Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial Enzyme I

Enzyme I (EI), the first component of the bacterial phosphotransfer signal transduction system, undergoes one of the largest substrate-induced interdomain rearrangements documented to date. Here, we characterize the perturbations generated by two small molecules, the natural substrate phosphoenolpyruvate (PEP) and the inhibitor α-ketoglutarate (αKG), on the structure and dynamics of EI using NMR, small-angle X-ray scattering (SAXS) and biochemical techniques. The results indicate unambiguously that the open-to-closed conformational switch of EI is triggered by complete suppression of micro- to millisecond dynamics within the C-terminal domain of EI. Indeed, we show that a ligand-induced transition from a dynamic to a more rigid conformational state of the C-terminal domain stabilizes the interface between the N- and C-terminal domains observed in the structure of the closed state, thereby promoting the resulting conformational switch and autophosphorylation of EI. The mechanisms described here may be common to several other multidomain proteins and allosteric systems.

cannot be explained in terms of the available static structures of EI, we show that a transition from a dynamic to a more rigid EIC structure, induced by PEP-binding, is responsible for the large conformational rearrangement. Our results provide direct evidence for large-scale interdomain rearrangements induced by a change in dynamics within one of the protein domains, further supporting the recently emerging idea of protein flexibility as a general mechanism for transmitting signals between distant sites in macromolecules or macromolecular assemblies. 12

Effect of PEP and αKG on the open-to-closed conformational equilibrium of EI
The effect of PEP and αKG on the global structure of EI was assessed by means of backbone ( 1 H N / 15 N) and side chain ( 1 H/ 13 C methyl ) chemical shift perturbation mapping and SAXS. Experiments were acquired on the wild-type protein (EI WT ) and on mutants of EI in which the active site H189 was replaced by Gln (EI Q ) or Ala (EI A ). The EI Q and EI A mutants allow the PEP-induced structural perturbations to be probed without interference caused by phosphorylation of H189, and have been extensively employed in structural and biochemical studies of EI. [13][14][15][16] Addition of 5 mM PEP to EI Q and EI A results in substantial changes in the 1 H-15 N and 1 H-13 C methyl transverse relaxation optimized (TROSY) correlation spectra ( Fig. 2a and Supplementary Fig. 1). As expected, larger chemical shift perturbations (Δ H/N,C ) are observed in the EIC domain and involve residues located in the PEP binding site and at the dimer interface (Fig. 2a). These perturbations closely resemble the PEP-induced chemical shift perturbations observed previously for isolated EIC ( Supplementary Fig. 2). 17 In addition, chemical shift perturbations are also observed in the EIN domain at sites located more than 20 Å away from the PEP binding site in the open EI structure (Fig. 2a). In particular, the largest perturbations on the EIN domain are observed in the linkers connecting the α and α/β subdomains, in the vicinity of the helical linker connecting EIN to EIC, at the interface between the α and α/β subdomains, and at the interfaces between EIN and EIC and between the two EIN subunits in the closed EI structure (Fig. 2a). All these regions are expected to experience chemical shift perturbations upon transition from the open to the closed form of EI. In contrast, addition of 10 mM αKG to EI WT , EI Q or EI A does not result in any significant chemical shift perturbation within the EIN domain ( Fig. 2b and Supplementary Fig. 1); the only chemical-shift changes observed are located within the EIC domain, and closely resemble the chemical shift perturbations induced by αKG on isolated EIC ( Supplementary Fig. 2). 8 Overall, the chemical shift perturbation data suggest that only PEP binding to EIC is able to trigger the transition from the open to the closed form of EI, while binding of αKG leaves the overall structure of EI unperturbed in the open state. To test this hypothesis SAXS data were acquired for EI WT , EI Q and EI A in the presence and absence of ligands. SAXS curves are linear time-averaged and ensemble weighted experimental measurements that are highly sensitive to the overall size and shape of a molecule. 18,19 Thus, SAXS is a good reporter of conformational equilibria involving large interdomain rearrangements.
The SAXS data were fit (up to q = 0.16 Å −1 ) with a helper program distributed with Xplor-NIH 20 (see Supplementary Methods) using a grid search (step size = 1%) in which increasing populations of the closed EI conformer were added to the starting system comprising EI in the open configuration. A plot of χ 2 versus population of closed state is shown in Fig. 3a. In general, raising the population of the closed EI state results in larger χ 2 values for the majority of the scattering curves, indicating that the SAXS data acquired for most of the EI samples are best fit by the open structure of the enzyme. However, exceptions to this trend are observed for the scattering curves obtained for EI Q and EI A in the presence of PEP where the χ 2 minimum is reached at closed-state populations of ~11 and ~62% for the EI Q -PEP and the EI A -PEP complexes, respectively (Figs. 3a and b), confirming that only the interaction of PEP with EIC is able to trigger the open-to-closed conformational transition of EI.
We note that although the SAXS curves in the absence of ligands are fit very well by the open structure of EI (χ 2 ≤ 1), small, systematic deviations between the experimental and calculated SAXS curves are observed for the EI A -PEP complex (Fig. 3b), for which the largest population of closed EI was obtained from the fits (Fig. 3a). This suggests that the crystal structure of the phosphorylated intermediate does not fully describe the solution structure of holo EI. Assessment of the small discrepancy between the structures of closed EI in the crystal and solution states is beyond the scope of the present work and will be the subject of future investigations.
The SAXS data in Figs. 3a and b indicate that PEP binding does not fully stabilize the closed state of EI, resulting in a dynamic equilibrium in which both open and closed forms are populated. This equilibrium is modulated by the nature of the amino acid side chain at position 189 in a manner that can be readily rationalized by simple steric and electrostatic considerations. In particular, addition of PEP to samples of EI WT results in rapid phosphorylation of the active site H189. Steric and electrostatic repulsion between the phosphoryl group of PEP and phosphorylated H189 destabilizes the closed form of EI (Fig.  4a), thereby keeping the enzyme in the open state ( Fig. 3a) with the phosphoryl group of H189 exposed and available for in-line phosphoryl transfer to HPr. For the EI Q -PEP complex, Q189 cannot be phosphorylated by PEP, and only minor contacts between PEP and the side chain of Q189 are formed (Fig. 4b). The weaker repulsion between Q189 and PEP results in partial stabilization of the closed state of EI, consistent with the larger population (~11%) of closed state obtained from analysis of the SAXS data for the EI Q -PEP complex (Fig. 3a). When the active site H189 is mutated to Ala, no contacts are formed between PEP and A189 (Fig. 4c) resulting in the largest population of closed state (~62%) derived from analysis of the SAXS data (Fig. 3a). Thus, we conclude that no direct contacts between PEP and the EIN domain are required to trigger the open-to-closed transition of EI.
The conclusions drawn from the SAXS analysis correlate well with the affinities of EI WT , EI A and EI Q for PEP and αKG. Enzyme kinetic assays indicate that the affinity of PEP for EI WT (K m ~ 330±20 µM) is two times lower than for EI Q (K m ~ 130±10 µM) and about an order of magnitude lower than for EI A (K m < 50 µM) (Fig. 5a), reflecting the fact that only the open form of EI can release the substrate/product from the binding pocket on the EIC domain. In contrast, NMR titration experiments show that mutations of H189 have no effect on the affinity of the EI for αKG (K D ~ 2.0±0.2 mM, Fig. 5b), confirming that this small molecule inhibitor does not perturb the overall structure of EI. It is also worth noting that the affinities of isolated EIC for PEP (K m ~ 370±30 µM) 17 and αKG (K D ~ 2.2±0.2 mM), 8 are the same (within experimental error) as the affinities of full-length EI WT for PEP and of EI WT , EI Q and EI A for αKG, in agreement with the conclusion from SAXS analysis that the EI WT -PEP and all the investigated EI complexes with αKG adopt a fully open conformation (Fig. 3a).

Effect of PEP and αKG on µs-ms dynamics of EI
The results presented in the previous section clearly indicate that the open-to-closed transition of EI is induced by PEP-binding to the EIC domain with no requirement for direct interactions between the substrate and EIN domain. On the other hand, αKG, which shares most of the same interactions as PEP with the EIC domain (Figs. 5c and d), does not affect the relative positioning of the EIN domain. Inspection of the various EI X-ray structures shows very little variability in the coordinates of the EIC domain (backbone r.m.s.d. to the mean coordinate positions of 2 Å for residues 261-572, Supplementary Fig. 3), irrespective of the presence of ligands bound to EI. It is therefore not evident how PEP-binding triggers the massive conformational rearrangement from the open to closed state.
Using 15 N NMR relaxation dispersion experiments, we recently showed that the PEP binding pocket of isolated EIC undergoes a dynamic conformational equilibrium on the submillisecond time-scale (k ex ~ 1,500 s −1 ) between a major ("expanded") state (population ~ 97%) and a minor state (population ~ 3%) that is more "compact" (in terms of several loops) and closely resembles the PEP-bound state. 17 To investigate whether PEP and αKG affect this conformational equilibrium in full-length EI, we analyzed the µs-ms dynamics of EI WT , EI Q and EI A , in the presence and absence of ligands, using 1 H-13 C Multiple-Quantum (MQ) 21 and 13 C Single-Quantum (SQ) 22 Carr-Purcell-Meinboom-Gill (CPMG) 23 relaxation dispersion spectroscopy. Experiments were acquired at 600, 800 and 900 MHz on the methyl groups of U-[ 2 H, 15 N]/Ile(δ1)-13 CH 3 -labeled and U-[ 2 H, 15 N]/Val,Leu-( 13 CH 3 / 12 C 2 H 3 )-labeled EI. Like 15 N-CPMG relaxation dispersion, the 13 C methyl relaxation dispersion experiments probe exchange dynamics between species with distinct chemical shifts on a time scale ranging from ~50 µs to ~10 ms. However, due to favorable relaxation properties, the absence of exchange with water and the 3-fold degeneracy of methyl proton chemical shifts, the 13 C methyl relaxation dispersion experiments yield data of superior quality for large macromolecular systems 24 such as the full-length 128 kDa EI.
In the absence of ligands, large exchange contributions to the transverse MQ-relaxation rates (MQ-R ex ) were detected for several Ile, Val and Leu side chains in EIC domain ( Fig. 6a and Supplementary Fig. 4). The majority of methyl groups experiencing conformational exchange are located within or in the vicinity of the β3α3 (residues 333-366) and β6α6 (residues 453-477) loops that were shown to be dynamic in isolated EIC (Fig. 6b). 17 In addition, large MQ-R ex values were observed for L264, V269 and I533 located in the vicinity of the long helical linker connecting the EIC and EIN domains (Figs. 6a and b). All the MQ and SQ relaxation dispersion curves of the 19 methyl groups exhibiting MQ-R ex values > 3 s −1 at 800 MHz were fit simultaneously to a model describing the interconversion of two conformational states (see Supplementary Methods). In the global fitting procedure, the exchange rate (k ex ) and the fractional population of the minor state (p B ) were optimized as global parameters, while the 13 C chemical shift differences between the two conformational states (Δω C ) were treated as methyl-specific parameters. MQ relaxation dispersion curves also depend upon the 1 H chemical shift differences between the two conformational states (Δω H , see Supplementary Methods). However, an initial residue-based fitting (in which k ex , p B , Δω C , and Δω H were all optimized as methyl-specific parameters) indicated that the contribution of Δω H to the observed dispersions is negligible for all analyzed methyl groups ( Supplementary Fig. 5). Therefore, to improve convergence of the calculation, the methyl-specific Δω H values were set to zero in the subsequent global fit of the relaxation dispersion curves. An example of the global fit is provided in Fig. 4c. Curves for all the 19 methyl groups as well as the optimized Δω C values are provided in Supplementary Fig. 6 and Supplementary Table 1, respectively. The overall exchange rate (sum of forward and backward rate constants) is 1,200 ± 50 s −1 , and agrees within experimental error with the k ex value obtained for the isolated EIC domain (1,500 ± 350 s −1 ). 17 The population of the minor species, however, is 13±5 %, which is considerably larger than the value of 3±1 % obtained from fitting the 15 N-CPMG data for isolated EIC. 17 Consistent with this latter finding, an overlay of the NMR spectra acquired for full-length EI and the isolated EIC domain shows that, in the absence of ligands, the NMR signals of EIC domain are more shifted towards the bound conformation in the full-length EI protein than in the isolated EIC domain (Fig. 7), indicating that the presence of the EIN domain promotes sampling by the EIC domain of the more compact conformational state that resembles the substrate-bound form.
Thus, the 13 C methyl -CPMG relaxation dispersion data presented above for free EI describe the same conformational equilibrium previously detected in the PEP-binding site of the isolated EIC domain by 15 N backbone CPMG relaxation dispersion experiments. 17 The 1 H-13 C methyl MQ CPMG relaxation dispersion experiments acquired on EI in the presence of ligands indicate that PEP and αKG affect the conformational equilibrium differently. In particular, analysis of the MQ-R ex data obtained at 800 MHz for the EI Q -PEP and EI A -PEP complexes shows that PEP-binding completely suppresses µs-ms dynamics within the EIC domain (Fig. 6A, and Supplementary Fig. 4). In this regard, the closed form of EI is stabilized by contacts between the α/β subdomain of EIN and the β3α3 and β6α6 loops of EIC, which have been observed to undergo dynamics in the free enzyme ( Fig. 6d and Supplementary Fig. 7). Structural stabilization of the β3α3 and β6α6 loops in the compact PEP-bound form allows optimal docking of the EIN domain onto the EIC domain, and is therefore responsible for triggering the open-to-closed transition of EI.
We also noticed that, although the chemical shift perturbations induced by PEP on the backbone 15 N resonances of isolated EIC closely match the residue specific Δω N parameters obtained from the fit of the 15 N relaxation dispersion data acquired on the C-terminal domain of the enzyme, 17 and a moderate correlation is observed between the methylspecific Δω C parameters derived from relaxation dispersion data on wild type EI versus the chemical shift perturbations induced by PEP on the 13 C methyl resonances of full-length EI Q and EI A (Δ C , Supplementary Fig. 8), the measured Δ C values for several methyl groups located within or in the proximity of the β3α3 loop are considerably larger than the corresponding Δω C values ( Supplementary Fig. 8). Taken together, these observations suggest that the compact state transiently sampled by the EIC domain in the absence of PEP while overall is similar to the PEP-bound state, differs from the PEP-bound structure in terms of side-chain packing within and in the proximity of the β3α3 loop ( Supplementary  Fig. 8). Interestingly, contacts between the α/β subdomain of EIN and the β3α3 loop of EIC account for ~20 % of the interdomain interface in closed EI (Supplementary Fig. 7). The different conformations adopted by the β3α3 loop in the compact states sampled by EIC in the absence (compact I ) and presence (compact II ) of PEP are consistent with our SAXS analysis indicating that the closed state of EI is significantly populated only in the presence of PEP (Fig. 3a).
αKG, on the other hand, is not able to provide full stabilization of the EIC domain, as evidenced by the fact that several methyl groups with MQ-R ex values > 3s −1 are still observed for the αKG complexes with EI WT , EI A and EI Q (Fig. 6c, and Supplementary Fig.  4). Specifically, large MQ-R ex values for the αKG complexes are observed for residues 331, 336, 342, 355 and 378 within or in the proximity of the β3α3 loop (Fig. 6b, and Supplementary Fig. 9) which does not make contacts with αKG but does interact with PEP (Figs. 5c and d, and Supplementary Fig. 9); likewise large MQ-R ex values are also observed for residues 482, 488 and 505 that are located close to the active site Cys 502 , which forms hydrophobic contacts with PEP and is involved in one hydrogen bond with the C 2 carbonyl group of αKG (Figs. 5c and d, and Supplementary Fig. 9). Other MQ-R ex values > 3 s −1 are detected for residues 264 and 269 that are spatially close to the linker helix. Thus, in the case of the complexes with αKG, the more dynamic state of the long β3α3 loop hampers effective docking of EIN over EIC, thereby destabilizing the closed EI configuration.
As a final note, although the EIN domain of EI A experiences significant PEP-induced chemical shift perturbations (Fig. 2a), and the SAXS data reveal that the closed EI form is highly populated in the presence of PEP (population > 50% for the EI A -PEP complex, Fig.  3), no relaxation dispersions are observed in the EIN domain (Figs. 6a and d), and a single set of cross-peaks is observed in the NMR spectra of all the investigated EI-PEP complexes. These observations indicate that the interdomain rearrangement in EI is too fast (>10,000 s −1 ) to be detected by CPMG-type relaxation dispersion measurements. The intricate coupling between the open-to-closed dynamics for EI and the expanded-to-compact conformational exchange within the EIC domain revealed by the present work is summarized in Fig. 8.

Discussion
Addressing the mechanisms whereby small molecule ligands modulate the interactions between protein domains is of paramount importance for understanding fundamental phenomena in biology such as protein switches and allostery. In this Communication, we have investigated the effect of two small molecules on the interaction between the EIN and EIC domains of EI, the first component of the bacterial phosphotransferase system. The open-to-closed conformational change induced by substrate binding to EI (Fig. 1a) is one of the largest ligand-induced interdomain rearrangements reported in the literature so far.
Although there is now a wealth of structural studies on EI and its complexes, 8,10,11,17,[25][26][27][28][29] the fundamental forces driving this conformational rearrangement were not evident. Here, we have shown that binding of the substrate PEP to the EIC domain of EI results in structural stabilization of the otherwise dynamic loops of EIC that form a large portion of the interface between the EIC and EIN domains in the structure of the closed state of EI. This finding provides a direct link between ligand binding and the open-to-closed conformational transition, and is a clear example of how conformational dynamics and small-molecule binding have co-evolved to confer the proper biological function upon this enzyme. Ligand-induced stabilization (or destabilization) of the interface between protein domains may be a common mechanism by which small-molecule binding propagates into large interdomain rearrangements, and may serve as the basis for interdomain communication in a recently discovered class of allosteric proteins where disorder-to-order transitions induced by ligand-binding within one domain were shown to be associated with changes in the spectral and thermodynamic properties of adjacent structural motifs. [30][31][32][33]

Protein expression and purification
Mutants of E. coli EI (H189Q and H189A) were made using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). EI (wild type and mutants) was expressed and purified as described previously. 17 U-[ 2 H, 15 N]/Val,Leu-( 13 CH 3 / 12 C 2 H 3 )-labeled and U-[ 2 H, 15 N]/ Ile(δ1)-13 CH 3 -labeled EI samples were prepared following standard protocols for specific isotopic labeling of the methyl groups of Ile, Leu and Val side chains. 34

Small-Angle X-ray scattering
SAXS data were collected on samples of EI (6 mg/mL corresponding to ~48 µM dimer) in 20 mM Tris buffer, pH 7.4, 100 mM NaCl, 10 mM DTT, 4 mM MgCl 2 , 1 mM EDTA, and 1 tablet of cOmplete, EDTA-free protease inhibitor cocktail (Roche). Ligands were added to a final concentration of 20 mM immediately before data acquisition. SAXS data were acquired at Beamline 12-IDC at the Advanced Photon Source (Argonne National Laboratory, Argonne, IL) at protein concentrations ranging from 1.5 to 6 mg/mL. Data collection was done using a Gold CCD detector positioned at 2 m from the sample capillary. Incident radiation with an energy of 18 keV was used, resulting in observable q-ranges of 0.014−0.24 Å −1 . Q-axis mapping was done using scattering from a silver behenate standard sample. A total of 20 sequential data frames with exposure times of 1.5 s was recorded with the samples kept at 25°C throughout the measurement. To prevent radiation damage, volumes of 100 µL of samples and buffers were oscillated during data collection using a flow-though setup. Individual data frames were masked, corrected for the detector sensitivity, radially integrated and normalized by the corresponding transmitted beam intensities. The final 1D scattering profiles and their uncertainties were calculated as means and standard deviations over the 20 individual frames.
Subtraction of the buffer data and analysis of the scattering profiles was carried out using the Xplor-NIH calcSAXS-bufSub helper program (see Supplementary Methods). 19 The SAXS data were fit using a linear combination of the experimental 3D structures of E. coli EI in the open (PDB code: 2KX9) 9 and closed (PDB code: 2HWG) 10 conformational states. Fits were performed for q values in the range 0.02-0.16 Å −1 . In this range, SAXS data are characterized by very high signal-to-noise ratio and are mainly dependent on the overall shape of the molecule (i.e. contributions from small rearrangements within the domains are negligible). 17 NMR spectra were recorded at 37°C on Bruker 900, 800 and 600 MHz spectrometers equipped with z-shielded gradient triple resonance cryoprobes. Spectra were processed using NMRPipe 35 and analyzed using the program SPARKY. 1 H-15 N TROSY 36 and 1 H-13 C methyl TROSY 24 experiments were acquired using previously described pulse schemes. Resonance assignments of the 1 H-15 N and 1 H-13 C methyl TROSY spectra of free EI were achieved by transferring the assignments obtained for the isolated EIN 37,38 and EIC 17, 39 domains on the spectra of full-length EI (Supplementary Figs. 10 and 11). Only non-stereospecific assignments were available for the methyl groups of the Leu and Val residues in the EIC domain. Therefore, methyl-TROSY correlations for such resonances were arbitrarily assigned as δ A and δ B (for Leu), and γ A and γ B (for Val). A total of 256 1 H-15 N and 227 1 H-13 C methyl correlations were assigned. Assignments of the NMR correlations for the EI-PEP and EI-αKG complexes were performed by titration experiments, following the change in cross-peak positions as a function of added ligand. Weighted combined 1 H N / 15 N (Δ H/N ) and 1 H/ 13 C (Δ H/C ) chemical shift perturbations resulting from the addition of 5 mM PEP and 10 mM αKG were calculated using the following equation: 40 Δ H/N,C = ((Δδ H W H ) 2 + (Δδ N,C W N,C ) 2 ) 1/2 , where W H, W N , and W C are weighing factors for the 1 H, 15 N and 13 C shifts, respectively (W H = |γ H /γ H | = 1, W N = |γ N /γ H | = 0.101, W C = |γ C /γ H | = 0.251); Δδ H , Δδ N and Δδ C are the 1 H, 15 N and 13 C chemical shift differences in ppm, respectively, between free and bound states; and γ H , γ N and γ C are the 1 H, 15 N and 13 C gyromagnetic ratios, respectively.

NMR spectroscopy
Pulse schemes for acquisition of 1 H-13 C Multiple Quantum (MQ) 21 and 13 C Single Quantum (SQ) 22 CPMG relaxation dispersion experiments have been described by Kay and coworkers. Experiments were performed at 600, 800 and 900 MHz with a fixed relaxation delay but a changing number of refocusing pulses to achieve different effective CPMG fields. 41 The transverse relaxation periods were set to 30 and 40 ms for experiments run on U[ 2 H, 15 N]/Val,Leu-( 13 CH 3 / 12 C 2 H 3 )-labeled EI and on U-[ 2 H, 15 N]/Ile(δ1)-13 CH 3 -labeled EI, respectively. The resulting MQ and SQ relaxation dispersion curves were fit to a twostate exchange model using the Carver-Richards equation 42 (see Supporting Methods).

Binding assays
The enzymatic activity of EI for the hydrolysis of PEP to inorganic phosphate and pyruvate was assayed spectrophotometrically at 37°C using the EnzChek Phosphate Assay Kit (Invitrogen) and a nanodrop 2000 spectrophotometer, as described previously. 17 Michaelis constants (K m ) for the PEP complexes with EI WT , EI A and EI Q were obtained by fitting the enzymatic velocities to the Michaelis-Menten equation.
The affinity of αKG for EI WT , EI A and EI Q was measured at 37°C by means of NMR titration experiments. Equilibrium dissociation constants (K D ) were obtained by fitting the changes in the weighted combined 1 H N / 15 N chemical shifts (Δ H/N , see NMR Spectroscopy) with increasing concentration of αKG using the equation: 43 (1) where Δ 0 is the weighted combined 1 H N / 15 N chemical shift at saturation, and P and L are the protein and ligand concentrations, respectively. The chemical shift perturbation curves for residues with Δ H/N values > 0.1 ppm in the presence of 10 mM αKG were fit simultaneously using the same K D value.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. The magnesium ion present in the X-ray structure is displayed as a yellow sphere.     Supplementary Fig. 8. (d) Summary of relaxation dispersion data plotted on the structure of the closed state of EI. 11 Color coding is the same as in b.

Figure 7. Comparison of the NMR spectra of EI and EIC in the free and bound forms
Overlay of the 1 H-15 N TROSY correlation spectra for isolated EIC in the absence (red) and presence (blue) of 50 mM PEP, and for full-length EI in absence (pink) and presence (cyan) of 50 mM PEP. Close-up views of the cross peaks for residues I426 and G427 are provided. The amide groups of I426 and G427 show very large 15 N relaxation dispersion in isolated EIC, and experience large PEP-induced chemical shift perturbations. 17 Interestingly, I426 and G427 are located more than 15 Å away from the PEP binding site in the structure of the EI-PEP complex, and the changes in chemical shift for these two residues are exclusively dependent upon the change in the populations of the expanded and compact conformational states of EIC induced by PEP-binding. 17 Considering that (i) for the isolated EIC in the absence of PEP the population of compact conformer is ~3%, 17 (ii) at saturating concentrations of PEP the population of compact EIC is ~100%, 17 and (iii) the observed chemical shift is a population weighted average of the chemical shifts of the expanded and compact states, the theoretical position of the cross-peaks for I426 and G427 in the presence of 13% compact state can easily be calculated from the position of the peaks in the spectra of isolated EIC (~3% compact state) and of the isolated EIC-PEP complex (~100% compact state). The positions of the calculated cross-peaks are reported as black crosses, and agree very well with the position of the cross peaks in the spectra of the full-length enzyme in the absence of ligand. These results provide an independent validation of the accuracy of the global fit of the MQ and SQ relaxation dispersion curves.

Figure 8. Coupling between open-to-closed and expanded-to-compact conformational equilibria of EI
The EIN and EIC domains are colored blue and red, respectively. The PEP molecule is colored green. k ex is the exchange rate constant for the conformational equilibrium, and K m the Michaelis constant for the EI-PEP complex. The compact state of EIC exists in two different conformations (compact I and compact II ) that differ in side chain packing within and in the proximity of the β3α3 loop (see main text). Populations of the different conformational states are indicated. Populations on the left side of the free/bound equilibrium correspond to the populations obtained from the fit of the relaxation dispersion data acquired on EI WT ; populations on the right side correspond to the populations obtained from the fit of the SAXS curves acquired for the EI Q -PEP complex.