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Detecting transient intermediates in macromolecular binding by paramagnetic NMR

Abstract

Macromolecular complex formation is governed by two opposing constraints of specificity and speed1,2. Kinetic3,4,5,6 and theoretical considerations suggest that significant rate enhancement can be achieved either by reducing the dimensionality of the search process1,7 or by the creation of a short-range attractive potential around the target site2. This implies the existence of transient intermediates involving non-specific binding modes. Here we show that intermolecular paramagnetic relaxation enhancement (PRE) provides a means of directly detecting the presence of, and investigating the nature of, low population transient intermediates under equilibrium conditions. Applying this approach, we characterize the search process whereby a sequence-specific transcription factor (the homeodomain of HOXD9) binds to non-cognate DNA sites as a means of enhancing the rate of specific association. The PRE data in the fast exchange regime reveal the presence of transient intermediates formed in a stochastic manner at non-cognate sites whose structure is similar to that of the specific complex. Two distinct search processes involving intra- as well as intermolecular translocations can be delineated. The intermolecular PRE method is general and can be readily applied to investigations of transient intermediates in many other macromolecular binding processes.

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Figure 1: Intermolecular PRE in an exchanging system.
Figure 2: Intermolecular PRE for the HOXD9 homeodomain/DNA complex in slow and fast exchange.
Figure 3: Summary of the intermolecular PRE profiles arising from dT-EDTA-Mn 2+ at sites 1 to 4 for the HOXD9 homeodomain/DNA complex in the slow and fast exchange regimes.
Figure 4: Intermolecular PRE arises from inter- and intramolecular translocation processes.

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Acknowledgements

This work was supported by the Intramural Program of the NIH, the NIDDK, and in part by the AIDS Targeted Antiviral Program of the Office of the Director of the NIH (G.M.C.).

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Correspondence to G. Marius Clore.

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Supplementary Notes

This file contains details of lineshape simulations, residual dipolar coupling data, NMR exchange experiments and PRE profiles to be linked to the online version of the paper. (PDF 1671 kb)

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Iwahara, J., Clore, G. Detecting transient intermediates in macromolecular binding by paramagnetic NMR. Nature 440, 1227–1230 (2006). https://doi.org/10.1038/nature04673

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