![]() ![]() ![]() Distinct cross-peaks are observed connecting the conformation that directly binds the partner ( U in panel E, conformational selection and N in panel F, induced fit).ġ3C- 1H HMQC spectrum (middle) of 400 μM ILVM- 13CH 3 R17* containing 800 μM I- 13CH 3 DnaK/ADP. The U-N interconversion is assumed to be too slow to give rise to detectable cross-peaks in this experiment. ( E, F) Schematic of 2D planes derived from a 13C- 1H magnetization exchange experiment recorded on a system where the binding reaction is described by conformational selection, U → U K ( E) or induced fit, N → U K ( F). Because a minor dip is not observed between N and UK (red) the flux through the reaction N → U K must be much smaller than via U → U K. ![]() A prominent minor state dip is observed in the U profile (green) at the position of the bound frequency, δ B, corresponding to a flux through the U → U K reaction. ( D) In contrast, if states N and U interconvert slowly then distinct CEST profiles can be observed for CS and IF binding mechanisms, as shown here for binding to the U state via conformation selection. If N and U interconvert rapidly compared to the kinetics of binding ( C), any perturbation to the magnetization of the bound state by application of a weak radiofrequency field is rapidly conveyed to N even if binding is only to U, resulting in identical CEST profiles for N (red) and U (green). NMR spin-relaxation methods can distinguish between IF and CS binding pathways so long as N and U conformations interconvert slowly (slower than the binding kinetics). Note, however, that the CEST experiment amplifies the fingerprint of exchange, so that a minor dip for a spin in state B can be observed even if the peak cannot be directly visualized in the spectrum ( p B <5%). Black and orange dashed lines between panels A and B show the correspondence between CEST and zz-exchange experiments via chemical shifts in states A and B. A major dip at the chemical shift of the spin in state A (−0.6 ppm) as well as a minor dip at the corresponding chemical shift in state B (0 ppm), can be observed. ( A) Schematic of a zz-magnetization exchange spectrum showing diagonal peaks derived from magnetization in exchanging states A and B, A ⇄ B, as well as cross-peaks (black dots) connecting the two states. ( B) Corresponding CEST profile for the exchange reaction in panel A that plots the normalized peak intensity of a spin in state A as a function of the offset at which a weak radiofrequency field is applied. ( D) The crystal structure of DnaK/ATP in the absence of substrate ( Kityk et al., 2012 Qi et al., 2013), showing that upon ATP hydrolysis the α-helical lid becomes docked on the β-sandwich domain concomitant with the undocking of NBD and SBD (PDB ID: 4B9Q). ( C) Space filling structure of the substrate binding domain (SBD) with a closed conformation of the helical lid, illustrating the pore that the substrate (red) threads through. The remainder of the peptide substrate is shown in red. ( B) An enlarged view of the substrate binding cleft showing key hydrophobic residues Ile 401, Val 436 and Ile 438 of DnaK as green sticks forming contacts with the substrate residue that occupies the central binding position (cyan sticks) (PDB ID: 1DKZ) ( Zhu et al., 1996). ( A) Cartoon representation of the NMR structure of DnaK/ADP ( Bertelsen et al., 2009) showing the N-terminal ATPase domain (NBD) and the C-terminal substrate binding domain which consists of a β-sandwich subdomain and an α-helical lid (PDB ID: 2KHO). ![]()
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