Helix 2 of the paired domain plays a key role in the regulation of DNA-binding by the Pax-3 homeodomain.

Pax3 contains two structurally independent DNA-binding domains, a paired domain (PD) and a homeodomain (HD). Biochemical and mutagenesis studies have shown that both domains are functionally interdependent. In particular, it has been shown that the PD can regulate the DNA-binding specificity and dimerization potential of the HD. To delineate Pax3 protein segments that are involved in the regulation of HD DNA-binding, a series of chimeric proteins were created in which the HD and linker region were gradually replaced with corresponding sequences from a heterologous HD protein, Phox. Characterization of chimeric proteins by electrophoretic mobility shift analysis (EMSA) suggests that a portion of the linker region contributes to the functional interaction between the PD and HD. In addition, stepwise removal of sequences from the Pax3 PD was used to define regions within this domain that are involved in the regulation of HD DNA-binding. EMSA of these proteins in the context of the chimeric Pax3/Phox backbone provided two key findings: (i) the C-terminal subdomain of the PD does not play a major role in the regulation of HD DNA-binding and (ii) the N-terminal subdomain and, in particular, the second alpha-helix are essential for modulation of HD DNA-binding. Significantly, deletion of helix 2 was found to be sufficient to uncouple regulation of HD DNA-binding by the PD.     

Characterization of the cation-binding properties of porcine neurofilaments

In the presence of physiological levels of Na+ (10 mM), K+ (150 mM), and Mg2+ (2 mM), dephosphorylated neurofilaments contained two Ca2+ specific binding sites with Kd = 11 microM per unit consisting of eight low, three middle, and three high molecular subunits, as well as 46 sites with Kd = 620 microM. Only one class of 126 sites with Kd = 740 microM was detected per unit of untreated neurofilaments. A chymotryptic fraction enriched in the alpha-helical domains of neurofilament subunits contained one high-affinity Ca2+-binding site (Kd = 3.6 microM) per domain fragment of approximately 32 kDa. This site may correspond to a region in coil 2b of the alpha-helical domain, which resembles the I-II Ca2+-binding site in intestinal Ca2+-binding protein. Homopolymeric filaments composed of the low or middle molecular weight subunits contained low-affinity Ca2+-binding sites with Kd = 37 microM and 24 microM, respectively, while the Kd values for the low-affinity sites in heteropolymeric filaments were 8-10-fold higher. Competitive binding studies, using the chymotryptic fraction to assay the high-affinity Ca2+-binding sites and 22Na+ to monitor binding to the phosphate-containing low-affinity sites, yielded Kd values for Al3+ of 0.01 microM and 4 microM, respectively. This suggests that the accumulation of Al3+ in neurons may be due in part to its binding to neurofilaments.     

Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli

The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis.     

Isolation and sequence-specific DNA binding of the Antennapedia homeodomain.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was overproduced in a T7 expression vector in Escherichia coli. The corresponding polypeptide of 68 amino acids was purified to homogeneity. The homeodomain was analysed by ultracentrifugation and assayed for DNA binding. The secondary structure of the isolated homeodomain was determined by nuclear magnetic resonance spectroscopy. DNA-binding studies indicate that the isolated homeodomain binds to DNA in vitro. It selectively binds to the same sites as a longer Antp polypeptide and a full-length fushi tarazu (ftz) protein. Therefore, the homeodomain represents the DNA-binding domain of the homeotic proteins.     

Solution structure of the Reps1 EH domain and characterization of its binding to NPF target sequences

The recently described EH domain recognizes proteins containing Asn-Pro-Phe (NPF) sequences. Using nuclear magnetic resonance (NMR) data, we determined the solution structure of the EH domain from the Reps1 protein and characterized its binding to linear and cyclic peptides derived from a novel targeting protein. The structure calculation included 1143 distance restraints and 122 angle restraints and resulted in structures with a root-mean-square deviation of 0.40 +/- 0.05 A for backbone atoms of superimposed secondary structural elements. The structure comprises two helix-loop-helix motifs characteristic of EF-hand domains. Titration data with NPF-containing peptides showed evidence of intermediate exchange on the NMR chemical shift time scale, which required an analysis that includes curve fitting to obtain accurate equilibrium constants and dissociation rate constants. The cyclic and linear peptides bound with similar affinities (Kd = 65 +/- 17 and 46 +/- 14 microM, respectively) and to the same hydrophobic pocket formed between helices B and C. The cyclic peptide formed a complex that dissociated more slowly (k(off) = 440 +/- 110 s(-1)) than the linear peptide (k(off) = 1800 +/- 250 s(-1)), but had little change in affinity because of the slower rate of association of the cyclic peptide. In addition, we characterized binding to a peptide containing a DPF sequence (Kd = 0.5 +/- 0.2 mM). The characterization of binding between the Reps1 EH domain and its target proteins provides information about their role in endocytosis.     

Segments of the POU domain influence one another''s DNA-binding specificity.

The ubiquitously expressed mammalian POU-domain protein Oct-1 specifically recognizes two classes of cis-acting regulatory elements that bear little sequence similarity, the octamer motif ATGCAAAT and the TAATGARAT motif. The related pituitary-specific POU protein Pit-1 also recognizes these two motifs but, unlike Oct-1, binds preferentially to the TAATGARAT motif. Yet in our assay, Pit-1 still binds octamer elements better than does the octamer motif-binding protein Oct-3. The POU domain is responsible for recognizing these diverse regulatory sequences through multiple DNA contacts that include the two POU subdomains, the POU-specific region, and the POU homeodomain. The DNA-binding properties of 10 chimeric POU domains, in which different POU-domain segments are derived from either Oct-1 or Pit-1, reveal a high degree of structural plasticity; these hybrid proteins all bind DNA well and frequently bind particular sites better than does either of the parental POU domains. In these chimeric POU domains, the POU-specific A and B boxes and the hypervariable POU linker can influence DNA-binding specificity. The surprising result is that the influence a particular segment has on DNA-binding specificity can be greatly affected by the origin of other segments of the POU domain and the sequence of the binding site. Thus, the broad but selective DNA-binding specificity of Oct-1 is conferred both by multiple DNA contacts and by dynamic interactions within the DNA-bound POU domain.     

Investigation of the forms of pig duodenal Ca2+-binding protein produced by limited tryptic hydrolysis.

Vitamin D-dependent Ca2+-binding protein from pig duodenum was hydrolysed with trypsin in the presence of Ca2+ and two products were obtained: T1, which differed from the native protein by loss of Ac-Ser-Ala-Gln-Lys from the N-terminus and Ile-Ser-Gln-OH from the C-terminus, and T2, which differed from T1 by loss of a C-terminal lysine. The hydrolysis inactivated one of the two high-affinity Ca2+-binding sites on the native protein, and the remaining site was stable in T1 but labile in T2 when the proteins were Ca2+-free. Binding studies showed that T1 had Kd values of 2.8 +/- 0.1 nM, 57 +/- 13 microM and 0.8 +/- 0.3 microM for Ca2+, Mg2+ and Mn2+ respectively, and T2 had Kd 2.2 +/- 0.3 nM for Ca2+. The affinity for Mn2+, together with the other Kd values, identified the site on T1 as the site on the native protein previously found to have Kd 0.6 microM for Mn2+, rather than one with Kd 50 microM for Mn2+. In contrast with both the native protein and another form of the protein with a single Ca2+-binding site, the intrinsic fluorescence of T1 and T2 was little affected by the addition of Ca2+. It was concluded that the active binding site in T1 and T2, and also the site in the native protein with the higher affinity for Mn2+, was probably in the C-terminal half of the molecule.     

The GCN4 bZIP targets noncognate gene regulatory sequences: quantitative investigation of binding at full and half sites

We previously reported that a basic region/leucine zipper (bZIP) protein, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes cognate target sites AP-1 (5'-TGACTCA-3') and cAMP-response element (CRE) (5'-TGACGTCA-3') but also binds selectively to noncognate DNA sites: C/EBP (CCAAT/enhancer binding protein, 5'-TTGCGCAA), XRE1 (xenobiotic response element, 5'-TTGCGTGA), HRE (HIF response element, 5'-GCACGTAG), and E-box (5'-CACGTG). In this work, we used electrophoretic mobility shift assay (EMSA) and circular dichroism (CD) for more extensive characterization of the binding of wt bZIP dimer to noncognate sites as well as full- and half-site derivatives, and we examined changes in flanking sequences. Quantitative EMSA titrations were used to measure dissociation constants of this hybrid, wt bZIP, to DNA duplexes: Full-site binding affinities gradually decrease from cognate sites AP-1 and CRE with Kd values of 13 and 12 nM, respectively, to noncognate sites with Kd values of 120 nM to low microM. DNA-binding selectivity at half sites is maintained; however, half-site binding affinities sharply decrease from the cognate half site (Kd = 84 nM) to noncognate half sites (all Kd values > 2 microM). CD shows that comparable levels of alpha-helical structure are induced in wt bZIP upon binding to cognate AP-1 or noncognate sites. Thus, noncognate sites may contribute to preorganization of stable protein structure before binding target DNA sites. This work demonstrates that the bZIP scaffold may be a powerful tool in the design of small, alpha-helical proteins with desired DNA recognition properties.     

Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy.

Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H2O and D2O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 +/- 0.5 and 4.6 +/- 0.5 water molecules coordinated to Eu3+ bound to the n1 site of unadenylylated enzyme, GS0, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 +/- 0.5 water molecules coordinated to Eu3+ at site n2 for both GS0 and GS12. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 +/- 1.5 A. Distances between a Tb3+ ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site n1 was observed to decrease from Kd = 0.35 +/- 0.09 microM for GS0 to Kd = 0.06 +/- 0.02 microM for GS12. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 +/- 0.9 microM and Kd = 2.6 +/- 0.7 microM for GS0 and GS12, respectively. Competition experiments indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from Kd = 0.05 +/- 0.02 microM for GS0 to Kd = 0.35 +/- 0.09 microM for GS12. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 +/- 1.3 microM for GS0 and Kd = 4.0 +/- 1.0 microM for GS12). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991, Biochemistry 30:1413-1418), supporting the hypothesis that adenylylation regulates GS by altering substrate and metal ion affinities.     

DNA binding affinity and sequence permutation preference of the telomere protein from Euplotes crassus

Telomere end binding proteins from diverse organisms use various forms of an ancient protein structure to recognize and bind with single-strand DNA found at the ends of telomeres. To further understand the biochemistry and evolution of these proteins, we have characterized the DNA binding properties of the telomere end binding protein from Euplotes crassus (EcTEBP). EcTEBP and its predicted amino-terminal DNA-binding domain, EcTEBP-N, were expressed in Escherichia coli and purified. Each protein formed stoichiometric (1:1) complexes with single-strand DNA oligos derived from the precisely defined d(TTTTGGGGTTTTGG) sequence found at DNA termini in Euplotes. Dissociation constants for DNA x EcTEBP and DNA x EcTEBP-N complexes were comparable: K(D-DNA) = 38 +/- 2 nM for the full-length protein and K(D-DNA) = 60 +/- 4 nM for the N-terminal domain, indicating that the N-terminal domain retains a high affinity for DNA even in the absence of potentially stabilizing moieties located in the C-terminal domain. Rate constants for DNA association and DNA dissociation corroborated a slightly improved DNA binding performance for the full-length protein (ka = 45 +/- 4 microM(-1) s(-1), kd = 0.10 +/- 0.02 s(-1)) relative to that of the N-terminal domain (ka = 18 +/- 1 microM(-1) s(-1), kd = 0.15 +/- 0.01 s(-1)). Equilibrium dissociation constants measured for sequence permutations of the telomere repeat spanned the range of 55-1400 nM, with EcTEBP and EcTEBP-N binding most tightly to d(TTGGGGTTTTGG), the sequence corresponding to that of mature DNA termini. Additionally, competition experiments showed that EcTEBP recognizes and binds the telomere-derived 14-nucleotide DNA in preference to shorter 5'-truncation variants. Compared with the results for multisubunit complexes assembled with telomere single-strand DNA from Oxytricha nova, our results highlight the relative simplicity of the E. crassus system where a telomere end binding protein has biochemical properties indicating one protein subunit caps the single-strand DNA.     

Effect of experimental hypoalbuminemia on the plasma protein binding of tolmetin

The purpose of this work was to study tolmetin plasma protein binding in an experimental model of hypoalbuminemia in the rat. Hypoalbuminemia was produced by repetitive plasmapheresis, achieving a 26.2 +/- 4.6% reduction in albumin circulating levels. Rats then received a 100 mg/kg oral tolmetin dose. Control rats received oral tolmetin 10, 56 or 100 mg/kg. Tolmetin plasma protein binding was determined by an ultrafiltration technique using an in vivo pharmacokinetic approach. Plasma protein binding data for the 3 doses studies in control animals could be described considering a single binding site with Kd = 21.9 +/- 2.1 microM and N = 0.98 +/- 0.05 sites per molecule of albumin. For hypoalbuminemic rats Kd was significantly increased (p < 0.05), while there was no significant change in the number of binding site per albumin molecule (Kd = 131.6 +/- 38.1 microM and N = 1.58 +/- 0.77). Our results show that hypoalbuminemia produces a disproportionate increase in the free fraction of tolmetin, not only by reducing albumin concentration, but also by a decrease in affinity. The mechanism responsible of such changes in affinity remains to be elucidated.     

Chimeric restriction enzymes: what is next?

Chimeric restriction enzymes are a novel class of engineered nucleases in which the non-specific DNA cleavage domain of Fokl (a type IIS restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely the helix-turn-helix motif, the zinc finger motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-F(N) was shown to find and cleave its target in vivo. This was tested by microinjection of DNA substrates and the enzyme into frog oocytes (Carroll et al., 1999). The injected enzyme made site-specific double-strand breaks in the targets even after assembly of the DNA into chromatin. In addition, this cleavage activated the target molecules for efficient homologous recombination. Since the recognition specificity of zinc fingers can be manipulated experimentally, chimeric nucleases could be engineered so as to target a specific site within a genome. The availability of such engineered chimeric restriction enzymes should make it feasible to do genome engineering, also commonly referred to as gene therapy.     

Isolation of two novel myb-like genes from Arabidopsis and studies on the DNA-binding properties of their products

Two novel myb-like genes (atmyb6 and atmyb7) were isolated from an Arabidopsis thaliana cDNA library. The entire proteins or the Myb domains encoded by the genes were expressed as fusion proteins in Escherichia coli. The DNA-binding domain of the murine c-Myb was also expressed in the same way for use in comparative studies. The fusion proteins were examined for their DNA-binding activity using the animal c-Myb DNA-binding site (MBS) and the binding site of the maize P gene product (PBS). The Myb domain of Atmyb6 bound to PBS more efficiently than to MBS. Complete Atmyb6 and Atmyb7 proteins preferentially bound to PBS but not MBS. This suggests that the in vitro binding consensus sequences for both Atmyb6 and Atmyb7 are similar to PBS. The binding of the Myb domain of Atmyb6 to both PBS and MBS raises the possibility that the protein recognizes multiple sequences in vivo. The third α-helix and three adjacent amino acids in the third repeat (R3) of c-Myb were replaced with the analogous sequence of Atmyb6 to create a chimeric Myb protein. This chimeric protein bound to PBS with a low affinity but failed to bind to MBS. Thus the binding pattern of the chimeric Myb protein is similar to that of the Atmyb6. This result suggests that the last 20 amino acids in the R3 repeat of Atmyb6 play a major role in DNA-binding.