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Low-barrier hydrogen bonds in enzyme cooperativity

Abstract

The underlying molecular mechanisms of cooperativity and allosteric regulation are well understood for many proteins, with haemoglobin and aspartate transcarbamoylase serving as prototypical examples1,2. The binding of effectors typically causes a structural transition of the protein that is propagated through signalling pathways to remote sites and involves marked changes on the tertiary and sometimes even the quaternary level1,2,3,4,5. However, the origin of these signals and the molecular mechanism of long-range signalling at an atomic level remain unclear5,6,7,8. The different spatial scales and timescales in signalling pathways render experimental observation challenging; in particular, the positions and movement of mobile protons cannot be visualized by current methods of structural analysis. Here we report the experimental observation of fluctuating low-barrier hydrogen bonds as switching elements in cooperativity pathways of multimeric enzymes. We have observed these low-barrier hydrogen bonds in ultra-high-resolution X-ray crystallographic structures of two multimeric enzymes, and have validated their assignment using computational calculations. Catalytic events at the active sites switch between low-barrier hydrogen bonds and ordinary hydrogen bonds in a circuit that consists of acidic side chains and water molecules, transmitting a signal through the collective repositioning of protons by behaving as an atomistic Newton’s cradle. The resulting communication synchronizes catalysis in the oligomer. Our studies provide several lines of evidence and a working model for not only the existence of low-barrier hydrogen bonds in proteins, but also a connection to enzyme cooperativity. This finding suggests new principles of drug and enzyme design, in which sequences of residues can be purposefully included to enable long-range communication and thus the regulation of engineered biomolecules.

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Fig. 1: Structural evidence for LBHBs in human transketolase catalysis and cooperativity.
Fig. 2: Computed structures and hydrogen-bond potentials for the human transketolase proton wire.
Fig. 3: Structural evidence for LBHBs in L. plantarum pyruvate oxidase catalysis and cooperativity.

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Data availability

The refined structural protein models and corresponding structure-factor amplitudes have been deposited in the Protein Data Bank under accession codes 6HAD (human transketolase variant E160Q in the resting state), 6HA3 (human transketolase variant E160Q in covalent complex with substrate F6P), 6RJB (human transketolase variant T382E in the resting state), 6RJC (E. coli transketolase apo-enzyme) and 6HAF (L. plantarum variant E59Q in complex with phosphate). All other data are available from the corresponding authors upon request.

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Acknowledgements

We thank J. P. Klinman, P. A. Frey, A. Warshel, R. Kluger, H. Stark, A. Chari, G. Bourenkhov, T. Schneider, G. Sheldrick, D. Hilvert, P. Neumann, D. Tawik, E. Pai, R. S. Prosser and G. Groenhof for discussions. We acknowledge access to beamlines P13 and P14 at DESY/EMBL Hamburg, Germany and local support by G. Bourenkhov and T. Schneider. This project was supported by grants from the Deutsche Forschungsgemeinschaft (FOR 1296/TP 3, to K.T.; MA 5063/2, to R.A.M.), the Göttingen Graduate Center for Neurosciences, Biophysics, and Molecular Biosciences (to K.T.), the Max-Planck-Institute for Biophysical Chemistry Göttingen (to K.T.), a PhD scholarship from the Chinese Scholarship Council of the Chinese Government (to S.D.) and a PostDoc scholarship from Fundación Ramón Areces, Spain (to J.U.).

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Authors and Affiliations

Authors

Contributions

K.T. and R.A.M. designed and coordinated the project. K.T. planned and supervised the structural experiments and functional analysis of the studied enzymes. S.D., F.R.v.P. and V.S. determined the crystal structures of human and E. coli transketolase variants and S.D. carried out all kinetic and thermodynamic assays on these enzymes. L.-M.F. and V.S. determined the crystal structure of pyruvate oxidase variant E59Q. L.-M.F. conducted kinetic experiments on all pyruvate oxidase variants discussed in this study. K.T. conducted nuclear magnetic resonance experiments. S.D., L.-M.F., F.R.v.P., V.S. and K.T. analysed and interpreted the structural and functional data. R.A.M. planned and supervised the computational experiments and carried out QM/MM calculations. M.P. conducted the molecular dynamics simulations. J.U. carried out QM/MM calculations. B.S. computed the proton density distributions. R.A.M. and K.T. wrote the paper with input from all authors.

Corresponding authors

Correspondence to Ricardo A. Mata or Kai Tittmann.

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The authors declare no competing interests.

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Peer review information Nature thanks Doeke Hekstra, Babis Kalodimos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Structure of wild-type human transketolase the E160Q variant.

a, Structure of the human transketolase dimer, highlighting the communication channel between the two active sites (coloured in magenta). The bound substrates (S1, S2) and ThDP cofactors (ThDP 1, ThDP 2) at the two active sites are labelled. b, Cofactor binding sites in transketolases from different organisms including Saccharomyces cereviseae (PDB code: 1GPU), E. coli (PDB code: 1QGD) and Zea mays (PDB code: 1ITZ). The thiamine cofactor (ThDP) and the conserved dicarboxylate motif consisting of two glutamates are highlighted. The hydrogen-bonding distances are indicated. Note the short distances between the two glutamate side chains, which suggests the putative formation of LBHBs in transketolase catalysis. The two subunits are coloured individually (blue and green), residues from the second subunit are labelled with an apostrophe. c, Structure of the covalent conjugate between cofactor ThDP and substrate F6P trapped at the active site of human transketolase variant E160Q. The structural model is superposed with the corresponding 2mFo – DFc electron density map (blue, contoured at 2σ). Note the pronounced out-of-plane distortion of the bond formed between sp2-hybridized C2 of the cofactor and C2x of the substrate relative to the aromatic ring plane of the thiazolium moiety shown in grey. The scissile C2x–C3x bond of the substrate is highlighted. Of further note is the elongated C2x–C3x bond of the substrate (bond length shown in red). d, Nuclear magnetic resonance (NMR) analysis of covalent reaction intermediates in the catalysis of wild-type human transketolase and the stated variants. Intermediates were isolated by an acid quench after 30 s of reaction time in which the enzyme was mixed with substrate F6P, and subsequently analysed by 1D 1H NMR spectroscopy using the fingerprint signals of standards as previously described24,43. Whereas wild-type transketolase and the E160Q and E160A variants accumulate the covalent substrate–cofactor conjugate F6P–ThDP, the E366Q and E165Q variants do not accumulate covalent intermediates to a measurable extent. ThDP, C2-unsubstituted cofactor; F6P–ThDP, covalent conjugate between F6P and ThDP; DHEThDP, dihydroxyethyl-ThDP carbanion–enamine (cleaved substrate–ThDP intermediate), isolated as conjugate acid43. The NMR experiments were independently repeated twice with similar results. e, Structural comparison between wild-type human transketolase (yellow) and the E160Q variant (grey). Structures of the superposed active sites show the substrate F6P covalently bound to cofactor ThDP, selected amino acid residues and water molecules. On the right is a magnified section showing the aminopyrimidine portion of cofactor ThDP, residues E366′ and E160/Q160, and three water (W) molecules. Structural displacements of selected atoms of residues E366′ and Q160 in the variant relative to those of corresponding residues in the wild-type enzyme are indicated.

Extended Data Fig. 2 Functional analysis of wild-type human transketolase and wire variants.

ad, Circular dichroism spectroscopic analysis of the apparent pKa of the E366′–thiamine proton shuttle in wild-type human transketolase and variants. Near-UV circular dichroism spectra of human transketolase were measured at different pH values. The bound ThDP cofactor gives rise to several charge-transfer signals, which correspond to distinct protonation and tautomeric states of the cofactor and activating residue E366′ (refs 44,45,46). The measured signal at 330 nm was fitted to Supplementary Equation 4 (one pKa, a, b, c) or Supplementary Equation 5 (two pKas, d). The thermodynamic constants are summarized in Extended Data Table 2. Quantum chemical calculations suggest that the band at 330 nm (negative signature) pertains to the aminopyrimidine form of ThDP with E366′ being negatively charged, whereas the band at 290 nm (negative signature) pertains to the aminopyrimidine form of ThDP with an uncharged residue at position 366 (E366′ protonated in wild-type transketolase, or Q366′ in the E366Q variant)46. The iminotautomeric form of ThDP gives rise to a positive signal centred around 295–300 nm. e, f, Pre-steady-state stopped-flow kinetic analysis of human transketolase catalysis using the cofactor charge-transfer signal at 325 nm as a spectroscopic probe. e, Stopped-flow transients monitored after mixing human transketolase (wild-type, E160Q and E160A) with substrate (10 mM F6P). In the case of wild-type transketolase, a monophasic process was observed and data were fitted (red line) with a monoexponential function (Supplementary Equation 6). For the variants the process is biphasic, and data were fitted (red line) with a double exponential function (Supplementary Equation 7). NMR analysis of the intermediates indicates that the observed spectroscopic changes of the bound cofactor correspond to formation of the covalent substrate–ThDP conjugate. In the case of the variants, a non-covalent intermediate—the substrate Michaelis complex—is transiently populated. f, Dependence of the apparent rate constants (wild-type, single phase; variants, second phase of reaction) of formation of the substrate–ThDP intermediate on concentration of the substrate. Data were fitted using Supplementary Equation 8. All measurements were carried out in triplicate and are shown as mean ± s.d. All experiments were independently repeated twice with similar results. Kinetic and thermodynamic constants are provided in Extended Data Table 2.

Extended Data Fig. 3 Kinetics of active-site communication in human transketolase.

a, Protonic and tautomeric equilibria of enzyme-bound ThDP with key species and their absorbance maxima indicated. b, Near-UV circular dichroism spectroscopic analysis reveals a chemical asymmetry in human transketolase (AP in one site, IP in the other site), as previously described for related ThDP enzymes45. Upon covalent binding of the substrate, the IP form is predominantly formed. c, Proposed chemical states of enzyme-bound ThDP in the different monomers of the human transketolase dimer. d, Kinetics of active-site communication as measured by temperature-jump analysis. The changes in the IP and AP signals of the resting-state enzyme were monitored at 294 nm (IP) and 325 nm (AP) after a temperature jump from 12 °C to 21 °C. Data were fitted with a monoexponential (AP) or double exponential (IP) function. In response to the temperature jump, the concentration of both the IP and AP forms decrease in favour of the APH+ species: [AP ↔ IP] + H+ ↔ APH+. The almost identical rate constants for the decrease in IP and AP suggest that the communication through the wire occurs considerably faster than the preceding protonation. The origin of the second phase observed for the IP form remains to be defined. e, In the E160Q variant, the communication between the active sites—as observed for the wild-type enzyme—is absent. Fifty transients were averaged in each case (d, e). Experiments were repeated twice with similar results.

Extended Data Fig. 4 Functional and structural analysis of human transketolase regulation variants.

a, Steady-state kinetic analysis of pseudo-phosphorylation variant T382E reveals an almost twofold increase in enzymatic activity relative to the wild-type enzyme, whereas the T382Q variant (isosteric to T382E, but uncharged) exhibits a tenfold decrease in activity. b, Transient kinetic analysis of substrate binding reveals an enhanced cooperativity in the T382E variant (Hill coefficient nH = 2.42 ± 0.36) compared to wild-type transketolase (nH = 1.56 ± 0.25). All measurements were carried out in triplicate and are shown as mean ± s.d. c, Circular dichroism spectroscopic analysis of the apparent pKa of the E366′–thiamine proton shuttle in human transketolase ‘pseudo-phosphorylation’ variant T382E. All measurements were carried out in triplicate and are shown as mean ± s.d. All experiments (ac) were independently repeated twice with similar results. All kinetic and thermodynamic constants are provided in Extended Data Table 2. d, Phosphorylation site T382 of human transketolase. A structural model of human transketolase is shown highlighting the phosphorylation site T382′, the cofactor ThDP at the active site as well as residues E366′, E160, E165′ and water molecules (W) of the proton wire. Note that T382′ contacts helix365–381 (in blue) that contributes E366′ to the proton wire. Chains A and B are coloured individually, and residues from chain B are marked with an apostrophe. Interatomic distances of critical interactions, including the LBHB between E366′ and E160, are provided. e, Structural analysis of the ‘pseudo-phosphorylation’ variant T382E of human transketolase. A structural model of human transketolase variant T382E is shown highlighting residue E382′ (corresponding 2mFo – DFc electron density is shown at a contour level of 3σ), the cofactor ThDP at the active site as well as residues E366′, E160, E165′ and water molecules (W) of the proton wire. Note the shortened interatomic distance for the LBHB interaction between E366′ and E160 (2.52 Å) compared to the wild-type enzyme (2.56 Å, see d). Chains A and B are coloured individually, and residues from chain B are marked with an apostrophe. Bottom, local hydrogen-bonding interactions of E382′ with neighbouring residues and water molecules.

Extended Data Fig. 5 Molecular dynamics simulations of the human transketolase dimer.

a, Top, cartesian coordinates root mean square deviation (r.m.s.d.) of Q367 relative to the original crystal structure using one of the partly occupied positions as reference. Bottom, difference of distances between a bridging water and the E165 and E165′ residues. The moving average over 10 ps is shown in red. From both graphics, it can be seen that the water oscillates through the symmetry axis. There is a large movement of Q367 between approximately 4.0 ns and 10.2 ns, during which it builds a hydrogen bond to another water molecule, deactivating the bridge. However, Q367 later returns to its original position. b, Density-based spatial clustering of applications with noise (DBSCAN) analysis of the trajectory, using the two quantities as descriptors for the times 0–4.0 ns and 10.2–16.5 ns. The method finds two clusters, pairing the distance difference around 1 Å to a 2.5 Å r.m.s.d., as well as −1 Å to a 1.0 r.m.s.d. approximately. The latter positions correspond to the partially occupied sites found in the crystal, thus confirming the oscillation. The oscillation takes place in the nanosecond regime, much faster than the reaction time of the catalysis. This observation confirms that a shuttling water could be involved in the proton wire. c, Proton positions for the human transketolase proton wire. The dependence of the structural features on an added proton and water molecule next to E165′ (controlled by the variable λ) are demonstrated, considering two different sampled conformations (top and bottom). Left, the dependence of the glutamate proton distances on λ (the blue area highlights the distance range for LBHBs, of around 1.3 Å); right, ball-and-stick representations of the QM/MM optimised structures at λ = 0 and 1. The hydrogens are shown as green balls and are labelled, from left to right, H1, H2 and H3.

Extended Data Fig. 6 Proton wire in E. coli transketolase and model of active-site communication in the transketolase dimer.

a, Communication channel between the two remote active sites of the E. coli transketolase homodimer showing selected amino acid residues, the substrate–thiamine intermediates and water molecules (PDB code: 2R8O). Hydrogen-bonding interactions are highlighted by dashed lines. The two subunits are coloured individually (blue and green), residues from the second subunit are labelled with an apostrophe. b, Structure of the proton wire in E. coli apo-transketolase (devoid of cofactor ThDP) showing wire residues E411, E160 and E165 and interacting water molecules. The corresponding 2mFo – DFc electron density map is shown at a contour level of 5σ. Interatomic distances for critical interactions are provided. Note the different protonation states of E165′ and E165 and the reduced intensity for a water molecule at the symmetry axis (Ws). c, Chemical model of the proton wire in E. coli apo-transketolase including the protonation states of wire residues E411, E160 and E165. The model includes a ‘swinging’ water and an ‘uneven proton’ at position 165, required for back-and-forth proton transfer. d, Proposed mechanism of active-site coupling in transketolase through the proton wire, which links and synchronizes catalysis of the two remotely located active sites in the dimer. Product release in one active site triggers substrate binding in the second site of the dimer, through proton exchange and reciprocal interconversion between the AP and IP tautomers of enzyme-bound ThDP in both active sites. The LBHBs are critical determinants of the wire and ensure fast back-and-forth oscillations of the transmitted proton through the wire.

Extended Data Fig. 7 Structure of wild-type POX in complex with substrate, and structure of the E59Q variant.

a, Structure of wild-type POX in complex with the carbanion–enamine Breslow intermediate (PDB code: 4FEG) showing the aminopyrimidine part of the bound cofactor and critical residues of the communication channel including E59′, H89′ and E60. The corresponding electron density maps are contoured at 5σ (2mFo – DFc, blue) and 2.5σ (H-omit mFo – DFc, magenta). The structural data suggest the formation of ordinary hydrogen bonds and separate charges at this particular catalytic stage. b, Structural comparison between wild-type pyruvate oxidase (yellow) and the E59Q variant (grey). Left, structures of the superposed active sites showing the substrate phosphate (Pi), the cofactor ThDP, selected amino acid residues and water molecules. Right, a magnified section showing the aminopyrimidine portion of cofactor ThDP and substrate phosphate, residues H89′ and E59′ (or Q59′) and two water (W) molecules. Structural displacements of selected atoms in the variant relative to those of corresponding residues in the wild-type enzyme are indicated.

Extended Data Fig. 8 Functional analysis of wild-type pyruvate oxidase and wire variants.

a, b, Stopped-flow kinetic analysis of substrate binding in pyruvate oxidase catalysis using substrate analogue methylacetyl phosphonate (MAP). The analogue forms a covalent conjugate with bound cofactor ThDP but is not further processed, enabling the exclusive analysis of substrate binding. a, Selected stopped-flow transients monitored after mixing pyruvate oxidase (wild-type, E60Q, E60A, H89N and H89A variants) with 10 mM MAP. b, Dependence of the observed first-order rate constants kapp on the MAP concentration. Data were fitted with Supplementary Equation 10 to obtain estimates of the substrate dissociation first-order rate constant koff (non-zero intercept with y-axis), substrate association second-order rate constant kon (slope) and substrate dissociation equilibrium binding constant KDapp (koff/kon). All measurements were carried out in triplicate and are shown as mean ± s.d. All experiments were independently repeated twice with similar results. Kinetic and thermodynamic constants are given in Extended Data Table 2. cf, Stopped-flow kinetic analysis of substrate binding and processing in pyruvate oxidase catalysis under single turnover conditions. c, Stopped-flow transients obtained upon mixing wild-type pyruvate oxidase with various concentrations of substrate pyruvate under anaerobic conditions, using the intrinsic absorbance of the bound cofactor FAD. d, Dependence of the calculated first-order rate constants kapp on the applied pyruvate concentration for wild-type pyruvate oxidase. Data were fitted with Supplementary Equation 12. Inset, a magnified section of the graph showing the substrate dependence in the concentration range 0–10 mM. Note the sigmoidal dependence, which indicates positive cooperativity (Extended Data Table 2). e, Dependence of the first-order rate constants kapp on the applied pyruvate concentration for the E59Q variant. Data were fitted with Supplementary Equation 12. f, Dependence of the first-order rate constants kapp on the applied pyruvate concentration for the wild-type enzyme and the H89N, H89A, E60Q and E60A. Data were fitted with Supplementary Equation 12. All measurements were carried out in triplicate and are shown as mean ± s.d. All experiments were independently repeated twice with similar results. Kinetic and thermodynamic constants are given in Extended Data Table 2.

Extended Data Table 1 X-ray crystallographic data collection and refinement statistics
Extended Data Table 2 Steady-state and pre-steady-state kinetic analysis of transketolase and pyruvate oxidase catalysis

Supplementary information

Supplementary Information

Supplementary Results and Supplementary Methods are combined in the Supplementary Information pdf. Supplementary Results contain a) Kinetic and structural analyses of human transketolase pseudo-phosphorylation variant T382E and variant T382Q and b) Kinetic analysis of pyruvate oxidase wild-type and variants. Supplementary Methods contain a) Functional analysis of enzymes and b) Computational details.

Reporting Summary

Video 1

Alternative models for coupled proton transfer in the communication wire of human transketolase. Animations illustrating the impact on the proton wire of removing/adding an hydronium ion near the symmetry axis. Depicted are the local minima structures obtained by QM/MM optimisation of the proton wire for different values of λ (as indicated in the Methods section).

Video 2

Alternative models for coupled proton transfer in the communication wire of human transketolase. Animations illustrating the impact on the proton wire of removing/adding an hydronium ion near the symmetry axis. Depicted are the local minima structures obtained by QM/MM optimisation of the proton wire for different values of λ (as indicated in the Methods section).

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Dai, S., Funk, LM., von Pappenheim, F.R. et al. Low-barrier hydrogen bonds in enzyme cooperativity. Nature 573, 609–613 (2019). https://doi.org/10.1038/s41586-019-1581-9

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