Restraints structure calculation

Using 18 trNOE-derived distance restraints and 13 trCCR-derived backbone torsion angle restraints, structure calculations using distance geometry and simulated annealing [48] resulted in a well-defined structure of the IKK/1-derived peptide bound to NEMO. The backbone structure is displayed in Fig. 7B and is compared with the result of the calculation carried out using the trNOE-derived distance restraints alone. It is obvious from Eig. 7 that only the combination of the trNOE- and trCCR-derived restraints results in the structure elucidation of the bound conformation of this peptide. 

Structure calculation algorithms in general assume that the experimental list of restraints is completely free of errors. This is usually true only in the final stages of a structure calculation, when all errors (e.g., in the assignment of chemical shifts or NOEs) have been identified, often in a laborious iterative process. Many effects can produce inconsistent or incorrect restraints, e.g., artifact peaks, imprecise peak positions, and insufficient error bounds to correct for spin diffusion. 

Figure 6 Steps in automated assignment. (1) Select the lowest energy structures from iteration / — 1 that are used to interpret the spectra. (2) For each peak, list all possible assignments compatible with the resonances within a frequency mnge. (3) Extract a distance for each assignment possibility from the ensemble of structures. (4) Use the distances to assign ambiguous NOEs. (5) Calibrate the peak volumes to obtain distance restraints. (6) Calculate structures based on the new restraints.

Considering all potential experimental and systematic errors of NOE/ROE crosspeak intensities, it is remarkable how robust the derived distance restraints still are. The reason Ues in the dependence of the cross-relaxation rate even if a cross-peak intensity is determined wrongly by a factor 2, the resulting distance restraint is only affected by the factor 1.12, which usually lies within the error range of distance restraints used in structure calculations. It should be further noted that the quaUty of a resulting structure is not so much determined by the... 

The full potenhal of RDCs, however, can be seen by the incorporation of RDC data in structure calculations. Several programs hke XPLOR-NIH, DISCOVER or GROM ACS allow the incorporation of RDCs as angular or combined angular and distance dependent restraints. Several studies on sugars have been reported (see, e.g. Ref [43] and references therein) and Fig. 9.8 shows the comparison of three structural models for the backbone of the cyclic undecapeptide cyclosporin A, derived from X-ray crystahography, ROE data in CDCI3 as the solvent, and RDCs and ROEs obtained in a PDMS/CDCfi stretched gel [22]. Due to the sensitivity to... 

As it was mentioned in Section 9.4.1, 3D structures generated by DG have to be optimized. For this purpose, MD is a well-suited tool. In addition, MD structure calculations can also be performed if no coarse structural model exists. In both cases, pairwise atom distances obtained from NMR measurements are directly used in the MD computations in order to restrain the degrees of motional freedom of defined atoms (rMD Section 9.4.2.4). To make sure that a calculated molecular conformation is rehable, the time-averaged 3D structure must be stable in a free MD run (fMD Sechon 9.4.2.5J where the distance restraints are removed and the molecule is surrounded by expMcit solvent which was also used in the NMR measurement Before both procedures are described in detail the general preparation of an MD run (Section 9.4.2.1), simulations in vacuo (Section 9.4.2.2) and the handling of distance restraints in a MD calculation (Section 9.4.2.3) are treated. Finally, a short overview of the SA technique as a special M D method is given in Sechon 9.4.2.6. 

The ubiquitin backbone structure has been determined using dipolar couplings as the sole source of structural restraints in the work reported by Hus et al. [105]. The resulting structure is practically identical to the NMR structure calculated from NOE and other... 

Fig. 7 Solution of structure calculations of the NBD-peptide bound to NEMO. A Only trNOE-derived distance restraints were used in the calculation. The structure is not defined by the distance restraints alone. B trNOE-derived distance restraints and trCCR-derived torsion angle restraints were combined. These restraints complement each other and are sufficient to define the NEMO-bound conformation of the IKK/8-derived peptide. In both cases ten solutions were superimposed...

Fig. 8 Bound conformations of the hexapeptide DRPVPY. Superposition of structures was performed within Insight II and is based on the backbone atoms of residues 4-6. A Family of 27 structures calculated using trNOE-derived restraints only. B Average of the 27 structures in panel A. Reproduced with permission from [125]. 2002 American Chemical Society...

The structural modeling of PG and PLV with the anti-parallel p-sheet form is carried out by the hybrid distance geometry-dynamic simulated annealing method115 as contained in the X-PLOR 3.1 program.116 For structural calculations, the proton-proton distance restraints and the torsion angle restraints ( =—139° and v /=135°) are derived from reference data by Wiithrich et al.ni Hydrogen-bond distance restraints are used for the N and atoms (2.7-3.3 A) in the secondary structure.118 120 The reference data of intra- and intermolecular proton-proton distances are shown in Fig. 19. 

Figure 1.2 (a) Superposition of 20 structures calculated from NMR restraints for the polypeptide backbone of the ribonuclease bamase (110 amino acid residues, see Chapter 19). (b) Superposition of the mean polypeptide backbone calculated from NMR and that from x-ray crystallography. 

In Fig. 1 we have highlighted with a dark background the different types of NMR methods that are used in drug-discovery projects. These include basic ID and 2D methods that are used to confirm the identity of peptides, determine their conformation, or derive restraint information used in 3D structure calculations (left side of Fig. 1). Methods to study binding interactions (middle section of Fig. 1) can be broadly categorized as being based on NOEs, diffusion, relaxation, or chemical shift changes. NOE-based methods include the transferred NOE... 

Structure calculations for the bound conformation of the ligand can be done in the absence of any knowledge on the actual structure of the receptor protein, as they involve only interproton distances of the ligand molecule. However, if the 3D structure of the protein is known, a model of the complex can be generated by using the tr-NOE distances as restraints to lock the otherwise flexible ligand in the bound conformation, which reduces the number of docking calculations considerably. 

For the structure calculation, 5000 binding poses of EpoA were generated by non-restraint docking of its NMR-derived conformation into the binding site of the EC-derived 3D structure of tubulin (complex TB). The docking protocol was designed to sample tubulin conformations that differed from the starting EC-derived... 

The tubulin-bound conformation of DDM in solution has been determined from tr-NOE data [112], Sample conditions were similar to those used previously to determine the bioactive conformation of EpoA. Distance restraints were obtained from a series of tr-NOE spectra recorded at several mixing times and were used in the structure calculation based on the complete relaxation matrix methodology [37], The NMR-derived bioactive conformation is quite similar to the crystal structure except for the conformation of the 8 lactone ring, that is close to a flattened chair in solution but a twisted boat in the crystal (Fig. 18). 

The nondegenerate geminal pairs are usually named according to their chemical shifts (e.g., downfield of ft ) rather than their stereochemical relationships (pro-R and pro-S). In structure calculations, this usually is dealt with by creating a pseudo-atom right between the pro-R and pro-S positions in 3D space. The NOE restraints are applied to the pseudoatom and not to the real atoms, and the distance limit is increased a bit to account for the ambiguity (we do not really know which restraint applies to which of the two positions in space). Similarly, a pseudoatom is created at the center of the three equivalent protons of a CH3 group, and the distance restraint is applied to the pseudoatom. 

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