Analysis of slow interdomain motion of macromolecules using NMR relaxation data.

The interpretation of NMR relaxation data for macromolecules possessing slow interdomain motions is considered. It is shown how the "extended model-free approach" can be used to analyze (15)N backbone relaxation data acquired at three different field strengths for Xenopus Ca(2+)-ligated calmodulin. This protein is comprised of two domains connected by two rigid helices joined by a flexible segment. It is possible to uniquely determine all "extended model-free" parameters without any a priori assumptions regarding their magnitudes by simultaneously least-squares fitting the relaxation data measured at two different magnetic fields. It is found that the two connecting helices (and consequently the domains) undergo slow motions relative to the conformation in which the two helices are parallel. The time scales and amplitudes of these "wobbling" motions are characterized by effective correlation times and squared-order parameters of approximately 3 ns and 0.7, respectively. These values are consistent with independent estimates indicating that this procedure provides a useful first-order description of complex internal motions in macromolecules despite neglecting the coupling of overall and interdomain motions.

Chemical shift mapped DNA-binding sites and 15N relaxation analysis of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K.

The K homology (KH) motif is one of the major classes of nucleic acid binding proteins. Some members of this family have been shown to interact with DNA while others have RNA targets. There have been no reports containing direct experimental evidence regarding the nature of KH module-DNA interaction. In this study, the interaction of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K (KH3) with its cognate single-stranded DNA (ssDNA) are investigated. Chemical shift perturbation mapping indicates that the first two helices, the conserved GxxG loop, beta 1, and beta 2, are the primary regions involved in DNA binding for KH3. The nature of the KH3-ssDNA interaction is further illuminated by a comparison of backbone 15N relaxation data for the bound and unbound KH3. Relaxation data are also used to confirm that the backbone of wild-type KH3 is structurally identical to that of the G26R mutant KH3, which was previously published. Amide proton exchange experiments indicate that the two helices involved in DNA binding are less stable than other regions of secondary structure and that a large portion of KH3 backbone amide hydrogens are protected in some manner upon ssDNA binding. The major backbone dynamics features of KH3 are similar to those of the structurally comparable human papillomavirus-31 E2 DNA binding domain. Secondary structure information for ssDNA-bound wild-type KH3 is also presented and shows that binding results in no global changes in the protein fold.

High precision solution structure of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K, a c-myc transcription factor.

Among it's many reported functions, heterogeneous nuclear ribonucleoprotein (hnRNP) K is a transcription factor for the c- myc gene, a proto-oncogene critical for the regulation of cell growth and differentiation. We have determined the solution structure of the Gly26-->Arg mutant of the C-terminal K-homology (KH) domain of hnRNP K by NMR spectroscopy. This is the first structure investigation of hnRNP K. Backbone residual dipolar couplings, which provide information that is fundamentally different from the standard NOE-derived distance restraints, were employed to improve structure quality. An independent assessment of structure quality was achieved by comparing the backbone15N T1/T2ratios to the calculated structures. The C-terminal KH module of hnRNP K (KH3) is revealed to be a three-stranded beta-sheet stacked against three alpha-helices, two of which are nearly parallel to the strands of the beta-sheet. The Gly26-->Arg mutation abolishes single-stranded DNA binding without altering the overall fold of the protein. This provides a clue to possible nucleotide binding sites of KH3. It appears unlikely that the solvent-exposed side of the beta-sheet will be the site of protein-nucleic acid complex formation. This is in contrast to the earlier theme for protein-RNA complexes incorporating proteins structurally similar to KH3. We propose that the surface of KH3 that interacts with nucleic acid is comparable to the region of DNA interaction for the double-stranded DNA-binding domain of bovine papillomavirus-1 E2 that has a three-dimensional fold similar to that of KH3.