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Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMR


Large-scale domain rearrangements in proteins have long been recognized to have a critical function in ligand binding and recognition, catalysis and regulation1,2,3,4,5. Crystal structures have provided a static picture of the apo (usually open) and holo usually closed) states. The general question arises as to whether the apo state exists as a single species in which the closed state is energetically inaccessible and interdomain rearrangement is induced by ligand or substrate binding, or whether the predominantly open form already coexists in rapid equilibrium with a minor closed species. The maltose-binding protein (MBP), a member of the bacterial periplasmic binding protein family6, provides a model system for investigating this problem because it has been the subject of extensive studies by crystallography7,8, NMR9,10,11 and other biophysical techniques11,12,13. Here we show that although paramagnetic relaxation enhancement (PRE) data for the sugar-bound form are consistent with the crystal structure of holo MBP, the PRE data for the apo state are indicative of a rapidly exchanging mixture (ns to μs regime) of a predominantly (95%) open form (represented by the apo crystal structure) and a minor (5%) partially closed species. Using ensemble simulated annealing refinement against the PRE data we are able to determine a 〈r-6〉 ensemble average structure of the minor apo species and show that it is distinct from the sugar-bound state.

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Figure 1: Domain reorientation of MBP on binding maltotriose.
Figure 2: PRE measurements on apo and holo MBP.
Figure 3: Simulated annealing refinement of the minor species of apo MBP.
Figure 4: Structure of the minor partly closed form of apo MBP.

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We thank J. Iwahara, A. Grishaev and A. Szabo for helpful discussions; and A. Grishaev and L. Guo for assistance with SAXS data collection and processing. Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science. BioCAT is a National Institutes of Health-supported Research Center. This work was supported by funds from the Intramural Program of the NIH, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and the AIDS Targeted Antiviral program of the Office of the Director of the NIH (to G.M.C.).

An ensemble of 50 sets of coordinates for the equilibrium mixture of major (95%) open and minor (5%) partly closed species of MBP, together with the PRE experimental restraints, are deposited in the Protein Data Bank with accession codes 2V93 and R2V93MR, respectively.

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Correspondence to G. Marius Clore.

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The file contains Supplementary Figures S1-S17 and Supplementary Table S1 with Legends, Supplementary Notes and additional references. (PDF 2358 kb)

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Tang, C., Schwieters, C. & Clore, G. Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMR. Nature 449, 1078–1082 (2007).

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