No abstract

The critical functions of the HIV-1 nucleocapsid protein NCp7 in genomic RNA packaging and reverse transcription, essentially rely on interactions with nucleic acids. A significant progress in the knowledge of these interactions has been recently achieved with the NMR-derived structures of NCp7 derivatives in complex with two short sequences of the HIV-1 psi packaging signal, namely ACGCC and the stem-loop 3 (SL3) motif. To further identify the key nucleotides in the formation of both NCp7-d(ACGCC) and NCp7-SL3 complexes, we quantitatively analyzed by steady-state and time-resolved fluorescence, the interaction of NCp7 with d(ACGCC) and SL3 mutants where each nucleotide in interaction with the protein has been systematically substituted. Moreover, by using several NCp7 derivatives, we investigated the contributions of Phe16, Trp37, and Trp61, and the various NCp7 domains, in the binding process. The binding of NCp7 appeared essentially driven by the interaction of the zinc finger domain and notably Trp37 with a G residue, irrespective of its location in the oligonucleotide. The involvement of Trp37 in the binding process depended on its location in the C-terminal finger motif and the proper folding of this motif. Phe16 in the N-terminal finger motif also strongly contributed to the binding energy, while in contrast, Trp61 in the C-terminal domain only marginally interacted with the oligonucleotides. The stem-loop structure of SL3 stabilized the binding of NCp7 by about -7 kJ/mol (at 0.1 M NaCl) by favoring the electrostatic binding of both N- and C-terminal domains. Finally, we found that NCp7 bound to nucleic acid single-stranded regions with the following preference: X(i)()TGX(j)() > X(i)()GXGX(j)() approximately X(i)()TXGX(j)() > X(i)()GX(j)() >> X(i)()X(j)(), where X corresponds to either A or C. This implies that recognition of nucleic acids by NCp7 may be achieved by a limited number of sites, and hence, no strong affinities are required in order to get a selective binding.

No abstract

The HIV-1 nucleocapsid protein, NCp7, is characterized by two CCHC zinc finger motifs which have been shown to stoichiometrically bind zinc in mature virions. Moreover, this binding of zinc proves to be critical in various NCp7 functions, especially in the encapsidation process. To further understand the central role of zinc binding to NCp7, we closely investigated the zinc binding properties of NCp7 and various deleted or substituted derivatives. To this end, the fluorescence of wither the naturally occurring Trp37 or the conservatively substituted Trp16 was used to monitor the binding of zinc to the N- and C-terminal finger motifs, respectively. At pH 7.5, the NCp7 proximal motif was found to bind zinc strongly with 2.8 x 10(14) M-1 binding constant about five times higher than the NCp7 distal motif. Moreover, the binding of zinc to one finger motif decreased the affinity of the second one, and this negative cooperativity was shown to be related to the spatial proximity of the zinc-saturated finger motifs. The binding seemed to be almost equally driven by entropy and enthalpy, and the binding information was essentially encoded by the finger motifs themselves whereas the other parts of the protein only played a marginal stabilization role. As expected, the Cys and His residues of the CCHC motifs were critical and competition between protons and zinc ions to these residues induced a steep pH-dependence of the zinc binding constants to both sites. Taken together, our data provide further evidence for the nonequivalence of the two NCp7 finger motifs.

No abstract

On the basis of the three-dimensional structure of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and of sequence comparison with the photosynthetic NAD(P)-dependent GAPDH of the chloroplast, a series of mutants of GAPDH from Bacillus stearothermophilus have been constructed. The results deduced from kinetic and binding studies suggest that the absence of activity of the wild-type GAPDH with NADP as a cofactor is the consequence of at least three factors: (1) steric hindrance, (2) electrostatic repulsion between the charged carboxyl group of Asp32 and the 2'PO4, and (3) structural determinants at the subunit interface of the tetramer. The best value for kcat/KM and KD for NADP was observed for the D32A-L187A-P188S mutant. This triple mutation leads to a switch in favor of NADP specificity but with a kcat/KM ratio 50- and 80-fold less than that observed for the wild type with NAD and for the chloroplast GAPDH with NADP, respectively. Substituting the invariant chloroplastic Thr33-Gly34-Gly35 for the B. stearothermophilus Leu33-Thr34-Asp35 residues on the double mutant Ala187-Ser188 does not improve significantly the affinity for NADP while substituting Ala32 for Asp32 on the double mutant does. Clearly, other subtle adjustments in the adenosine subsite are needed to reconcile the presence of the carboxylate group of Asp32 and the 2'-phosphate of NADP. Kinetic studies indicate a change of the rate-limiting step for the mutants. This could be the consequence of an incomplete apo-holo transition.(ABSTRACT TRUNCATED AT 250 WORDS)

TAL1 (also known as SCL) is expressed in >40% of human T cell acute lymphoblastic leukemias (T-ALLs). TAL1 encodes a basic helix-loop-helix transcription factor that can interfere with the transcriptional activity of E2A and HEB during T cell leukemogenesis; however, the oncogenic pathways directly activated by TAL1 are not characterized. In this study, we show that, in human TAL1–expressing T-ALL cell lines, TAL1 directly activates NKX3.1, a tumor suppressor gene required for prostate stem cell maintenance. In human T-ALL cell lines, NKX3.1 gene activation is mediated by a TAL1–LMO–Ldb1 complex that is recruited by GATA-3 bound to an NKX3.1 gene promoter regulatory sequence. TAL1-induced NKX3.1 activation is associated with suppression of HP1-α (heterochromatin protein 1 α) binding and opening of chromatin on the NKX3.1 gene promoter. NKX3.1 is necessary for T-ALL proliferation, can partially restore proliferation in TAL1 knockdown cells, and directly regulates miR-17-92. In primary human TAL1-expressing leukemic cells, the NKX3.1 gene is expressed independently of the Notch pathway, and its inactivation impairs proliferation. Finally, TAL1 or NKX3.1 knockdown abrogates the ability of human T-ALL cells to efficiently induce leukemia development in mice. These results suggest that tumor suppressor or oncogenic activity of NKX3.1 depends on tissue expression.

Low molecular mass heparin (5.1 kDa) forms a tight complex with mucus proteinase inhibitor, the physiologic neutrophil elastase inhibitor of the upper respiratory tract. This binding strongly enhances the intrinsic fluorescence of the inhibitor and the rate of neutrophil elastase inhibitor association. One mole of this heparin fragment binds 1 mol of inhibitor with a Kd of 50 nM. From the variation of Kd with ionic strength, it is inferred that (i) 85% of the heparin--inhibitor binding energy i due to electrostatic interactions, (ii) about seven ionic interactions are involved in heparin--inhibitor binding. strength, it is inferred that (i) 85% of the heparin--inhibitor binding energy is due to electrostatic interactions, (ii) about seven ionic interactions are involved in heparin--inhibitor binding. and (iii), about one-third of low quantum yield of Trp30, the single tryptophan residue of the inhibitor, blue-shifts its maximum emission wavelength by 6 nm, decreases the acrylamide quenching rate constant by a factor of 4, and increases the mean intensity weighted lifetime by a factor of 2.5. These important spectroscopic changes evidence a heparin--induced conformational change of the inhibitor which buries Trp30 in a very hydrophobic environment. Heparin accelerates the inhibition of elastase in a concentration-dependent manner. When both enzyme and inhibitor are saturated by the polymer, the second-order association rate constant is 7.7 x 10(7) M-1 s-1, a value that is 27-fold higher than that measured with the free partners. This finding may have important physiologic and therapeutic bearing.