Gottfried Otting

The structure of the antennapedia homeodomain determined by nmr spectroscopy in solution comparison with prokaryotic repressors

The structure of the Antennapedia homeodomain from Drosophila melanogaster was determined by nuclear magnetic resonance spectroscopy in solution. It includes three well-defined helices (residues 10-21, 28-38, and 42-52) and a more flexible fourth helix (residues 53-59). Residues 30-50 form a helix-turn-helix motif virtually identical to those observed in various prokaryotic repressors. Further comparisons of the homeodomain with prokaryotic repressors showed that there are also significant differences in the molecular architectures. Overall, these studies support the view that the third helix of the homeodomain may function as the DNA recognition site. The elongation of the third helix by the fourth helix is a structured element that so far appears to be unique to the Antennapedia homeodomain.

Protein - DNA contacts in the structure of a homeodomain - DNA complex determined by nuclear magnetic resonance spectroscopy in solution

The 1:1 complex of the mutant Antp(C39––S) homeodomain with a 14 bp DNA fragment corresponding to the BS2 binding site was studied by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. The complex has a molecular weight of 17,800 and its lifetime is long compared with the NMR chemical shift time scale. Investigations of the three‐dimensional structure were based on the use of the fully 15N‐labelled protein, two‐dimensional homonuclear proton NOESY with 15N(omega 2) half‐filter, and heteronuclear three‐dimensional NMR experiments. Based on nearly complete sequence‐specific resonance assignments, both the protein and the DNA were found to have similar conformations in the free form and in the complex. A sufficient number of intermolecular 1H‐1H Overhauser effects (NOE) could be identified to enable a unique docking of the protein on the DNA, which was achieved with the use of an ellipsoid algorithm. In the complex there are intermolecular NOEs between the elongated second helix in the helix‐turn‐helix motif of the homeodomain and the major groove of the DNA. Additional NOE contacts with the DNA involve the polypeptide loop immediately preceding the helix‐turn‐helix segment, and Arg5. This latter contact is of special interest, both because Arg5 reaches into the minor groove and because in the free Antp(C39––S) homeodomain no defined spatial structure could be found for the apparently flexible N‐terminal segment comprising residues 0‐6.

Heteronuclear filters in two-dimensional [1H,1H]-NMR spectroscopy: combined use with isotope labelling for studies of macromolecular conformation and intermolecular interactions.

The use of heteronuclear filters enables the editing of complex 1 H nuclear magnetic resonance (NMR) spectra into simplified subspectra containing a lesser number of resonance lines, which are then more easily amenable to detailed spectral analysis. This editing is based on the creation of heteronuclear two-spin or multiple-spin coherence and discrimination between protons that do or do not participate in these heteronuclear coherences. In principle, heteronuclear editing can be used in conjunction with one-dimensional or multidimensional 1 H-NMR experiments for studies of a wide variety of low-molecular-weight compounds or macromolecular systems, and is implicitely applied in a wide range of heteronuclear NMR experiments with proton detection (e.g. Bax et al. 1983; Griffey & Redfield, 1987). In the present article we shall focus on the use of heteronuclear filters in two-dimensional (2D) [ 1 H, 1 H]-NMR experiments. The selection of the material covered was primarily motivated by its impact on the practice of protein structure determination in solution, and on NMR studies of intermolecular interactions with biological macromolecules. Section 2 surveys potential applications of heteronuclear filters in this area. The remainder of the article is devoted to an introduction of the theoretical principles used in heteronuclear filters, and to a detailed description of the experimental implementation of these measurements. In writing the review we tried to minimize redundancy with the recent article in Quarterly Review of Biophysics by Griffey & Redfield (1987) and to concentrate on experiments that were introduced during the period 1986–9.

The structure of the homeodomain and its functional implications

The three-dimensional structure of the homeodomain, as determined by nuclear magnetic resonance spectroscopy, reveals the presence of a helix-turn-helix motif, similar to the one found in prokaryotic gene regulatory proteins. Isolated homeodomains bind with high affinity to specific DNA sequences. Thus, the structure-function relationship is highly conserved in evolution.

Determination of scalar coupling constants by inverse Fourier transformation of in-phase multiplets

Abstract A generally applicable new method for the determination of scalar coupling constants for spins with a single coupling partner, in particular 3JHNα. in polypeptides, is described. It has the special advantage for use in protein structure determinations that no extra NMR experiments need to be recorded, and that NOE distance constraints and dihedral angle constraints from 3JHNα can be derived from the same data sets. The scalar coupling constants are extracted from the in-phase multiplets of homonuclear [1H, 1H] NOESY spectra or heteronuclear [15N, 1H ] COSY spectra through inverse Fourier transformation of the data points representing a cross peak along ω2, and a subsequent nonlinear least-squares fit in the time domain. Practical applications are described for recombinant hirudin and the basic pancreatic trypsin inhibitor.

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.

Editing of 2D 1H NMR spectra using X half-filters. combined use with residue-selective 15N labeling of proteins

The introduction of efficient sequential assignment techniques for obtaining sequence-specific H NMR assignments in proteins (1, 2) has provided a basis for determination of the three-dimensional molecular structure in solution (3, 4), as well as for detailed studies of the molecular dynamics (e.g., (5)) and of intermolecular interactions (4). Limitations on the use of this approach arise if two or several cross peaks in the 2D H NMR spectra overlap. The degree of spectral overlap increases quite naturally with increasing molecular size , but if the H resonances are not well dispersed complete sequence -specific assignment of the spectrum may be difficult even for small proteins. As was also pointed out by others (6-9), the situation can be improved by residue-selective isotope labeling with15N or t3C. In such preparations the protons directly bound to 15N or 13C can be singled out on the basis of the large heteronuclear spin-spin coupling constants JXH. Simplified spectra have thus been obtained which correspond either to the difference between recordings with and without broadband decoupling of the X nucleus (6, 7), or with and without ir(15N) refocusing pulse (8), or were recorded with a heteronuclear zero-quantum filter (9). In this communication we describe the use of X(w,) and X(w2) half-filters for editing the 2D H NMR spectra of X-labeled molecules. Compared to difference spectroscopy techniques the half-filters have the advantage of providing two subspectra corresponding, respectively, to the X-labeled H peaks and to all other peaks. Compared to the heteronuclear zero-quantum filter (9) the sensitivity of the corresponding half-filter is improved by a factor 2. X half-filters can be employed with a wide variety of 2D NMR experiments, including 2D correlated spectroscopy (COSY), 2D total correlation spectroscopy (TOCSY), and 2D nuclear Overhauser enhancement spectroscopy (NOESY) (Fig. 1). Clearly, their use is not limited to proteins and there is a wide spectrum of potential applications with other macromolecules and with small molecules , in particular for studies of intermolecular interactions. In the experimental schemes for obtaining the desired simplified spectra with 15Nlabeled proteins, we inserted 15N half-filters consisting of a [-T/2-7r(H, 15N)-T/2r(15N)-] pulse sequence, either before the evolution period (w, half-filter) or immediately before detection (w2 half-filter) of the conventional 2D H NMR experiments

Structure determination of the Antp (C39----S) homeodomain from nuclear magnetic resonance data in solution using a novel strategy for the structure calculation with the programs DIANA, CALIBA, HABAS and GLOMSA.

The structure of a mutant Antennapedia homeodomain, Antp(C39----S), from Drosophila melanogaster was determined using a set of new programs introduced in the accompanying paper. An input dataset of 957 distance constraints and 171 dihedral angle constraints was collected using two-dimensional n.m.r. experiments with the 15N-labeled protein. The resulting high quality structure for Antp(C39----S), with an average root-mean-square deviation of 0.53 A between the backbone atoms of residues 7 to 59 in 20 energy-refined distance geometry structures and the mean structure, is nearly identical to the previously reported structure of the wild-type Antp homeodomain. The only significant difference is in the connection between helices III and IV, which was found to be less kinked than was indicated by the structure determination for Antp. The main emphasis of the presentation in this paper is on a detailed account of the practical use of a novel strategy for the computation of nuclear magnetic resonance structures of proteins with the combined use of the programs DIANA, CALIBA, HABAS and GLOMSA.

Secondary structure determination for the Antennapedia homeodomain by nuclear magnetic resonance and evidence for a helix-turn-helix motif.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was studied in aqueous solution by nuclear magnetic resonance (NMR). Sequence‐specific resonance assignments have been obtained for the complete polypeptide chain of 68 amino acid residues. The secondary structure determined from nuclear Overhauser effects (NOE) and information about slowly exchanging amide protons includes three helical segments consisting of the residues 10‐21, 28‐38 and 42‐52, respectively. Combination of the presently available NMR data with computer modeling provided preliminary evidence for the presence of a helix‐turn‐helix motif in the homeodomain. Near the turn, this supersecondary structure appears to be very similar to the DNA binding site in the 434 and P22 c2 repressors, but both helices in the homeodomain include 2‐3 additional residues when compared with these prokaryotic DNA‐binding proteins.

Protein hydration studied with homonuclear 3D1H NMR experiments

SummaryHomonuclear 3D1H NOESY-TOCSY and 3D1H ROESY-TOCSY experiments were used to resolve and assign nuclear Overhauser effect (NOE) cross peaks between the water signal and individual polypeptide proton resonances in H2O solutions of the basic pancreatic trypsin inhibitor. Combined with a novel, robust water-suppression technique, positive and negative intermolecular NOEs were detected at 4°C. The observation of positive NOEs between water protons and protein protons enables more precise estimates of the very short residence times of the water molecules in the hydration sites on the protein surface.