Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: Differences in solution and crystal forms of maltodextrin bindingprotein loaded with beta-cyclodextrin

Protein function is often regulated by conformational changes that occur in response to ligand binding or covalent modification such as phosphorylatio n. In many multidomain proteins these conformational changes involve reorie ntation of domains within the protein. Although X-ray crystallography can b e used to determine the relative orientation of domains, the crystal-state conformation can reflect the effect of crystal packing forces and therefore may differ from the physiologically relevant form existing in solution. He re we demonstrate that the solution-state conformation of a multidomain pro tein can be obtained from its X-ray structure using an extensive set of dip olar couplings measured by triple-resonance multidimensional NMR spectrosco py in weakly aligning solvent. The solution-state conformation of the 370-r esidue maltodextrin-binding protein (MBP) loaded with beta-cyclodextrin has been determined on the basis of one-bond N-15-H-N, N-15-C-13' Cr-13(alpha) -13C', two-bond C-13'-H-N, and three-bond C-13(alpha)-H-N dipolar couplings measured for 280, 262, 276, 262, and 276 residues, respectively. This conf ormation was generated by applying hinge rotations to various X-ray structu res of MBP seeking to minimize the difference between the experimentally me asured and calculated dipolar couplings. Consistent structures have been de rived in this manner starting from four different crystal forms of MBP. The analysis has revealed substantial differences between the resulting soluti on-state conformation and its crystal-state counterpart (Protein Data Bank accession code 1DMB) with the solution structure characterized by an 11(+/- 1)degrees domain closure. We have demonstrated that the precision achieved in these analyses is most likely limited by small uncertainties in the intr adomain structure of the protein (ca 5 degrees uncertainty in orientation o f internuclear vectors within domains). Ln addition, potential effects of i nterdomain motion have been considered using a number of different models a nd it was found that the structures derived on the basis of dipolar couplin gs accurately represent the effective average conformation of the protein. (C) 2000 Academic Press.