREs reveals that the discrimination between these two (specific) sites is due to a favorable delta(delta S0obs) which overcompensates an unfavorable delta(delta H0obs), i.e., the sequence specificity is in this case entropy driven. Thus, entropic effects are of decisive importance for the affinity as well as the specificity in GR-DNA interactions. The molecular basis for measured equilibrium and thermodynamic parameters is discussed on the basis of published structures of GR DBD-GRE and ER DBD-ERE complexes.

Thermodynamics of the glucocorticoid receptor-DNA interaction: binding of wild-type GR DBD to different response elements.

We used fluorescence spectroscopy to study the chemical equilibria between an 82-residue protein fragment containing the core conserved region of the glucocorticoid receptor DNA-binding domain (GR DBD) and a palindromic glucocorticoid response element (GRE), a consensus GRE half-site, a consensus estrogen response element (ERE) half-site, and two intermediate half-sites (GRE2 and ERE2). Equilibrium parameters were determined at 20 degrees C and buffer conditions that approximate intracellular conditions. The association constants for GR DBD binding to the GRE (5'TGTTCT3') and GRE2 (5'TGTCCT3') half-sites at 85 mM NaCl, 100 mM KCl, 2 mM MgCl2, and 20 mM Tris-HCl at pH 7.4 and low concentrations of an antioxidant and a nonionic detergent are (1.0 +/- 0.1) x 10(6) M-1 and (5.1 +/- 0.2) x 10(5) M-1, respectively. The association constants for binding to the ERE (5'TGACCT3') and ERE2 (5'TGATCT3') half-sites are < 10(5) M-1. The implications of these numbers for the specificity and affinity for the binding of the intact GR to DNA are discussed. Comparison of GR DBD binding to a GRE half-site and a palindromic GRE sequence allowed us to estimate the cooperativity parameter, omega obs = 25-50, for GR DBD binding to GRE. The thermodynamics of the GR DBD interaction with a GRE half-site were also investigated by determining the temperature dependence of the observed association constant. The nonlinear dependence in ln Kobs as a function of 1/T is consistent with a change in standard heat capacity, delta Cp degree obs = 1.0 +/- 0.2 kcal mol-1 K-1.(ABSTRACT TRUNCATED AT 250 WORDS)