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M Nilges

Publications and source records attributed to M Nilges.

At least 37 records · Page 2Linked to original sources
PubMed1999

The PH superfold: a structural scaffold for multiple functions.

Pleckstrin homology (PH) domains form a structurally conserved family that is associated with many regulatory pathways within the cell. Domains with a nearly identical fold are found in other families that share no sequence similarity, suggesting the existence of a stable PH superfold. The PH domains generally function as regulated membrane-binding modules that bind to inositol lipids and respond to upstream signals by targeting the host proteins to the correct cellular sites. The other domains with a similar fold, such as the phosphotyrosine binding domains, recognize protein ligands.

Amino Acid Motifs↗
PubMed1999

The structure in solution of the b domain of protein disulfide isomerase.

Protein disulfide isomerase (PDI) is a multifunctional protein of the endoplasmic reticulum, which catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. It consists of four domains designated a, b, b and a. Both a and a domains contains an active site with the sequence motif -Cys-Gly-His-Cys-involved directly in thiol-disulfide exchange reactions. As expected these domains have structures very similar to the ubiquitous redox protein thioredoxin. A low-resolution NMR structure of the b domain revealed that this domain adopts a fold similar to the PDI a domain and thioredoxin [Kemmink, J., Darby, N.J., Dijkstra, K., Nilges, M. and Creighton, T.E. (1997) Curr. Biol. 7, 239-245]. A refined ensemble of solution structures based on the input of 1865 structural restraints shows that the structure of PDI b is well defined throughout the complete protein except for about 10 residues at the C-terminus of the sequence. 15N relaxation data show that these residues are disordered and not part of this structural domain. Therefore the domain boundaries of PDI can now be fixed with reasonable precision. Structural comparison of the PDI b domain with thioredoxin and PDI a reveals several features important for thiol-disulfide exchange activity.

Amino Acid Sequence↗
PubMed1999

StarDOM: from STAR format to XML.

StarDOM is a software package for the representation of STAR files as document object models and the conversion of STAR files into XML. This allows interactive navigation by using the Document Object Model representation of the data as well as easy access by XML query languages. As an example application, the entire BioMagResBank has been transformed into XML format. Using an XML query language, statistical queries on the collected NMR data sets can be constructed with very little effort. The BioMagResBank/XML data and the software can be obtained at http://www.nmr.embl-heidelberg.de/nmr/StarDOM linge@embl-heidelberg.de

Databases, Factual↗
PubMed1999

Influence of non-bonded parameters on the quality of NMR structures: a new force field for NMR structure calculation.

The effects of different non-bonded parameters of force fields for NMR structure calculation on the quality of the resulting NMR solution structures were investigated using Interleukin 4 as a model system. NMR structure ensembles were calculated with an ab initio protocol using torsion angle dynamics. The calculations were repeated with five different non-bonded energy functions and parameters. The resulting ensembles were compared with the available X-ray structures, and their quality was assessed with common structure validation programs. In addition, the impact of torsion angle restraints and dihedral energy terms for the sidechains and the backbone was studied. The further improvement of the quality by refinement in explicit solvent was demonstrated. The optimal parameters, including those necessary for water refinement, are available in the new version of the PARALLHDG force field.

Interleukin-4↗
PubMed1998

The three-dimensional structure of a type I module from titin: a prototype of intracellular fibronectin type III domains.

BACKGROUND: Titin is a huge protein ( approximately 3 MDa) that is present in the contractile unit (sarcomere) of striated muscle and has a key role in muscle assembly and elasticity. Titin is mainly composed of two types of module (type I and II). Type I modules are found exclusively in the region of titin localised in the A band, where they are arranged in a super-repeat pattern that correlates with the ultrastructure of the thick filament. No structure of a titin type I module has been reported so far. RESULTS: We have determined the structure of a representative type I module, A71, using nuclear magnetic resonance (NMR) spectroscopy. The structure has the predicted fibronectin type III fold. Titin-specific conserved residues are either located at the putative module-module interfaces or along one side of the protein surface. Several proline residues that contribute to two stretches in a polyproline II helix conformation are solvent-exposed and line up as a continuous ribbon extending over more than two-thirds of the module surface. Homology models of the type I module N-terminal to A71 (A70) and the double module A70-A71 were used to discuss possible intermodule interactions and their role in module-module orientation. CONCLUSIONS: As residues at the module-module interfaces are highly conserved, we speculate that similar interactions govern all of the interfaces between type I modules in titin. This conservation would lead to a regular multiple array of similar surface structures. Such an arrangement would allow arrays of contiguous type I modules to expose multiple proline stretches in a highly regular way and these may act as binding sites for other thick filament proteins.

Amino Acid Sequence↗
PubMed1998

Crystallography & NMR system: A new software suite for macromolecular structure determination.

A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.

Computer Simulation↗
PubMed1998

Heteronuclear relaxation study of the PH domain of beta-spectrin: restriction of loop motions upon binding inositol trisphosphate.

The structural dynamics of protein ligand binding sites is one factor determining the specificity towards related ligands. In this context, the spectrin PH domain, which binds to a number of phosphatidylinositol lipid head groups, was investigated with respect to the dynamics of the binding loops. The latter were found to be of intermediate flexibility on a picosecond to nanosecond time-scale in the free protein and become more rigid upon ligand binding. Significant 15N and proton chemical shift changes occur in the binding loops. The internal correlation time, determined from 15N heteronuclear relaxation data using the standard model-free approach, decreases upon ligand binding. For several residues a concomitant rise in the generalized order parameter is observed. This is interpreted as a dampening effect of the ligand on a slow loop motion, while a fast component is not affected. Molecular dynamics simulations were performed to further investigate this situation. In fact, two time-scales of loop motions in the free state are observed in a 9 ns molecular dynamics trajectory. Agreement with generalized order parameters obtained from the experiment improves when a subtrajectory is analyzed that excludes rare dihedral transitions.

Binding Sites↗
PubMed1998

Are there non-trivial dynamic cross-correlations in proteins?

The analysis of internal motion in ensembles of flexible molecules in coordinate space requires the removal of overall motion by a least-squares fitting procedure of the Cartesian coordinates. It has been demonstrated that the choice of the atom set used for fitting influences the picture of the internal motion of BPTI. We have performed essential dynamics analyses of a 1 ns molecular dynamics trajectory of the single-stranded DNA-binding protein from the Pf3 phage using either all alpha-carbon atoms or the least mobile ones for fitting the trajectory prior to the analysis. We found that covariances of atoms separated by long distances were significantly reduced in the latter case; the overall overlap of essential spaces was still high. In the second part we present a method that does not introduce and bias caused by overall motion: principal component analysis in distance space. Non-trivial dynamic cross-correlations were preserved in distance space, which answers the question posed in the title in the affirmative. However, cross-correlations were throughout smaller than those detected by standard essential dynamics analyses.

Bacteriophages↗
PubMed1998

Essential spaces defined by NMR structure ensembles and molecular dynamics simulation show significant overlap.

Large concerted motions of proteins which span its "essential space," are an important component of protein dynamics. We investigate to what extent structure ensembles generated with standard structure calculation techniques such as simulated annealing can capture these motions by comparing them to long-time molecular dynamics (MD) trajectories. The motions are analyzed by principal component analysis and compared using inner products of eigenvectors of the respective covariance matrices. Two very different systems are studied, the beta-spectrin PH domain and the single-stranded DNA binding protein (ssDBP) from the filamentous phage Pf3. A comparison of the ensembles from NMR and MD shows significant overlap of the essential spaces, which in the case of ssDBP is extraordinarily high. The influence of variations in the specifications of distance restraints is investigated. We also study the influence of the selection criterion for the final structure ensemble on the definition of mobility. The results suggest a modified criterion that improves conformational sampling in terms of amplitudes of correlated motion.

Computer Simulation↗
PubMed1997

Solution structure of the spectrin repeat: a left-handed antiparallel triple-helical coiled-coil.

Cytoskeletal proteins belonging to the spectrin family have an elongated structure composed of repetitive units. The three-dimensional solution structure of the 16th repeat from chicken brain alpha-spectrin (R16) has been determined by NMR spectroscopy and distance geometry-simulated annealing calculations. We used a total of 1035 distance restraints, which included 719 NOE-based values obtained by applying the ambiguous restraints for iterative assignment (ARIA) method. In addition, we performed a direct refinement against 1H-chemical shifts. The final ensemble of 20 structures shows an average RMSD of 1.52 A from the mean for the backbone atoms, excluding loops and N and C termini. R16 is made up of three antiparallel alpha-helices separated by two loops, and folds into a left-handed coiled-coil. The basic unit of spectrin is an antiparallel heterodimer composed of two homologous chains, beta and alpha. These assemble a tetramer via a mechanism that relies on the completion of a single repeat by association of the partial repeats located at the C terminus of the beta-chain (two helices) and at the N terminus of the alpha-chain (one helix). This tetramer is the assemblage able to cross-link actin filaments. Model building by homology of the "tetramerization" repeat from human erythrocyte spectrin illuminates the possible role of point mutations which cause hemolytic anemias.

Amino Acid Sequence↗
PubMed1997

Refined structure, DNA binding studies, and dynamics of the bacteriophage Pf3 encoded single-stranded DNA binding protein.

The solution structure of the 18-kDa single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been refined using 40 ms 15N- and 13C-edited NOESY spectra and many homo- and heteronuclear J-couplings. The structures are highly precise, but some variation was found in the orientation of the beta-hairpin denoted the DNA binding wing with respect to the core of the protein. Backbone dynamics of the protein was investigated in the presence and absence of DNA by measuring the R1 and R2 relaxation rates of the 15N nuclei and the 15N-1H NOE. It was found that the DNA binding wing is much more flexible than the rest of the protein, but its mobility is largely arrested upon binding of the protein to d(A)6. This confirms earlier hypotheses on the role of this hairpin in the function of the protein, as will be discussed. Furthermore, the complete DNA binding domain of the protein has been mapped by recording two-dimensional TOCSY spectra of the protein in the presence and absence of a small amount of spin-labeled oligonucleotide. The roles of specific residues in DNA binding were assessed by stoichiometric titration of d(A)6, which indicated for instance that Phe43 forms base stacking interactions with the single-stranded DNA. Finally, all results were combined to form a set of experimental restraints, which were subsequently used in restrained molecular dynamics calculations aimed at building a model for the Pf3 nucleoprotein complex. Implying in addition some similarities to the well-studied M13 complex, a plausible model could be constructed that is in accordance with the experimental data.

Crystallography, X-Ray↗
PubMed1997

Automated NOESY interpretation with ambiguous distance restraints: the refined NMR solution structure of the pleckstrin homology domain from beta-spectrin.

We have used a novel, largely automated, calculation method to refine the NMR solution structure of the pleckstrin homology domain of beta-spectrin. The method is called ARIA for Ambiguous Restraints for Iterative Assignment. The starting point for ARIA is an almost complete assignment of the proton chemical shifts, and a list of partially assigned NOEs, mostly sequential and secondary structure NOEs. The restraint list is then augmented by automatically interpreting peak lists generated by automated peak-picking. The central task of ARIA is the assignment of ambiguous NOEs during the structure calculation using a combination of ambiguous distance restraints and an iterative assignment strategy. In addition, ARIA calibrates ambiguous NOEs to derive distance restraints, merges overlapping data sets to remove duplicate information, and uses empirical rules to identify erroneous peaks. While the distance restraints for the structure calculations were exclusively extracted from homonuclear 2D experiments, ARIA is especially suited for the analysis of multidimensional spectra. Applied to the pleckstrin homology domain, ARIA generated structures of good quality, and of sufficiently high accuracy to solve the X-ray crystal structure of the same domain by molecular replacement. The comparison of the free NMR solution structure to the X-ray structure, which is complexed to D-myo-inositol-1,4,5-triphosphate, shows that the ligand primarily induces a disorder-order transition in the binding loops, which are disordered in the NMR ensemble but well ordered in the crystal. The structural core of the protein is unaffected, as evidenced by a backbone root-mean-square difference between the average NMR coordinates and the X-ray crystal structure for the secondary structure elements of less than 0.6 A.

Binding Sites↗
PubMed1997

The folding catalyst protein disulfide isomerase is constructed of active and inactive thioredoxin modules.

BACKGROUND: Protein disulfide isomerase (PDI), a multifunctional protein of the endoplasmic reticulum, catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. Dissection of this protein into its individual domains has confirmed the presence of the a and a' domains, which are homologous to thioredoxin, having related structures and activities. The a and a' domains both contain a -Cys-Gly-His-Cys- active-site sequence motif. The remainder of the molecule consists primarily of two further domains, designated b and b' which are thought to be sequence repeats on the basis of a limited sequence similarity. The functions of the b and b' domains are unknown and, until now, the structure of neither domain was known. RESULTS: Heteronuclear nuclear magnetic resonance (NMR) methods have been used to determine the global fold of the PDI b domain. The protein has an alpha/beta fold with the order of the elements of secondary structure being beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-beta5+ ++-alpha4. The strands are all in a parallel arrangement with respect to each other, except for beta4 which is antiparallel. The arrangement of the secondary structure elements of the b domain is identical to that found in the a domain of PDI and in the ubiquitous redox protein thioredoxin; the three-dimensional folding topology of the b domain is also very similar to that of these proteins. CONCLUSIONS: Our determination of the global fold of the b domain of PDI by NMR reveals that, like the a domain, the b domain contains the thioredoxin motif, even though the b domain has no significant amino-acid sequence similarities to any members of the thioredoxin family. This observation, together with indications that the b' domain adopts a similar fold, suggests that PDI consists of active and inactive thioredoxin modules. These modules may have been adapted during evolution to provide PDI with its complete spectrum of enzymatic activities.

Amino Acid Sequence↗
PubMed1997

Functional diversity of PH domains: an exhaustive modelling study.

BACKGROUND: Pleckstrin homology (PH) domains are found in many proteins involved in signal transduction or cytoskeletal organization. The general function for the domain is still unclear; phospholipid binding of some PH domains and a strong electrostatic polarization in the experimental structures suggest a role in localization on membranes. We have analyzed the electrostatic properties and the spatial amino acid distribution from homology models of the entire PH domain family. RESULTS: Despite the sequence divergence, the quality of the models is sufficient for our study. Most PH domains have an electrostatic polarization similar to the experimental structures. but roughly half of the PH domains linked to a Dbl homology domain have very different electrostatic properties. We also found a striking electrostatic complementarity in some internal PH domain repeats. The analysis of the spatial distribution of amino acids identified residues in the phospholipid-binding site of the spectrin and dynamin PH domains as specific for these domains. CONCLUSIONS: The mostly conserved electrostatic polarization supports a general function in binding to phospholipid membranes. But the presence of PH domains with opposite polarity suggests that ligands and functions have diverged during evolution. We also demonstrate homology modelling as a general sequence analysis tool that can yield significantly more information than conventional analysis.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1997

Tertiary structure prediction using mean-force potentials and internal energy functions: successful prediction for coiled-coil geometries.

We report a preliminary study of the use of mean-force potentials (MFPs) for predicting protein tertiary structure. For three leucine zipper sequences, we have calculated ensembles of structures spanning all possible backbone conformations consistent with the canonical coiled-coil geometry. MFPs were measured with the program PROSA. The MFP alone was poor at discriminating the native structure from very divergent structures, and the global minimum of the MFP sometimes occurred far from the native structure. We found that adding an internal energy function (a subset of the CHARMM potential that describes only interactions between backbone atoms), the resultant total energy (CHARMM+PROSA) performed much better; in each case, there was a clear positive correlation between total energy and root-mean-square deviation (RMSD) from the experimental structure, and the lowest-energy structures were about 1 A RMSD from the experimental structures. Thus, we conclude that the combined potential is a powerful method for predicting leucine zippers and is very promising for general 3D structure prediction.

Leucine Zippers↗
PubMed1997

Ambiguous distance data in the calculation of NMR structures.

The use of molecular dynamics for simulated annealing optimization of structures calculated from NMR data is reviewed. I focus on ways of directly using and automatically assigning ambiguous peaks from nuclear Overhauser enhancement experiments during the structure calculation.

Magnetic Resonance Spectroscopy↗
PubMed1997

1H and 15N NMR resonance assignments and secondary structure of titin type I domains.

Titin/connectin is a giant muscle protein with a highly modular architecture consisting of multiple repeats of two sequence motifs, named type I and type II. Type I modules have been suggested to be intracellular members of the fibronectin type III (Fn3) domain family. Along the titin sequence they are exclusively present in the region of the molecule located in the sarcomere A-band. This region has been shown to interact with myosin and C-protein. One of the most noticeable features of type I modules is that they are particularly rich in semiconserved prolines, since these residues account for about 8% of their sequence. We have determined the secondary structure of a representative type I domain (A71) by 15N and 1H NMR. We show that the type I domains of titin have the Fn3 fold as proposed, consisting of a three- and a four-stranded beta-sheet. When the two sheets are placed on top of each other to form the beta-sandwich characteristic of the Fn3 fold, 8 out of 10 prolines are found on the same side of the molecule and form an exposed hydrophobic patch. This suggests that the semiconserved prolines might be relevant for the function of type I modules, providing a surface for binding to other A-band proteins. The secondary structure of A71 was structurally aligned to other extracellular Fn3 modules of known 3D structure. The alignment shows that titin type I modules have closest similarity to the first Fn3 domain of Drosophila neuroglian.

Amino Acid Sequence↗