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Solvent networks tune thermodynamics of oligosaccharide complex formation in an extended protein binding site Sonja Kunstmann, Ulrich Gohlke, Nina K. Broeker, Yvette Roske, Udo Heinemann, Mark Santer, and Stefanie Barbirz J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b03719 • Publication Date (Web): 25 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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Journal of the American Chemical Society

Solvent networks tune thermodynamics of oligosaccharide complex formation in an extended protein binding site Sonja Kunstmann1,2,#, Ulrich Gohlke3,#, Nina K. Broeker1,#, Yvette Roske3, Udo Heinemann3,4*, Mark Santer2,* and Stefanie Barbirz1,* 1

Physikalische Biochemie, Universität Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany. Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany 3 Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str. 10, 13125 Berlin, Germany. 4 Institut für Chemie und Biochemie, Freie Universität, Takustr. 6, 14195 Berlin, Germany. 2

Supporting Information Placeholder

Introduction

ABSTRACT: The principles of protein-glycan binding are still not well understood on a molecular level. Attempts to link affinity and specificity of glycan recognition to structure suffer from the general lack of model systems for experimental studies and the difficulty to describe the influence of solvent. We have experimentally and computationally addressed energetic contributions of solvent in protein-glycan complex formation in the tailspike protein (TSP) of E. coli bacteriophage HK620. HK620TSP is a 230 kDa native trimer of right-handed, parallel beta-helices that provide an extended, rigid binding site for bacterial cell surface O-antigen polysaccharides. A set of high-affinity mutants bound hexa- or pentasaccharide O-antigen fragments with very similar affinities even though hexasaccharides introduce an additional glucose branch into an occluded protein surface cavity. Remarkably different thermodynamic binding signatures were found for different mutants; however, crystal structure analyses indicated that no major oligosaccharide or protein topology changes had occurred upon complex formation. This pointed to a solvent effect. Molecular dynamics simulations using a mobility-based approach revealed an extended network of solvent positions distributed over the entire HK620TSP binding site. However, free energy calculations showed that a small water network inside the glucosebinding cavity had the most notable influence on the thermodynamic signature. The energy needed to displace water from the glucose binding pocket depended on the amino acid at the entrance, in agreement with the different amounts of enthalpy-entropy compensation found for introducing glucose into the pocket in the different mutants. Studies with small molecule drugs have shown before that a few active water molecules can control protein complex formation. HK620TSP oligosaccharide binding shows that similar fundamental principles also apply for glycans, where a small number of water molecules can dominate the thermodynamic signature in an extended binding site.

Non-covalent protein-glycan recognition is a key event in many intercellular communication processes linked to phys1-3 iology, immunology and pathogenesis. Interactions of carbohydrates with proteins take place in aqueous environments and between molecules with amphiphilic surfaces. This means that a full understanding of protein-glycan complex formation is ultimately linked to the general challenge of describing solvent dynamics, energetics and cooperativity 4-6 at biological interfaces. However, most fundamental studies on solvent effects are motivated by drug design whereas the number of carbohydrate complexes studied is remarkably 7-10 smaller. This mainly results from the limited availability of glycan ligands, despite a noteworthy need to match experi11-13 mental data with computational approaches. Moreover, most analyses focus on rather small carbohydrate interaction surfaces as found in lectins. Only recently, glycans in more extended sites or long-range effects upon glycan binding

Scheme 1. Structure of E. coli O18A O-antigen repeata

a

→2)-α-L-Rhap-(1→6)-α-D-Glcp-(1→4)-α-D-Galp((3→1)-βThe O18A1 subtype has an additional α-D-Glcp-(1→6)-branch (red) at the reducing end. D-GlcNAcp)-(1→3)-α-D-GlcNAcp-(1→.

1 ACS Paragon Plus Environment

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entire glycan ligand is in contact with the protein surface in a 25 Å binding groove on the central parallel β-helix fold (Figure 1A). This binding mode differs from many lectins which often interact with only a small number of monosaccharide 33 building blocks. The affinity of HK620TSP towards a hexasaccharide ligand is highly dependent on the amino acid residue at position 372, one of the two endoglycosidase active site residues. Glutamate in the wild-type has a submillimolar hexasaccharide KD. In contrast, exchange for glutamine or alanine significantly decreased the hexasaccharide KD into the submicromolar range. HK620TSP hexasaccharide complex formation is an enthalpically driven process in the E372A mutant, but has a more favorable entropic contribu27 tion for the E372Q mutant. These distinct thermodynamic signatures were interpreted as a consequence of water release from the E372Q mutant after rotation of a glutamine side chain. However, the lack of structural information on the ligand-free protein impeded a meaningful explanation in case of the E372A mutant. In this work, we studied HK620TSP as a model to analyse contributions of small patches of water molecules to the thermodynamic signature of oligosaccharide binding. We combined isothermal titration calorimetry, X-ray crystallography, molecular dynamics and free energy calculations. Using a set of different glycan ligands and HK620TSP mutants we show that distinct amounts of enthalpy-entropy compensation upon oligosaccharide-binding are linked to specific solvation structures in an occluded glucose-binding cavity.

Figure 1: Figure caption. (A) HK620TSP monomer (cartoon) with hexasaccharide (cyan sticks) in the binding site (blue frame). (B) Distribution of water molecules in the hexasaccharide binding site of HK620TSP with an alanine exchange at position E372. “Loop”: residues 471-476 shielding a Glcbinding pocket. Water molecules (crosses) and selected side chains are superimposed from the oligosaccharide complex (thick sticks, green) or from the structure without ligand (thin sticks, purple). In the E372A mutant hexasaccharide complex, two water molecules (green spheres) take the position of the wild-type glutamate (overlaid for comparison as black sticks) and mediate hexasaccharide contacts.

Results Crystal structure analysis of HK620TSP high-affinity mutants In this work we used a set of mutants in which both HK620TSP active site residues E372 and D339 were replaced by the respective amide or alanine in all combinations (Ta27 ble S1). We solved 21 crystal structures of these HK620TSP variants or wild-type protein in the presence or absence of two different types of oligosaccharide ligands in a resolution range of 1.06-2.10 Å (Tables S1 and S2). First, we examined complexes with hexasaccharide ligands prepared from lipopolysaccharide (LPS) preparations of the HK620 phage host 34 E. coli O-serogroup O18A1 as described earlier. Second, we co-crystallized a pentasaccharide ligand prepared for this work from an E. coli O-serogroup O18A strain (see Figures S1 and S2 in the Supporting Information). The pentasaccharide ligand lacks a branching glucose compared to the hexasaccharide (Scheme 1). All crystal structures of mutant variants and wild-type protein of the 230 kDa HK620TSP native trimer were very similar and had penta- and hexasaccharides in identical binding positions (Figure S3).

14,15

have been described. Whereas the protein part is often structurally well-defined, describing glycan and solvent dynamics remain a major challenge with both experimental and 9,10,13,16 computational approaches. X-ray crystal structure analysis is a straightforward way to experimentally define water positions on the protein surface; however description of contiguous solvent networks is often hampered by low 6,17-19 resolution. Assignment of water networks is a main challenge in drug design, and several model systems using a variety of ligands have been thoroughly analysed in this 5,17,20-22 respect. Bacteria display a rich pool of glycan structures on their surfaces, which play an important role in pathogenesis and as 23-25 targets in antibiotic therapy. Bacteriophages recognize these glycan structures with their tailspike proteins (TSP) and the resulting oligosaccharide-protein complexes are well 14,26-29 suited models for studying origins of glycan affinity. The TSP of bacteriophage HK620 is an endo-Nacetylglucosaminidase that cleaves the O-antigen of its E. coli host surface lipopolysaccharide as the initial step of the bac30-32 teriophage infection cycle. HK620TSP forms a homotrimer of 230 kDa with parallel β-helix architecture and three 27,31 specific oligosaccharide-binding sites. In HK620TSP, the

Major changes in side-chain positions of the different HK620TSP variants upon oligosaccharide complex formation only occurred in the center of the binding site (residues 339400) surrounding the high-affinity mutation site. As previously described, when glutamine is at position 372, in all mutational backgrounds it undergoes a side chain rotation to bind the ligand and displaces a water molecule, resulting in high affinity compared to the wild-type glutamate at this po-


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with H-bonds to main chain atoms of apolar residues shaping the interior walls. Upon hexasaccharide binding, four of these water molecules are expelled by the incoming glucose.

Thermodynamics of oligosaccharide binding to HK620TSP HK620TSP wild-type pentasaccharide complexes lacking the glucose side chain branch showed a twofold-reduced dissociation constant (KD = 75 µM) (Figure S7 and Table S3) compared to that found for hexasaccharide complexes with fluo27 rescence spectroscopy (KD = 129 µM) . Compared to a highaffinity mutant, pentasaccharide binding to wild-type was dominated by faster off-rates but only slightly reduced ligand association rates (Table 1 and Figure S8). Pentasaccharide binding to wild-type HK620TSP produced substantially more heat than hexasaccharide binding. Filling of the glucose binding pocket hence must contribute an enthalpic barrier in the wild-type; hence analysis of hexasaccharide complex formation was not possible with isothermal titration calorimetry (ITC).

Figure 2. Figure caption. Thermodynamics of complex formation with hexasaccharide (6mer, filled glucose-binding pocket) or pentasaccharide (5mer, empty glucose-binding pocket) in (A) HK620TSP E372Q and (B) E372A mutants. See Table S3 for the full isothermal titration calorimetry data set.

Table 1. Stopped-flow fluorescence HK620TSP-pentasaccharide binding TSP variant D339A/E372Q Wild type

kineticsa

kdiss -1 /s

kass -3 -1 -1 /10 M s

KD / µM

0.087

572

0.15

7.37

130

57

of

However, upon binding to the high-affinity mutants E372A or E372Q, ITC analyses showed no major differences in affinity between penta- and hexasaccharide (Figure 2, Table S3 and Figure S7). Remarkably, the E372A mutant showed a similar enthalpy-entropy distribution with both ligands, although with the hexasaccharide three additional H-bonds were formed in the glucose-filled cavity. Fixation of glucose inside the binding pocket therefore seemed to be enthalpically neutral in the alanine mutant, i.e. glucose and water behaved as cognate ligands. In contrast, in the E372Q mutant pentasaccharide binding showed a notably increased enthal-1 pic contribution (ΔΔH -16 kJ mol ), compared to the hexasaccharide. The glucose branch of the latter again was stabilized in the binding pocket via three additional H-bonds, but two H-bonds were lost that fixed the reducing end GlcNAc of the pentasaccharide (Figure S9). However, the enthalpic gain upon pentasaccharide binding was balanced by a substantial -1 loss of favorable entropy (ΔΔS 17 kJ mol ).

a

See Figure S8 in the Supporting Information for all kinetic traces 27

sition. In the present study, this water molecule and all binding site rearrangements were found conserved throughout all HK620TSP variants carrying the E372Q high-affinity substitution and in complex with both ligand types. Even more, crystal structures in the present work now revealed that in the other high-affinity variants with an alanine substitution at position 372 and in the presence of a hexasaccharide the carboxylate oxygen positions of the wild-type glutamate were substituted with two water molecules (Figure 1B). Side chain rearrangements in the E372A mutants were found for the imidazole ring of H374, that rotates about 60° when binding the ligand , whereas all in all other mutational backgrounds the H374 ring only undergoes positional shifts without rotation (Figure S4).

Given a similar degree of ligand fixation via H-bonds in all different complexes we do not expect major differences in

Table 2. Enthalpy and entropy differences of pentaand hexasaccharide binding to HK620TSP TSP variant

In general, in all crystal structures less solvent was observable in the oligosaccharide-binding site in the absence of the ligand, with 21.6±10.3 water molecules on average in all apo structures. As expected these numbers were highly resolu9 tion dependent (Figure S5). In contrast and irrespective of resolution, in the 18 ligand bound complexes the number of water molecules had doubled with 43.1±6.2 and 40.0±4.4 on average for penta- and hexasaccharide complexes, respectively. About 30 % of these water positions provided highly conserved H-bonds between protein and ligand throughout all complexes (Figure S6). Even more, 75 % of the crystallographic water positions showed high positional conservation regardless of their mutational background (Figure S5). In all pentasaccharide complexes these positions were conserved to more than 90 %. A network of five water molecules can be found inside the binding pocket for the branching glucose

-1

a

b

-1

ΔΔH / kJ mol

Δ(TΔS) / kJ mol

E372Q

15.9

17.0

E372Q/D339N

13.8

14.8

E372Q/D339A

14.4

15.3

E372A

6.4

4.3

E372A/D339N

8.0

6.7

E372A/D339A

6.4

7.4

E372QΔloop

6.6

-3.2

a

ΔΔH = ΔHhex-ΔHpent (cf. Table S3), TΔSpent (at 25 °C, cf. Table S3)


b

Δ(TΔS) = TΔShex-


Page 4 of 18

entropy contributions from the ligand. Rather, fixation of a 1-6-linked glucose would even impose an additional entropic penalty onto the system. We therefore hypothesized that a positive entropy contribution upon HK620TSP E372Q hexasaccharide binding is due to the solvent dissociation from the glucose-binding cavity. From this, it follows that in case of pentasaccharide binding the solvent entropy contribution was not observed because the glucose-binding pocket remained entirely solvated. This contradicts the initial hypothesis that had assigned both entropic and enthalpic penalties to the single water molecule outside the pocket that gets 27 expelled by the rotating glutamine upon ligand binding. This rotation and water displacement occurred in all pentaand hexasaccharide complexes. In contrast, for the E372A mutant we did not observe a difference in entropy between penta- and hexasaccharide binding. Apparently in this case, the thermodynamic signature was neither dominated by fixation of the 1-6 linked glucose nor by desolvation of the glucose-binding cavity.

creased the entropic penalty. Rather, we propose that the loop deletion changed the solvation characteristics of the glucose-binding pocket. As a consequence water molecules inside the pocket no longer contributed favorably to the entropy term upon their release during ligand binding. In addition, the HK620TSP Δloop hexasaccharide complex had a -1 slightly more favorable enthalpy term (ΔΔH -2.7 kJ mol ), although compared to a protein with an intact loop, two Hbonds are missing. The HK620TSPΔloop-pentasaccharide complex lacks only one of these H-bonds, but has a considerably reduced enthalpic term compared to the hexasaccharide -1 complex (ΔΔH 6.6 kJ mol ), which now resembles the ΔΔH value found for the E372A mutant with an intact loop. In the HK620TSPΔloop mutant this value however was no longer fully compensated by the corresponding entropy difference, resulting in more notable affinity differences for penta- and hexasaccharides (cf. Table 2). Opening the binding pocket therefore at least in part annulled the full EEC found for inserting glucose into an intact cavity.

In summary, the distinct thermodynamic signatures found for hexa- and pentasaccharide binding to HK620TSP mutants did not influence overall binding free energies. This means that enthalpy differences between hexa- and pentasaccharide binding were fully compensated for by the entropy differences (Table 2). Notably, the absolute amounts of enthalpyentropy compensation (EEC) were significantly higher in E372Q than in E372A mutational backgrounds and irrespective of the residue at position 339. EEC is highly sensitive to small changes in the ligand environment and dominated by 35 solvent reorganization. We therefore conclude that EEC differences occurring in HK620TSP high-affinity mutants E372A and E372Q were related to different solvation properties of the glucose binding pocket.

Solvation structure of the HK620TSP binding site In the crystal structures of the HK620TSP variants, especially in the absence of the ligand, different numbers of water positions became resolved (Table S5 and Figure S10). We performed molecular dynamics (MD) simulations for 50 and 100 ns with HK620TSP E372A in the absence or presence of a ligand and found high exchange rates for most water molecules. We calculated water positions from relative H-bond occupancies of more than 2 % during the simulation run time, which could reproduce about half of the crystal structure positions (Table S5 and Figure S11). To further analyze solvent effects in oligosaccharide binding we wanted to obtain a more complete picture of protein surface solvation. We therefore applied a water mobility-based approach to compute water positions on the different HK620TSP oligosaccharide complexes. This method was previously shown to suc-

Deletion of the HK620TSP glucose-cavity covering loop To further analyze whether the glucose-binding cavity dictated the solvent-related thermodynamic signatures of oligosaccharide binding, we used the HK620TSP E372Q mutant and deleted residues N471 and S472 of the loop that covers the glucose binding pocket (HK620TSP Δloop). As a consequence, in the absence of ligands, the loop became disordered (residues 473-476). With both ligands, HK620TSP Δloop displayed identical oligosaccharide-binding modes but increased flexibility at adjacent ends of the loop (residues 468-483). Whereas no electron density was detectable for the loop in the hexasaccharide complex, with the pentasaccharide bound the loop was distorted and proline 469 occupied the glucose position (Figure 3A). HK620TSP Δloop had slightly reduced binding affinities towards the oligosaccharides (Figure 3B and Table S4). No major changes in the enthalpy-entropy distribution occurred upon pentasaccharide binding, which is reasonable given that the glucose-binding pocket is not occupied in this case. This emphasizes that the loop deletion has no important influence on other parts of the binding site. However, compared to the intact protein, HK620TSPΔloop-hexasaccharide complex formation was less favorable in entropy. This complex showed no interpretable electron density for the loop, excluding the possibility that anchoring the loop had in-

Figure 3: Figure caption. (A) HK620TSPΔloop (orange) lacking the glucose-binding pocket shielding residues N471 and S472 in complex with pentasaccharide superimposed onto the intact protein in complex with hexasaccharide (blue). Penta- (thick sticks) and hexasaccharide ligand (thin sticks) are shown in white. (B) Thermodynamics of HK620TSPΔloop complex formation with hexasaccharides or pentasaccharides (bars in pale color) compared to E372Q complexes with intact loops (bars in full color) (cf. Figure 2). See Table S4 for the full isothermal titration calorimetry data set.


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cessfully predict hydration structures by accumulating information on water trajectories from MD simulations with an 36 identity-based clustering algorithm. As computationally more affordable models we used truncated HK620TSP monomers (amino acids 205-521) and simulated for 10 ns in a TIP3P explicit water box. The short simulations showed stable backbone conformations and no ligand detachment (Figure S12). From these trajectories we predicted mobility-based solvation structures as putative hydration positions connected by H-bonds (Figure 4 and Figures S13 and S14). This reproduced more than 60 % of the water positions from crystal structures. Moreover, when solvent positions were connected using a distance threshold of 3.5 Å, the resulting networks were notably extended over the entire binding site (Figure S15). Large clusters of connected water positions were present in the ligand-free mutants. These networks were well linked between the upper and the central part of the binding site in the mutants, whereas in the wild-type protein the two parts were not linked (Figure 4). Upon ligand binding, cluster size and network connectivity decreased in the highaffinity mutants and the networks separated in two parts (Figure 4B and Figure S14). In contrast, in the low-affinity wild-type protein, oligosaccharide binding increased the number of water positions in network clusters and the two separated network parts became linked. The mobility-based water positional analysis hence suggested a highly mobile solvent with unrestricted access to all parts of the HK620TSP oligosaccharide-binding site.

Figure 4: Figure caption. Water networks in the HK620TSP binding site from MD simulations and mobility analysis. (A) Typical network calculated for the ligand-free wild-type protein (see Figure S14 for all networks in the presence of ligands and mutants) Dashed lines show potential H-bonds between water molecules, which are colored according to the number of partners within the network: 0=green, 1=yellow, 2=orange, 3=red, 4=purple, 5=blue, 6=brown, 7=black. The loop covering the glucose binding pocket is shown as red surface. The five water positions in the glucose binding groove are shown as enlarged spheres. (B) Relative contribution of water clusters of different size to the networks in wild-type (WT) and mutants in the presence of pentasaccharides (“5”) or hexasaccharides (“6”) or with no ligand (“nl”). Clusters were defined as non-interrupted subnets containing different numbers of water molecules (1=red; 2=orange; 3=light green; 5-10= dark green; 11-25, cyan; 26-75=blue). with the TIP3P water model throughout. With the TIP5P model no notable differences in the ΔΔGFE were found for a test set of water molecules that were displaceable by glucose (Table S8). For the wild-type negative ΔΔGFE values were obtained for all positions except W#0 in the absence of the ligand (Table 3 and Figure 5B). However, they became markedly positive in the presence of the pentasaccharide. This reflects a reduced solvent stabilization by H-bonds in the pocket, in agreement with the mobility analysis predicting larger distances between these water positions in the pentasaccharide complex (Figure S14). In contrast, in the alanine mutant, ΔΔGFE of glucose binding pocket waters were very similar in the complex and the unbound form. Mobility analysis here proposed an even tighter water network inside the glucose pocket for the pentasaccharide complex. In the glutamine mutant, both positive and negative ΔΔGFE values were found in the absence of an oligosaccharide. With the pentasaccharide bound, again ΔΔGFE became mostly positive, albeit to smaller amounts. In conclusion we did not observe notable differences in the positional networks predicted for the glucose binding pocket in the absence or presence of the pentasaccharide.

Free energy calculations of glucose-binding pocket desolvation The calculated position-based networks lack information on occupancies and cooperative behavior that link network connectivity to solvation energetics. To probe solvent positions in the HK620TSP glucose binding pocket for their energetic contribution, we calculated free energies of desolvation ΔΔGFE with the double decoupling (DDC) method with37 in an atomistic simulation setup. This approach allows evaluating the free energy needed to remove a water molecule from a protein surface location relative to the energy needed to remove it from the bulk. It thus represents a measure of how easily a water molecule tends to be immobilized at a certain site within a given solvent network and protein environment. We defined five water positions in the glucose binding pocket that according to mutational background and ligand present form different H-bond networks (Figure 5A and Table S6). W#1-W#4 are replaced by glucose whereas W#0 is conserved also in the presence of glucose, mediating unsaturated backbone contacts between residues G468 and S429.

For a qualitative estimate of the total desolvation free energy we summed up individual ΔΔGFE on all positions in the pocket of each structure (Figure 5B). In the ligand-free proteins, these ΣΔΔGFE showed that pocket desolvation was clearly favorable for the E372A mutant but unfavorable for the wild-type, with the E372Q mutant lying in between. In contrast, water molecules remaining in the pocket while a pentasaccharide ligand was present, showed positive ΣΔΔGFE, especially for the wild-type, implying that fixing of water here comes with the highest penalty.

The five water sites were always occupied in the crystal structures of pentasaccharide complexes, whereas in ligandfree structures between two and five waters were found. Moreover, all five glucose binding pocket water positions were predicted in the mobility-based analyses (cf. Figure S17). Repeated double decoupling calculations on W#0 -1 showed a statistical error of 3 kJ mol and no impact of backbone flexibility on ΔΔGFE (Figure S16). Water was described



Table 3. Desolvation free energies of water molecules in the glucose-binding pocket from double decoupling calculations

Identifying “active water” in the HK620TSP oligosaccharide binding site HK620TSP has a large oligosaccharide binding interface and at least 40 water positions were resolved in crystal structures of complexes with the glycan ligand. This reflects the situation at cryo-temperatures and this number is additionally influenced by resolution, crystal lattice and the manual 6,39,40 refinement. MD simulations in contrast are limited by sampling rates upon determination of H-bonded solvent 8,41,42 positions and the use of an appropriate water model. When comparing sets of structural water molecules in HK620TSP E372A simulated with TIP3P, TIP4P or TIP5P we found that no model outperformed the other, and only half of the crystal structure positions were correctly predicted (cf. Table S5 and Figure S11). This emphasizes that the first hydration shell on the HK620TSP glycan binding surface has specific properties which are not well addressed by the different water models. Here potential functions are optimized against bulk water properties and are insufficient to fully picture the complex solvent behavior on varying biomolecu8 lar surfaces. For example, the TIP5P model was shown to have a large impact on dynamics of unfolded regions of model peptides but could well describe polysaccharide hydration.

-1 a,b

ΔΔGFE / kJ mol c

Water #

ligand-free

c,d

5mer

c,d

6mer

HK620TSP wild type 0

2.44

13.8

16.3

1

-3.33

6.89

-

2

-12.17

10.17

-

3

-8.14

16.79

-

4

-7.44

12.82

-

HK620TSP E372Q 0

8.04

12.95

5.43

1

-8.64

2.10

-

2

7.36

8.41

-

3

-7.38

-9.63

-

4

-1.37

7.25

-

Page 6 of 18

43,44

HK620TSP E372A 0

-3.70

-3.92

5.50

1

10.16

8.72

-

2

-2.13

-4.15

-

3

9.43

12.11

-

4

-4.21

-0.93

-

5*

-

-7.62

-

Even more, our data illustrate that force field improvements have to target the directionality of H-bonds near surfaces in order to more accurately describe non-bulk electro45,46 statics of water. Eventually, the mobility-based approach provided the most complete prediction of crystal water positions in the HK620TSP glycan binding interface. Here, waterprotein interactions are not taken into account explicitly, in contrast to longer simulations employing H-bond occupancies. The predicted mobility-based solvent positions thus could show all sites capable of forming networks. Additional information on network properties must come from other approaches.

a

See Table S7 for crystal structure residue numbers, simub lation IDs and rmsd values. ΔΔGFE calculations were repeated five times on E372Q-apo W#0 for the test of convergence -1 c with an average error of 3 kJ mol (cf. Figure S16) Asterisk marks an additional water position #5 found in the E372A d pentasaccharide complex crystal structure. Italic values indicate positions added from MD simulation.

In case of HK620TSP we used double decoupling calculations to probe “active water” positions. They showed that each individual water molecule in the glucose binding pocket was sensitive to the surrounding solvent network, varying with ligand status or mutational background. As a consequence the ΔΔGFE varied both in sign and magnitude. Here, especially for the conserved W#0 that is not replaced by glucose we found positive ΔΔGFE. Other studies also reported 47 pronounced positive values , which may be attributed to the inaccurate representation of interactions between polar and non-polar residues when using a non-polarizable force 48 field. Here, for protein-bound, conserved water, TIP5P was 8 described as the most reliable water model. It yielded pronounced negative ΔΔGFE for a water that could not be replaced by a ligand, whereas TIP3P could not confirm this trend. In our work, W#0 was conserved in all crystal structures. Moreover, it showed markedly increased residence times compared to mobile W#1-W#4 during 50 to 100 ns simulations employing both TIP3P or TIP5P water models (Table S9). However, the conserved W#0 in the different mutational backgrounds showed either higher or lower but still positive ΔΔGFE with TIP5P when compared to TIP3P (cf.

Discussion In the present work we have analyzed a large glycan binding site that has a distinct thermodynamic signature for solvent rearrangements upon oligosaccharide binding. As previously described for other systems, these effects are dominated by a few solvent molecules in rather small subsites 21 which thus contribute to the “active solvation”. For HK620TSP we propose that such an active solvation is present in its glucose binding pocket. We could fully associate the entropic and enthalpic differences observed to solvent rearrangements inside and around this pocket because we did not observe changes in protein or ligand rearrangements between HK620TSP E372A and E372Q mutants upon pentaor hexasaccharide complex formation. Furthermore, very similar ΔCp values were found for hexasaccharide binding to the alanine and glutamine mutant implying similar Van-der27,38 Waals contact areas between the two mutants.


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Table S8). This shows that in the HK620TSP glucose binding pocket TIP5P did not produce negative ΔΔGFE for W#0.

Solvent contributions to thermodynamic signature of HK620TSP oligosaccharide binding

Further test calculations on mobile water in the pocket did not reveal major differences for ΔΔGFE obtained with either TIP3P or TIP5P. For the hydrophilic mobile water network in the rigid, slightly hydrophobic glucose binding pocket TIP3P should thus be an appropriate choice of water model (Figure S18). It has increased mobility and diffusivity, facilitating 43 statistical sampling especially in restricted environments. Accordingly, we can describe the energy contributions of single water molecules as part of a given water network because we do not account for interactions with the non-polar interior of the glucose binding pocket and leave the remaining pocket waters unrestrained. Interestingly, the calculated energies showed that water positions already defined by crystal structures had a substantial impact on the overall desolvation free energies. This emphasizes that in HK620TSP their properties will dominate the thermodynamic signature of ligand binding upon filling the glucose-binding pocket. A similar effect was observed for hydrated protein cavities with 49 grand canonical Monte Carlo simulations. In contrast, for other systems also the importance of low-occupancy solvent 50 sites for hydration thermodynamics has been discussed.

Solvent rearrangements in the glucose-binding pocket dominate the different thermodynamic signatures of HK620TSP penta- and hexasaccharide complex formation. We can distinguish two cases: i) Solvent enclosure with a strong gain in entropy upon water displacement, accompanied by an enthalpic expense due to water dissociation from 52 the hydrophobic surface, i.e. a classical hydrophobic effect. This would be the case with glutamine at the glucosebinding pocket entrance. ii) Fewer restrictions on solvent inside the cavity, in this case the gain in entropy is negligible upon ligand binding as observed for the alanine mutant. The different amounts of enthalpy-entropy compensation (EEC) found for the HK620TSP mutants hence must also reflect their different solvation structures in the glucose binding pocket (cf. Table 2). EEC is commonly found when monitoring complex formation in ITC as a consequence of solvent reorganization in response to conformational changes during 35,53 ligand binding. Recently, mutations in the human carboanhydrase active site also showed distinct EEC patterns for two types of an arylsulfonamide ligand, one water-replacing 38 and one non-water replacing. These were assigned to the entropies and enthalpies of individual waters in the binding site, showing that disruption of water networks in the site made favorable entropy contributions. A similar effect occurred in the HK620TSP glutamine mutant, although we have not yet assigned individual entropic and enthalpic contributions to hydration free energies in the glucose-binding pocket.

The double decoupling calculations hence revealed pronounced desolvation free energy variations for water in the HK620TSP glucose-binding pocket depending on the residue at the entrance. Although further calculations are needed for the HK620TSP system to define enthalpy and entropy con50,51 tributions, we can state that with glutamine, more energy was needed to desolvate the pocket compared to alanine. In the wild-type protein, an even tighter bound water network was found in the glucose-binding pocket, in agreement with a high energetic penalty for hexasaccharide binding.

EEC occurs due to the hydrophobic effect that can be understood as a consequence of altered H-bond dynamics close 4,52 to surfaces. We used the tool MobyWat and found distinct water positional distributions on the HK620TSP surface. For HK620TSP we have not further classified the resulting potential water networks into either static or dynamic parts. This has been done in a histone-chaperone model complex and high affinities were assigned to a more static water network surrounding the complex indicating higher kinetic barriers 54,55 for solvent exchange. In case of HK620TSP all highaffinity mutants had a smaller number of connections in the mobility-based water networks compared to the wild type when bound to oligosaccharides (cf. Figure 4). We assume that this indicates a more static water network in the highaffinity complexes that are consequently linked to elevated dissociation barriers that reflect how easily a water network 21 is restored when the ligand is removed. Particularly, in kinetic analyses of pentasaccharide binding we found a pronounced decrease in the ligand dissociation rates for the high-affinity mutant when compared to wild type (Figure S8).

Figure 5: Figure caption. (A) Crystallographic solvent positions in the HK620TSP glucose-binding pocket used for calculation of desolvation free energies ΔΔGFE with the double decoupling (DDC) method (cf. Table 3). The glucose position found for hexasaccharide complexes is shown as thin sticks. (B) Desolvation free energies of glucose-binding pocket water molecules in the absence of a ligand or in the presence of the pentasaccharide. ΣΔΔGFE were calculated from values in Table 3.

As a result, the double decoupling calculations employed for HK620TSP could also detect different free energies of glucose pocket desolvation because this statistical mechanics approach relies on subtle differences in the dynamics of water at the solvent positions in the pocket. Here, a further analysis is necessary for the alanine and the glutamine mutant to understand EEC differences found when filling the



glucose binding pocket (cf. Table 1). It is tempting to speculate here that a more bulk-like behavior of water can be found in the alanine mutant that consequently does not influence filling of the pocket to large extent. In contrast, with glutamine at the entrance – and most probably also with glutamate in the wild-type - water is more enclosed inside the pocket and builds a rather static network that is energetically less favorable to dissociate.

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uid nitrogen. Diffraction data sets were collected at 100 K at beamline BL14.1 at the synchrotron-radiation source BESSY II, Helmholtz-Zentrum Berlin. Data processing, refinement, and model statistics are summarized in Table S2. Water positions were added automatically to all models using 57 ARP/wARP and checked manually throughout refinement with all occupancies set to 1. The water molecules present in the final models are within 3.4 Å (i.e. H-bond distance) from at least one polar protein or carbohydrate atom or another water site, have similar B values as the atoms they are hydrogen-bonded to and are within 2Fo-Fc electron density contoured at 0.9 sigma. The final model coordinates have been deposited at the Protein Data Bank with accession numbers 6G0X, 4XKW, 4XKV, 4XLA, 4XL9, 4XLF, 4XLE, 4XLC, 4XMY, 4YEL, 4XLH, 4XM3, 4XN3, 4XN0, 4YEJ, 4XR6, 4XNF, 4XOR, 4XOP, 4XON, 4XOT, 4XQF, 6GVP, 6GVR. Figures were gen58 erated with PyMOL.

Furthermore, the crystal structures of E372A variants showed an additional water molecule at the entrance that might act as a bridge between the solvent inside and outside the pocket (Figure S19). An additional water molecule was also found in the glucose binding pocket of some E372A pentasaccharide complexes (cf. Table 3 and Figure S19). In the double decoupling analyses, adding the ΔΔGFE value for this sixth water position did not markedly change the sum of all -1 ΔΔGFE given an error of about 3 kJ mol of the calculation. Adding more water thus did not influence the solvation properties of the E372A glucose binding pocket to a large extent.

Binding analyses. Fluorescence stopped-flow analyses 26 have been described. Briefly, the fluorescence change (λex = 295 nm, λem = 350 nm) upon mixing equal volumes of oligosaccharide and HK620TSP solutions in standard buffer was monitored with a SX-18MV (Applied Photophysics, Leatherhead, U.K.) stopped-flow spectrofluorimeter. The final protein concentration after mixing was 0.23 µM in all measurements. All points are the mean of 15 individual measurements with a standard deviation of less than 1 %. The 27 experimental set up for the ITC has been published.

In summary, for HK620TSP a physically significant picture emerges linking water dynamics on a protein surface to the different hydrophobic effects observable in calorimetry. Altered water network dynamics have been proposed to be related to reduced ligand pull-down capacities of mutant chaperone-histone complexes and hence to reduced affinity, however, thermodynamics of complex formation have not 54 been described for this system. A distribution of water molecules with different H-bond dynamics close to a protein surface is also in line with recent approaches to describe entropies as probability distributions with the Kulback-Leibler 56 divergence. The present thermodynamic study of the HK620TSP glucose-binding pocket is a useful starting point to further quantify such water network effects that may be especially prevalent in many solvent-rich low-affinity protein-glycan binding sites.

Theory. For molecular dynamics (MD) simulations, HK620TSP structures were parameterized with the 59 AMBER03 force field for proteins and the GLYCAM06 force 60 field for glycans. Simulations were conducted in TIP3P, TIP4P or TIP5P explicit water for 10, 50 or 100 ns as described 14 elsewhere. Conserved water positions were obtained by treating simulations without periodic boundary conditions with the trjconv –pbc nojump implementation of 61 Gromacs/4.5.5. Hydrogen bond based water positions were obtained by VMD analysis of hydrogen bonds with occupancies >2 % and averaged accordingly. Mobile water positional analysis was performed with MobyWat (for a full description 36 see Figure S13). Simulations were analyzed in prediction mode with protein as target and all water molecules of the simulation as water with a distance limit of 5 Å. Desolvation free energies were calculated from 10 ns simulations in 21 λsteps for coulomb and van der Waals values independently 37 with the double decoupling method as described. The error of the individual ΔΔGFE on each water molecule was estimated from five independent calculations on water position 0 (Figure S16).

Methods Materials. E. coli strain O18A (DSM #10809) was purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germa27,34 ny) with Oligosaccharide purification has been described. All HK620TSP were N-terminally shortened lacking the capsid adaptor domains. Protocols for protein purification and 31 mutation have been published elsewhere. TSP molar subunit concentrations are given. All experiments were carried out in standard buffer (50 mM sodium phosphate pH 7.5, 1 mM EDTA) if not stated otherwise. Crystal structure analysis. All HK620TSP variants crystallized in space group P321 at room temperature by sittingdrop vapor-diffusion mixing 3 µL of protein solution (8 mg/mL in 40 mM Tris-HCl pH 7.8, 200 mM NaCl, 2 mM EDTA) with equal volumes of precipitant solution (3.5 M sodium formiate, 0.1 M Tris-HCl, pH 8.5). For cocrystallization of enzyme and ligands 0.6 µL of a 15 mM stock solution of the O18A1 hexa- or O18A pentasaccharide in water was added. Crystals appeared after two days, were harvested from the crystallization drop and shock-frozen in liq-

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Crystallographic data collection; ligand superimpositions; side chain conformations and water positions in the binding site; ITC thermograms; fluorescence stopped-flow traces; MD trajectories; solvent positions from MD simulations with TIP3P, TIP4P, TIP5P water; validation procedures for water


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mobility analyses; free energy calculation set ups. A PyMOL session file with all crystal structures has been deposited at https://doi.org/10.7910/DVN/2APUI4.

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AUTHOR INFORMATION

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Corresponding Authors

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*[email protected] *[email protected] *[email protected]

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Author Contributions ‡ S.K., U.G. and N.K.B. contributed equally.

Notes

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The authors declare no competing financial interests.

ACKNOWLEDGMENT (16)

S.B. was funded by the Deutsche Forschungsgemeinschaft [grant number BA 4046/1-2]. S.K. received a Max Planck Society grant [IMPRS Multiscale Bio-Systems]. We thank the staff at BESSY II (Helmholtz-Zentrum Berlin) and Hans König for support.

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Table of Contents artwork



→2)-α-L-Rhap-(1→6)-α-D-Glcp-(1→4)-α-D-Galp((3→1)-β-D-GlcNAcp)-(1→3)-α-D-GlcNAcp-(1→. The O18A1 subtype has an additional α-D-Glcp-(1→6)-branch (red) at the reducing end. 84x34mm (300 x 300 DPI)

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Figure caption. (A) HK620TSP monomer (cartoon) with hexasaccharide (cyan sticks) in the binding site (blue frame). (B) Distribution of water molecules in the hexasaccharide binding site of HK620TSP with an alanine exchange at position E372. “Loop”: residues 471-476 shielding a Glc-binding pocket. Water molecules (crosses) and selected side chains are superimposed from the oligosaccharide complex (thick sticks, green) or from the structure without ligand (thin sticks, purple). In the E372A mutant complex, two water molecules (green spheres) take the position of the wild-type glutamate (overlaid for comparison as black sticks) and mediate hexasaccharide contacts. 160x167mm (300 x 300 DPI)



Thermodynamics of complex formation with hexasaccharide (6mer, filled glucose-binding pocket) or pentasaccharide (5mer, empty glucose-binding pocket) in (A) HK620TSP E372Q and (B) E372A mutants. See Table S3 for the full isothermal titration calorimetry data set. 46x25mm (300 x 300 DPI)


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HK620TSP∆loop (orange) lacking the glucose-binding pocket shielding residues N471 and S472 in complex with pentasaccharide superimposed onto the intact protein in complex with hexasaccharide (blue). B. Thermodynamics of HK620TSP∆loop complex formation with hexasaccharides or pentasaccharides (bars in pale color) compared to E372Q complexes with intact loops (bars in full color) (cf. Figure 2). See Table S4 for the full isothermal titration calorimetry data set. 42x21mm (300 x 300 DPI)



Water networks in the HK620TSP binding site from MD simulations and mobility analysis. (A) Typical network calculated for the ligand-free wild-type protein (see Figure S13 for all networks in the presence of ligands and mutants) Dashed lines show potential H-bonds between water molecules, which are colored according to the number of partners within the network: 0=green, 1=yellow, 2=orange, 3=red, 4=purple, 5=blue, 6=brown, 7=black. The loop cover-ing the glucose binding pocket is shown as red surface. (B) Relative contribution of water clusters of different size to the networks in wild-type (WT) and mutants in the presence of pentasaccharides (“5”) or hexasaccharides (“6”) or with no ligand (“nl”). Clusters were defined as non-interrupted sub-nets containing different numbers of water molecules (1=red; 2=orange; 3=light green; 5-10= dark green; 11-25, cyan; 26-75=blue). 32x12mm (300 x 300 DPI)


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(A) Crystallographic solvent positions in the HK620TSP glucose-binding pocket used for calculation of desolvation free energies ∆∆GFE with the double decoupling (DDC) method (cf. Table 4). The glucose position found for hexasaccharide complexes is shown as thin sticks. (B) Desolvation free energies of glucose-binding pockets in absence of a ligand or in the presence of the pentasaccharide. Σ∆∆GFE were calculated from values in Table 4. 45x24mm (300 x 300 DPI)



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