Linda Tennant

Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA

We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone1 HN, 15N,13 Cα and13 Cβ and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects.

Specific interaction of the first three zinc fingers of TFIIIA with the internal control region of the Xenopus 5 S RNA gene

A DNA plasmid encoding the first 101 amino acid residues of the Xenopus 5 S RNA gene-specific transcription factor IIIA (TFIIIA) was derived by polymerase chain reaction amplification of this region from the cDNA for TFIIIA. This polypeptide includes the first three zinc fingers of the TFIIIA DNA binding domain. The polypeptide was expressed in Escherichia coli and purified to greater than 95% homogeneity. The three finger polypeptide binds the internal control region of the 5 S RNA gene with sequence specificity and high affinity. Binding is metal-dependent and treatment of the polypeptide with EDTA abolishes binding. Polypeptide-DNA complexes exhibit a dissociation constant of 5.6(+/- 0.9) nM, while that for full-length Xenopus TFIIIA is 2.2(+/- 0.4) nM, measured under the same conditions. This suggests that the majority of the free energy of TFIIIA binding resides in these amino-terminal zinc fingers. The polypeptide protects 21 base-pairs of the internal control region from attack by DNase I, with protection from nucleotides +75 to +95 of the 120 base-pair gene. This region includes the C-block promoter element and several guanine residues that are essential for TFIIIA binding. Methylation interference experiments suggest that the mode of binding of the polypeptide and TFIIIA are similar. The minimal DNA sequences required for polypeptide binding were determined using a series of synthetic double-stranded deoxyribo-oligonucleotides. A 13 base-pair oligonucleotide spanning nucleotides +80 to +92 of the 5 S RNA gene retained specific and high-affinity binding, although the latter was reduced sixfold relative to longer DNA fragments. Polypeptides containing fingers 1 and 2, or fingers 2, 3 and 4 of TFIIIA do not exhibit sequence-specific DNA binding. Overall, these studies provide strong support for a model in which the first three zinc fingers of TFIIIA bind with high affinity between nucleotides +80 and +92 of the internal control region of the 5 S RNA gene.

Probing protein structure using biochemical and biophysical methods: Proteolysis, matrix-assisted laser desorption/ionization mass spectrometry, high-performance liquid chromatography and size-exclusion chromatography of p21Waf1/Cip1/Sdi1

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53 tumor suppressor-dependent cell growth control in humans and other organisms, mediates G1/S-phase arrest through inhibition of cyclin-dependent kinases (Cdks). The enzymatic activity of these kinases is essential for progress through the cell division cycle and one level of cell cycle regulation is exerted through inhibition of Cdks by a family of small proteins, including p21. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2-terminus. Using proteolytic mapping, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, HPLC and size-exclusion chromatography, we show that p21, active as a Cdk inhibitor, exists in an extended, non-globular conformation in the absence of its biological target and that p21 lacks the hallmarks of stable secondary and tertiary structure. We have developed an efficient approach to obtain detailed proteolytic maps that takes advantage of the high accuracy and sensitivity of MALDI mass spectrometry. Our method allows a proteolytic map to be obtained from a single mass spectrum for fragments produced from a single proteolytic reaction.

1H resonances of proximal histidine in CO complexes of hemoglobins provide a sensitive probe of coordination geometry

A straightforward strategy for assignment of the CϵH, CδH and NδH proton resonances of the proximal histidine ligand in diamagnetic complexes of monomeric hemoglobins and myoglobins is reported. These resonances are subject to large ring current shifts and are highly sensitive to coordination geometry. There are no significant differences between the CO complexes of myoglobin, leghemoglobin or hemoglobin α‐subunits in proximal His coordination geometry or hydrogen bonding to the backbone at Leu F4. Ring current calculations show that the His F8 coordination geometry in the CO complexes of myoglobin and hemoglobin α‐subunits is very similar in crystal and solution.

1H, 15N and 13C resonance assignments for the first three zinc fingers of transcription factor IIIA

SummaryThe first three zinc fingers (ZF1-3) of transcription factor IIIA (TFIIIA) from Xenopus have been shown to contribute the majority of the binding energy to the intact TFIIIA-DNA interaction [Liao et al. (1992) J. Mol. Biol., 223, 857–871]. We have expressed a 92-amino acid polypeptide containing the three N-terminal zinc fingers of TFIIIA. This three-fingered polypeptide has been isotopically labeled with 15N and 13C in E. coli and purified to homogeneity. Assignment of backbone 1H, 15N, aliphatic 1H and 13C and aromatic 1H and 13C resonances of ΔNZF1-3 has been obtained using a combination of single-, double-and triple-resonance multidimensional NMR experiments. The secondary structures for each finger have been determined from NOE connectivities, 3JNHα values and chemical shifts. The results show that each finger folds into a canonical β-sheet-helix zinc finger structural motif, while the linkers adopt an extended structure. The helix between the two histidine ligands in ZF3 is distorted by zinc coordination, to accommodate the presence of four intervening amino acids instead of three as in ZF1 and ZF2.

1H NMR studies of heme pocket conformation in zinc-substituted leghemoglobin, a diamagnetic analog of deoxyleghemoglobin.

Reconstitution of apoleghemoglobin with zinc protoporphyrin IX is reported. NMR spectra show that the reconstitution is orientation specific and that there is no detectable heme isomerism or conformational heterogeneity. Resonances of heme substituents and distal and proximal amino acid protons have been assigned. Only minor differences in porphyrin-protein packing occur between zinc leghemoglobin and the CO complex of ferrous leghemoglobin. The zinc is five-coordinate and is ligated by the proximal histidine. Comparisons with diamagnetic six-coordinate complexes show that the distal His-61 and Leu-65 side chains move away from the binding site upon coordination of exogenous ligands. Conformational changes are minimal when the ligand is O2.

Letter to the Editor: Assignment of 1H, 13C and 15N resonances of the I-domain of human leukocyte function associated antigen-1

The interaction between the integrin leukocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) is primarily mediated via an inserted domain (I-domain) of approximately 190 amino acids (for review see Gahmberg, 1997), which is a common feature of all leukocyte integrins. X-ray structures of the LFA-1 and Mac-1 I-domains in the presence of EDTA or divalent cations are available (see, for example, Qu and Leahy, 1995), and we have recently completed an NMR structure determination (Legge et al., 2000) which was based on the sequencespecific assignments of the LFA-1 I-domain reported in the present paper. These assignments provide the basis for further studies on LFA-1 I-domain/ICAM-1 interactions.