Search PubMed⌕ Search

Biomedical subjects

M Nilges

Publications and source records attributed to M Nilges.

At least 55 records · Page 3Linked to original sources
PubMed1997

Floating stereospecific assignment revisited: application to an 18 kDa protein and comparison with J-coupling data.

We report a floating chirality procedure to treat nonstereospecifically assigned methylene or isopropyl groups in the calculation of protein structures from NMR data using restrained molecular dynamics and simulated annealing. The protocol makes use of two strategies to induce the proper conformation of the prochiral centres: explicit atom 'swapping' following an evaluation of the NOE energy term, and atom 'floating' by reducing the angle and improper force constants that enforce a defined chirality at the prochiral centre. The individual contributions of both approaches have been investigated. In addition, the effects of accuracy and precision of the interproton distance restraints were studied. The model system employed is the 18 kDa single-stranded DNA binding protein encoded by Pseudomonas bacteriophage Pf3. Floating chirality was applied to all methylene and isopropyl groups that give rise to non-degenerate NMR signals, and the results for 34 of these groups were compared to J-coupling data. We conclude that floating stereospecific assignment is a reliable tool in protein structure calculation. Its use is beneficial because it allows the distance restraints to be extracted directly from the measured peak volumes without the need for averaging or adding pseudoatom corrections. As a result, the calculated structures are of a quality almost comparable to that obtained with stereospecific assignments. As floating chirality furthermore is the only approach treating prochiral centres that ensures a consistent assignment of the two proton frequencies in a single structure, it seems to be preferable over using pseudoatoms or (R(-6)) averaging.

Amino Acid Sequence↗
PubMed1997

The structure of a novel insecticidal neurotoxin, omega-atracotoxin-HV1, from the venom of an Australian funnel web spider.

A family of potent insecticidal toxins has recently been isolated from the venom of Australian funnel web spiders. Among these is the 37-residue peptide omega-atracotoxin-HV1 (omega-ACTX-HV1) from Hadronyche versuta. We have chemically synthesized and folded omega-ACTX-HV1, shown that it is neurotoxic, ascertained its disulphide bonding pattern, and determined its three-dimensional solution structure using NMR spectroscopy. The structure consists of a solvent-accessible beta-hairpin protruding from a disulphide-bonded globular core comprising four beta-turns. The three intramolecular disulphide bonds from a cystine knot motif similar to that seen in several other neurotoxic peptides. Despite limited sequence identity, omega-ACTX-HV1 displays significant structural homology with the omega-agatoxins and omega-conotoxins, both of which are vertebrate calcium channel antagonists; however, in contrast with these toxins, we show that omega-ACTX-HV1 inhibits insect, but not mammalian, voltage-gated calcium channel currents.

Amino Acid Sequence↗
PubMed1997

Solution structure of the DNA-binding domain and model for the complex of multifunctional hexameric arginine repressor with DNA.

The structure of the monomeric DNA-binding domain of the Escherichia coli arginine repressor, ArgR, determined by NMR spectroscopy, shows structural homology to the winged helix-turn-helix (wHTH) family, a motif found in a diverse class of proteins including both gene regulators and gene organizers from prokaryotes and eukaryotes. Biochemical data on DNA binding by intact ArgR are used as constraints to position the domain on its DNA target and to derive a model for the hexamer-DNA complex using the known structure of the L-arginine-binding domain. The structural independence of the wHTH fold may be important for multimeric DNA-binding proteins that contact extended DNA regions with imperfect match to consensus sequences, a feature of many wHTH-domain proteins.

Amino Acid Sequence↗
PubMed1996

Molecular replacement with NMR models using distance-derived pseudo B factors.

The statistical significance of molecular-replacement solutions with models derived from NMR data is strongly enhanced if pseudo B factors that reflect expected atomic coordinate errors are introduced. These B factors are derived from atomic distances of an ensemble of NMR models and an averaged model. A recently determined X-ray structure of a Pleckstrin homology domain:ligand complex has been used as a test case for molecular replacement with NMR templates. The feasibility of the models for molecular replacement has been studied in two steps: (i) correctness of solutions, verified by correct rotational angles and translations; (ii) statistical significance (detection) of solutions, measured by R factors and correlation coefficients in the translation function. None of the models with uniform B factors were detectable in the translation function whereas two models with distance-derived B factors gave statistically significant R factors and correlation coefficients. The potential impact of distance-derived B factors on the detectability of molecular-replacement solutions was further tested by using B factors that were derived from spatial distances between each NMR model and the X-ray structure. It is concluded that the introduction of distance-derived B factors can be an essential component of many future molecular-replacement problems that use NMR models as templates.

Journal Article↗
PubMed1996

Structure determination of the N-terminal thioredoxin-like domain of protein disulfide isomerase using multidimensional heteronuclear 13C/15N NMR spectroscopy.

As a first step in dissecting the structure of human protein disulfide isomerase (PDI), the structure of a fragment corresponding to the first 120 residues of its sequence has been determined using heteronuclear multidimensional NMR techniques. As expected from its primary structure homology, the fragment has the thioredoxin fold. Similarities and differences in their structures help to explain why thioredoxins are reductants, whereas PDI is an oxidant of protein thiol groups. The results confirm that PDI has a modular, multidomain structure, which will facilitate its structural and functional characterization.

Amino Acid Sequence↗
PubMed1996

High resolution NMR solution structure of the leucine zipper domain of the c-Jun homodimer.

The solution structure of the c-Jun leucine zipper domain has been determined to high resolution using a new calculation protocol designed to handle highly ambiguous sets of interproton distance restraints. The domain comprises a coiled coil of parallel alpha-helices in which most of the hydrophobic residues are buried at the highly symmetrical dimer interface; this interface extends over 10 helical turns and is the most elongated protein domain solved to date using NMR methods. The backbone fold is very similar to that seen in crystal structures of the GCN4 and Jun-Fos leucine zippers; however, in contrast with these crystal structures, the Jun leucine zipper dimer appears to be devoid of favorable intermolecular electrostatic interactions. A polar asparagine residue, located at the dimer interface, forms the sole point of asymmetry in the structure; furthermore, the side chain of this residue is disordered due to motional averaging. This residue, which is highly conserved in the leucine zipper family of transcription factors, provides a destabilizing influence that is likely to facilitate the rapid exchange of zipper strands in vivo.

Amino Acid Sequence↗
PubMed1996

Three-dimensional structure and stability of the KH domain: molecular insights into the fragile X syndrome.

The KH module is a sequence motif found in a number of proteins that are known to be in close association with RNA. Experimental evidence suggests a direct involvement of KH in RNA binding. The human FMR1 protein, which has two KH domains, is associated with fragile X syndrome, the most common inherited cause of mental retardation. Here we present the three-dimensional solution structure of the KH module. The domain consists of a stable beta alpha alpha beta beta alpha fold. On the basis of our results, we suggest a potential surface for RNA binding centered on the loop between the first two helices. Substitution of a well-conserved hydrophobic residue located on the second helix destroys the KH fold; a mutation of this position in FMR1 leads to an aggravated fragile X phenotype.

Asparagine↗
PubMed1996

Structure calculation from NMR data.

NMR calculation methods have kept pace with the rapid extension of NMR experiments to larger molecules. By including additional data and effects of local dynamics in the refinement, we can obtain a more complete picture of the molecule in solution. The structure determination process is being aided by new methods to solve some aspects of spectral assignment during the structure calculation.

Animals↗
PubMed1995

The leucine zippers of the HLH-LZ proteins Max and c-Myc preferentially form heterodimers.

c-Myc and Max are members of a subfamily of the helix-loop-helix transcription-regulating proteins. Their function is mediated by switches in the dimerization partners; c-Myc does not homodimerize in vivo but competes with Mad, another member of the subfamily, to form heterodimers with Max, leading to either activation or repression of transcription. Max is also able to form homodimers. In an attempt to identify which regions of the proteins carry the information to determine specific recognition of the dimerization partner, we have investigated the dimerization properties of synthetic peptides corresponding to the leucine zipper sequence of Max and c-Myc using circular dichroism and nuclear magnetic resonance techniques. We show that the heterodimer is obtained readily by simply mixing the peptides and that at neutral pH it is more stable than the homodimer of the Max leucine zipper. We have shown in a previous paper [Muhle-Goll, C. et al. (1994) Biochemistry 33, 11296-11306] that the leucine zipper of c-Myc does not form stable homodimers under these conditions. Thus, the leucine zipper regions of these two proteins by themselves display the same behavior as the entire proteins. However, even the heterodimer is less stable than dimers of leucine zippers of the basic leucine zipper family such as GCN4 and Fos-Jun. The specificity of the interaction between different monomers can be explained by polar interactions. We investigate the structural role of the polar and charged residues in the hydrophobic interface by molecular-modeling studies.

Amino Acid Sequence↗
PubMed1995

Structure of the binding site for inositol phosphates in a PH domain.

Phosphatidylinositol bisphosphate has been found to bind specifically to pleckstrin homology (PH) domains that are commonly present in signalling proteins but also found in cytoskeleton. We have studied the complexes of the beta-spectrin PH domain and soluble inositol phosphates using both circular dichroism and nuclear magnetic resonance spectroscopy, and X-ray crystallography. The specific binding site is located in the centre of a positively charged surface patch of the domain. The presence of 4,5-bisphosphate group on the inositol ring is critical for binding. In the crystal structure that has been determined at 2.0 A resolution, inositol-1,4,5-trisphosphate is bound with salt bridges and hydrogen bonds through these phosphate groups whereas the 1-phosphate group is mostly solvent-exposed and the inositol ring has virtually no interactions with the protein. We propose a model in which PH domains are involved in reversible anchoring of proteins to membranes via their specific binding to phosphoinositides. They could also participate in a response to a second messenger such as inositol trisphosphate, organizing cross-roads in cellular signalling.

Amino Acid Sequence↗
PubMed1995

Solution structure of the single-stranded DNA binding protein of the filamentous Pseudomonas phage Pf3: similarity to other proteins binding to single-stranded nucleic acids.

The three-dimensional structure of the homodimeric single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been determined using heteronuclear multidimensional NMR techniques and restrained molecular dynamics. NMR experiments and structure calculations have been performed on a mutant protein (Phe36 --> His) that was successfully designed to reduce the tendency of the protein to aggregate. The protein monomer is composed of a five-stranded antiparallel beta-sheet from which two beta-hairpins and a large loop protrude. The structure is compared with the single-stranded DNA binding protein encoded by the filamentous Escherichia coli phage Ff, a protein with a similar biological function and DNA binding properties, yet quite different amino acid sequence, and with the major cold shock protein of Escherichia coli, a single-stranded DNA binding protein with an entirely different sequence, biological function and binding characteristics. The amino acid sequence of the latter is highly homologous to the nucleic acid binding domain (i.e. the cold shock domain) of proteins belonging to the Y-box family. Despite their differences in amino acid sequence and function, the folds of the three proteins are remarkably similar, suggesting that this is a preferred folding pattern shared by many single-stranded DNA binding proteins.

Coliphages↗
PubMed1995

Refined solution structure of the Tyr41-->His mutant of the M13 gene V protein. A comparison with the crystal structure.

The three-dimensional solution structure of mutant Tyr41-->His of the single-stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13 has been refined in two stages. The first stage involved the collection of additional NOE-based distance constraints, which were then used in eight cycles of back-calculations and structure calculations. The structures of the gene V protein dimers were calculated using simulated annealing, employing restrained molecular dynamics with a geometric force field. In the second stage of the refinement procedure, solvent was explicitly included during the dynamic calculations. A total of 30 structures was calculated for the protein, representing its solution structure in water. The first calculation step significantly improved the convergence of the structures, whereas the subsequent simulations in water made the structures physically more realistic. This is, for instance, illustrated by the number of hydrogen bonds formed in the molecule, which increased considerably upon going to aqueous solution. It is shown that the solution structure of the mutant gene V protein is nearly identical to the crystal structure of the wild-type molecule, except for the DNA-binding loop (residues 16-28). This antiparallel beta-hairpin is twisted and partially folded back towards the core of the protein in the NMR structure, whereas it is more extended and points away from the rest of the molecule in the X-ray structure. Unrestrained molecular dynamics calculations suggest that this latter conformation is energetically unstable in solution.

Amino Acid Sequence↗
PubMed1995

Structure of the dsRNA binding domain of E. coli RNase III.

The double-stranded RNA binding domain (dsRBD) is a approximately 70 residue motif found in a variety of modular proteins exhibiting diverse functions, yet always in association with dsRNA. We report here the structure of the dsRBD from RNase III, an enzyme present in most, perhaps all, living cells. It is involved in processing transcripts, such as rRNA precursors, by cleavage at short hairpin sequences. The RNase III protein consists of two modules, a approximately 150 residue N-terminal catalytic domain and a approximately 70 residue C-terminal recognition module, homologous with other dsRBDs. The structure of the dsRBD expressed in Escherichia coli has been investigated by homonuclear NMR techniques and solved with the aid of a novel calculation strategy. It was found to have an alpha-beta-beta-beta-alpha topology in which a three-stranded anti-parallel beta-sheet packs on one side against the two helices. Examination of 44 aligned dsRBD sequences reveals several conserved, positively charged residues. These residues map to the N-terminus of the second helix and a nearby loop, leading to a model for the possible contacts between the domain and dsRNA.

Amino Acid Sequence↗
PubMed1995

Calculation of protein structures with ambiguous distance restraints. Automated assignment of ambiguous NOE crosspeaks and disulphide connectivities.

The distances derived from nuclear Overhauser effect (NOE) spectra are usually converted into three-dimensional structures by computer algorithms loosely termed distance geometry. To a varying degree, these methods require that the distance data is unambiguously assigned to pairs of atoms. Typically, however, there are many NOE crosspeaks that cannot be assigned without some knowledge of the structure. These crosspeaks have to be assigned in an iterative manner, using preliminary structures calculated from the unambiguous crosspeaks. In this paper, I present an alternative to this iterative approach. The ambiguity of an NOE crosspeak is correctly described in terms of the distances between all pairs of protons that may be involved. A simple restraining term is defined in terms of "ambiguous" distance restraints that can allow all possible assignments. A new minimization procedure based on simulated annealing is described that is capable of using highly ambiguous data for ab initio structure calculations. In particular, it is feasible to specify the restraint list directly in terms of the proton chemical shift assignment and the NOE peak table, without having assigned NOE crosspeaks to proton pairs. While the primary aim of this paper is determining the global fold of proteins from NMR data, similar strategies can be used for other types of ambiguous distance data. The application to one example, disulphide bridges with unknown connectivity, is described. Model NOE data were generated from the X-ray crystal structure of a small protein with known chemical shift assignments. Varying degrees of ambiguity in the data were assumed. The method obtained the correct polypeptide fold even when all distance restraints were ambiguous. Thus, the new approach may facilitate structure calculations with data derived from very overlapped spectra. It is also a step towards automating the calculation of structures from NMR data. This could prove especially valuable for data derived from three- and four-dimensional experiments. The approach may also prove useful for model building studies and tertiary structure prediction.

Disulfides↗
PubMed1994

The dimerization stability of the HLH-LZ transcription protein family is modulated by the leucine zippers: a CD and NMR study of TFEB and c-Myc.

In the HLH-LZ protein family, the helix-loop-helix DNA-binding dimerization domain is followed in the sequence by a leucine zipper motif. The precise function of this second dimerization domain is still unclear, since the HLH motif of a subset of this family has been shown to be necessary and sufficient for dimerization. However, deletion and mutagenesis studies of the leucine zipper in various HLH-LZ proteins have shown a clear influence of this motif on homo- and heterodimerization. In this paper, we present a structural characterization of synthetic peptides encompassing the leucine zipper sequences of c-Myc and TFEB, using circular dichroism, analytical ultracentrifugation, and nuclear magnetic resonance. We show that the different ability of the synthetic leucine zippers of c-Myc and TFEB to homodimerize at neutral pH reflects the different dimerization properties reported for the entire proteins. The TFEB protein is known to form homodimers. c-Myc, on the other hand, does not homodimerize in vivo, but is mostly found in heterodimeric complexes with Max, another protein of the HLH-LZ family. Accordingly, our results show that the TFEB peptide homodimerizes at neutral pH whereas the Myc peptide dimerizes to a comparable amount only at acidic pH and high ionic strength. Both synthetic peptides are far less stable than leucine zippers of the b-ZIP family. The relative stability of the two leucine zippers and the factors which stabilize the dimer formation are discussed.

Amino Acid Sequence↗
PubMed1994

A model of the complex between single-stranded DNA and the single-stranded DNA binding protein encoded by gene V of filamentous bacteriophage M13.

A contact analysis and a series of restrained molecular dynamics simulations were employed to derive a model of the complex between single-stranded DNA and the single-stranded DNA-binding protein encoded by gene V of the filamentous phage M13. The study is based on the recently elucidated solution structure of the Tyr41-->His mutant of the protein. Electron microscopy studies, indicating that the complex forms a flexible, left-handed helical coil with a diameter of 8 to 9 nm and an average pitch of 9 nm, were taken into consideration. The contact analysis served to determine the helix parameters that permit the energetically most favourable packing of protein molecules. Then a protein super-helix was built, into which two extended strands of DNA were modelled using restrained molecular dynamics. Specific constraints were included to ensure that the DNA would position itself into the binding groove of the protein. These constraints are based on recent NMR spin label experiments which offered a direct identification of the amino acids of the protein present in the DNA-binding domain. We present a model for the complex which is in full agreement with the existing reliable biophysical and biochemical data. A description of the protein-protein interface is given and the protein-DNA interaction is discussed in view of the derived model. In addition, we demonstrate that, on the basis of the available experimental data, and not imposing the left-handedness of the nucleoprotein complex, it is feasible to build also a plausible model for the complex which exhibits the opposite, i.e. right-handed, helical sense. This nucleoprotein structure features characteristics highly similar to those of the left-handed helix.

Bacteriophage M13↗