Two-conformer equilibrium of maltose-binding protein in the absence of ligand from residual dipolar coupling analysis

Prior analyses found good agreement between numerous residual dipolar couplings (RDCs) measured in the apo-state of maltose binding protein (MBP) and its X-ray crystal structure. However, paramagnetic relaxation enhancement (PRE) measurements on the same system reported on the presence of a small population of partially closed states in the absence of ligand, with somewhat different relative orientations of its N- and C-terminal domains. We present a protocol for RDC fitting to such a dynamic system that yielded quantitative validation of the PRE results. Our analysis is based on a multi-conformer singular value decomposition (SVD) RDC fitting procedure that provides a straightforward method for quantifying conformational equilibria, provided that high-quality RDCs and accurate coordinates for invariant domains of the protein are available, such as may apply for allosterically regulated systems. For MBP, the analysis reveals interdomain dynamics that can be fit to a two-state equilibrium, with the major state very close to the apo-state X-ray structure and a minor conformer near the center of an ensemble of partially closed structures, previously derived by PRE. The holo-state of MBP was found to agree, to within RDC measurement precision, with a single relative orientation of its two domains. The multi-tensor multi-conformer software is available to all users as an interactive web application at https://spin.niddk.nih.gov/bax-apps/nmrserver/dc/svdm.html.