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  1. Evaluation Summary:

    This work attempts to extract information about protein thermodynamics from X-ray crystallography data, which is a challenging problem. This work presents a comprehensive examination of the structural transitions associated with small molecule binding to proteins. The heterogenous pattern of order parameter changes in response to ligand binding implies that the approach is identifying new information. This work offers insights into ligand binding affinity and specificity mechanisms, suggesting that distal (allosteric) perturbations represent a possible avenue to modulate protein function.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  2. Reviewer #1 (Public Review):

    Essential functions of biomacromolecules such as enzyme catalysis, receptor activation or inhibition, etc. depend on the thermodynamics and kinetics of ligand binding. Recent NMR data support the model that conformational entropy of protein side chains directly impacts ligand binding affinity. Also, it has been shown that conformational entropy is responsible for modulating positive and negative cooperativity. However, NMR data on complex systems are still sparse, and it is unclear whether the conclusions derived from the analysis of NMR order parameters are generally applicable. Here, the authors use a multiconformer modeling of time- and space-averaged electron density to determine the conformational heterogeneity in matched pairs (holo and apo) of crystallographic datasets and evaluate protein responses to ligand binding. In their analysis, the authors include both anharmonic and harmonic (i.e., static and dynamic) disorder using the qFit software package as well as crystallographic order parameters. The most important observation is that ligand binding globally affects the structural dynamics of proteins. When the enthalpic contribution dominates in and near the ligand-binding sites (i.e., rigidification of the binding pocket due to protein-ligand interactions), the conformational dynamics are redistributed throughout the remainder of the protein, and distal sites become more disordered. Although the hypothesis on the redistribution of conformational free energy upon ligand binding has been previously contemplated, the direct experimental proof has been difficult to achieve using thermodynamic methods alone. The latest NMR techniques, however, enable one to estimate the conformational entropy from the calculation of site-specific order parameters for methyl-bearing side chains. Therefore, the distinct merit of this paper is to demonstrate the direct link between the disorder observed in the X-ray coordinates and the conformational entropy as measured by NMR spectroscopy. The connection between these orthogonal techniques leads to the conclusion that conformational entropy must modulate ligand binding phenomena.

    Overall, the paper is exciting and offers new insights into ligand binding affinity and specificity mechanisms, suggesting that distal (allosteric) perturbations represent a possible avenue to modulate protein function. There are, however, a few weaknesses. The authors have stated some of them in the discussion section, i.e., the possibility of underestimating motions from frozen samples. Additionally, the authors offer a concise explanation for the redistribution of the conformational disorder. In fact, from the analysis of the holo/apo pairs, the authors deduced that packing optimization and rearrangements within the protein cores may be responsible for the redistribution of heterogeneity upon ligand binding. The current analysis of the data does not allow the reader to appreciate these conclusions entirely. Is there a way to look at the packing of the structures, both the apo and holo state? Additionally, the authors suggest that possible hydrogen bond rearrangements may also occur upon ligand binding. However, this aspect is poorly developed or discussed in the current version of the paper. Another shortcoming is the lack of consideration for other electrostatic interactions involving charged side chains that often dominate ligand binding affinity. Despite these shortcomings, the authors' work is solid.

  3. Reviewer #2 (Public Review):

    Overall, this is a comprehensive examination of the structural transitions associated with small molecule binding to proteins. A database of protein-small molecule complexes has been well curated from the PDB and the criteria explicitly stated. The resulting structures are carefully analyzed and compared and the observations very clearly presented. Since a continuum of response is seen, an excellent statistical framework is provided. Many new insights into the structural (energetic) and entropic contributions to ligand binding thermodynamics are discovered and synoptically put together. The main observation is the heterogeneity of response of proteins to small molecule ligand binding. One imagines that this will become a classic step forward in our understanding of the thermodynamics of molecular recognition by proteins and will be of great utility for biochemists, structural biologists and drug developers.

  4. Reviewer #3 (Public Review):

    Protein function emerges from a fine balance between enthalpic and entropic contributions of the various interactions between constituent atoms. While protein structures derived from X-ray diffraction data often yield rich information about enthalpic contributions, extraction of entropic information remains challenging. Here, Wankowicz et al. present a three step approach to quantitatively map changes in conformational heterogeneity for side chains across hundreds of different proteins in response to ligand binding. First, the authors create a well-curated, high resolution set of paired apo and holo structures from the PDB. The models were re-refined against the published data, and then multiconformer models were generated with qFit. The authors calculated crystallographic order parameters for most residues across all pairs. In cases where the binding site was found to rigidify upon ligand binding, a compensatory increase in disorder was observed at distant sites-an observation consistent with free energy considerations as well as with results of single protein studies conducted using other methods.

    This work is interesting as it is the first systematic attempt to make such measurements from X-ray data at scale, spanning hundreds of different crystal structures bound to a variety of ligands. This approach leverages existing data, and could thus prove to be hypothesis generating in specific cases related to goals such as drug design and the identification of allosteric pathways/sites. Importantly, the development of this approach directly builds upon previous work (Fenwick et al. 2013) and represents a conceptual step towards the general goal of estimating of configurational entropy from X-ray crystallography data.

    While the authors are candid about the limitations of the study and describe a plan to overcome some of the issues they describe, this work does have some weaknesses which should be addressed. From a technical perspective, definitions of different types of residues (binding site vs. distant) can seem arbitrary, so explaining the criteria thoroughly and showing that the results are robust to variation in these definitions is important. Conceptually, this work suffers somewhat from a lack of comparison with other studies of configurational entropy and, more generally, propagated structural change in proteins. Some quantitative comparison with data from the literature for specific cases is warranted.