Structure of a low-population intermediate state in the release of an enzyme product

Thank you for sending your work entitled “Structure of a low-population intermediate state in the release of an enzyme product” for consideration at eLife. Your article has been handled by John Kuriyan (Senior editor and Reviewing editor) and three additional reviewers.

The reviewers agree that this is a fine piece of work that is overall very well performed and the conclusions logically drawn. The results further highlight the importance of characterizing lowly populated conformational states. Nevertheless, the reviewers are also in agreement that some additional data would be needed to make the conclusions more compelling. We ask that you respond by email to the points raised below, paying particular attention to the major points. Your response should tell us how you could address these issues in a realistic timeframe. A final decision on your paper will be made after the reviewers have considered your response.

Ensembles of structures of the enzyme lysozyme were determined by combining NMR-detected RDCs and MD simulations to structurally characterize an intermediate species along the enzymatic pathway. In particular, the paper analyzes NMR data using a method called ensemble-average molecular dynamics simulations. The analysis was carried out on the ligand-free and the ligand-bound states of the enzyme. The data show that the ground state (∼87%) exists in the locked state (the one wherein the product is still bound to the enzyme) and in equilibrium with an energetically excited state (populated ∼13% of the time), which allows product release (termed the unlocked state). The two states are related by an opening/closure of the ligand-binding cleft by ∼10 degrees.

The unlocked state with the bound ligand is a compact conformation that differs from the locked state by a global motion, with global RMSD values of about 1.5 Å relative to the X-ray structure. The unlocked conformations lose the tight interactions that are stabilized in the locked state and gain new interactions on the external surface of the protein. These interactions, which mainly involve hydrogen bonds between donors and acceptors groups from the ligand and the protein surface, are highly variable and heterogeneous in the unlocked conformations. It is hypothesized that the unlocked state represents an intermediate state that is critical for fast product release. The authors then identified a stabilizing hydrogen bond that can be formed only in the unlocked state (Figure 4A), which involves side-chain atoms of residue N44 E35. The ability of the N44A variant to release triNAG from its bound state was assessed by SPR. The dissociation constants of the wild-type and the N44A variant to be, respectively, 4.19 sec-1 and 1.31 sec-1. This provides a degree of cross-validation that this state is in fact important for release.

Major points:

1) One weakness identified by the reviewers is that it is only a speculation that the ligand is more loosely bound in the “unlocked” state. The authors provide no firm basis for this assertion. The paper provides only one mutant to test the key concept that there is an unlocked state. Based on the available information, it seems that one should be able to design a mutant that stabilizes the unlocked state. If so, then one could: (1) confirm the existence of the state directly by NMR, (2) measure the binding constants (kinetics and equilibrium) of the ligand for the unlocked state by SPR to show that it is indeed faster and with a weaker binding affinity. From a computational point of view, it should be possible to calculate the binding free energy of the ligand for the locked and unlocked state, and show that the affinity is reduced in the latter. Doing this additional work is substantial, but it would really demonstrate that the conclusions are correct.

2) It might be thought that the RDC approach for detecting rare states would be inferior to other approaches, like relaxation dispersion or PREs. In the case of relaxation dispersion one relies on the fact that the minor state is only transiently formed so that its effective linewidth is very large. Thus, the exchange process gives rise to a net increased linewidth in the ground state that can be detected. In a similar manner, as long as the excited state brings the NMR probes close to the spin label, a PRE effect is observed in the ground state. By contrast, however, RDCs contain information about each state and it is unlikely that the rare state has RDCs that are so different relative to the ground state, making this approach potentially less sensitive. Can the authors comment on the sensitivity of their method in general to studies of rare conformations? How much lower than 15% can one go and how robust are the states that are determined?

3) In this regard, the authors do address the robustness with their N44A mutant and this is an important control. It would be very interesting to record RDC on this mutant (free form) and show that the same profile as in Figure 1A is observed. Second, it would then be of great interest to add the product to the N44A mutant, measure RDC and repeat the calculations. One would predict that the rare conformer would become less populated, less than 15%, and that would provide further important proof of the methodology that jives with the functional (release) studies that they perform. These controls are not particularly onerous but they would be very convincing. At issue here to some extent is the reliability of RDCs for dynamics, and there has been much debate in the literature about this (especially when one considers the fact that methods for estimating RDCs from structure are only approximate, especially for electrostatic alignment, or methods for dealing with the effects of averaging between multi-states again can be complex). This work opens up an approach for looking at rare states, and further evidence of utility is important.

4) Although not necessary, have the authors considered experiments to show that the initial binding step of substrate is unaffected by the N44A mutation, as would be predicted since the ground state is unaffected?

1) One weakness identified by the reviewers is that it is only a speculation that the ligand is more loosely bound in the “unlocked” state. The authors provide no firm basis for this assertion. The paper provides only one mutant to test the key concept that there is an unlocked state. Based on the available information, it seems that one should be able to design a mutant that stabilizes the unlocked state. If so, then one could: (1) confirm the existence of the state directly by NMR, (2) measure the binding constants (kinetics and equilibrium) of the ligand for the unlocked state by SPR to show that it is indeed faster and with a weaker binding affinity. From a computational point of view, it should be possible to calculate the binding free energy of the ligand for the locked and unlocked state, and show that the affinity is reduced in the latter. Doing this additional work is substantial, but it would really demonstrate that the conclusions are correct.

We are grateful for these key suggestions. To take account of them, we have used the structure of the intermediate that we calculated to rationally design a new mutant meant to stabilise the unlocked state. In the new mutant, N46Q/V110Q, a strong glutamine-glutamine interaction is inserted with the purpose to effectively block the ‘unlocked’ state in its conformation (see Figure 4–figure supplement 3A).

We have verified the folding of the new mutant by NMR (see Figure 4–figure supplement 3B) and measured the binding constants of the ligand for the unlocked state by SPR to show that it corresponds to a weaker binding affinity (see Figure 4–figure supplement 3C).

While the Kd of the wild type is about 9 uM, the Kd of the N46Q/V110Q mutant is high almost beyond detection, indicating that the mutant essentially does not bind the substrate. These experimentally measured binding constants are consistent with the observation that, considering that the free energy of the free state is the same, the binding free energy of the locked state is larger than that of the unlocked state because the free energy of the former is lower than that of the latter (Figure 1).

In order to validate the design procedure of the mutational variants with modified stability of the unlocked state, we have performed new RDC measurements of the N44A variant and used them to determine the corresponding free energy landscape (see Figure 4–figure supplement 2). We have not repeated this NMR analysis for the N46Q/V110Q mutant since the measurements of the binding constants provided already clear indications of the success of the design procedure. If the editors, however, believe that we should perform the additional NMR analysis also on the second mutant we will be happy to do so.

2) It might be thought that the RDC approach for detecting rare states would be inferior to other approaches, like relaxation dispersion or PREs. In the case of relaxation dispersion one relies on the fact that the minor state is only transiently formed so that its effective linewidth is very large. Thus, the exchange process gives rise to a net increased linewidth in the ground state that can be detected. In a similar manner, as long as the excited state brings the NMR probes close to the spin label, a PRE effect is observed in the ground state. By contrast, however, RDCs contain information about each state and it is unlikely that the rare state has RDCs that are so different relative to the ground state, making this approach potentially less sensitive. Can the authors comment on the sensitivity of their method in general to studies of rare conformations? How much lower than 15% can one go and how robust are the states that are determined?

We recognise the importance of these considerations. In the revised version of the manuscript we have addressed the issue of the sensitivity of the approach that we have used, and briefly reviewed previous publications in which we have demonstrated that it can be employed to characterise populations as low as those studied here (De Simone et al., JACS 131, 3810-3811, 2009; J. Chem., Theor. Comput., 7, 4189-4195, 2011; Biochemistry 52, 6684-6694, 2013; Biochemistry 52, 6480-6486, 2013).

3) In this regard, the authors do address the robustness with their N44A mutant and this is an important control. It would be very interesting to record RDC on this mutant (free form) and show that the same profile as in Figure 1A is observed. Second, it would then be of great interest to add the product to the N44A mutant, measure RDC and repeat the calculations. One would predict that the rare conformer would become less populated, less than 15%, and that would provide further important proof of the methodology that jives with the functional (release) studies that they perform. These controls are not particularly onerous but they would be very convincing. At issue here to some extent is the reliability of RDCs for dynamics, and there has been much debate in the literature about this (especially when one considers the fact that methods for estimating RDCs from structure are only approximate, especially for electrostatic alignment, or methods for dealing with the effects of averaging between multi-states again can be complex). This work opens up an approach for looking at rare states, and further evidence of utility is important.

To take account of this point, we performed additional 1H-15N RDC measurements on the N44A mutant and carried out new restrained molecular dynamics simulations to determine its free energy landscape. The new results (see Figure 4–figure supplement 2a) demonstrate that the unlocked state is not populated in the N44A mutant, as expected from its rational design and the measurement of the binding constants reported in the original version of the manuscript.

4) Although not necessary, have the authors considered experiments to show that the initial binding step of substrate is unaffected by the N44A mutation, as would be predicted since the ground state is unaffected?

We have checked whether the binding step is affected by the N44A mutation.

The destabilisation of the intermediate in the N44A mutant brings down the k_off by a factor 3, while changing the K_d by a factor 1/3. The k_on varies by a factor 2, indicating that the pathways of capture and release are not completely distinct and thus perturbing the pathway for release affects in part also that of capture.