Letter | Published:

Dynamic allostery can drive cold adaptation in enzymes

Naturevolume 558pages324328 (2018) | Download Citation


Adaptation of organisms to environmental niches is a hallmark of evolution. One prevalent example is that of thermal adaptation, in which two descendants evolve at different temperature extremes1,2. Underlying the physiological differences between such organisms are changes in enzymes that catalyse essential reactions3, with orthologues from each organism undergoing adaptive mutations that preserve similar catalytic rates at their respective physiological temperatures4,5. The sequence changes responsible for these adaptive differences, however, are often at surface-exposed sites distant from the substrate-binding site, leaving the active site of the enzyme structurally unperturbed6,7. How such changes are allosterically propagated to the active site, to modulate activity, is not known. Here we show that entropy-tuning changes can be engineered into distal sites of Escherichia coli adenylate kinase, allowing us to quantitatively assess the role of dynamics in determining affinity, turnover and the role in driving adaptation. The results not only reveal a dynamics-based allosteric tuning mechanism, but also uncover a spatial separation of the control of key enzymatic parameters. Fluctuations in one mobile domain (the LID) control substrate affinity, whereas dynamic attenuation in the other domain (the AMP-binding domain) affects rate-limiting conformational changes that govern enzyme turnover. Dynamics-based regulation may thus represent an elegant, widespread and previously unrealized evolutionary adaptation mechanism that fine-tunes biological function without altering the ground state structure. Furthermore, because rigid-body conformational changes in both domains were thought to be rate limiting for turnover8,9, these adaptation studies reveal a new model for understanding the relationship between dynamics and turnover in adenylate kinase.

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We thank A. Mujamdar, A. Schön, and K. Tripp for technical assistance and instrumentation. Funding from NIH (R01-GM063747), NSF (MCB-1330211), and Johns Hopkins University is acknowledged.

Reviewer information

Nature thanks G. Phillips and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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  1. Department of Biology, Johns Hopkins University, Baltimore, MD, USA

    • Harry G. Saavedra
    • , James O. Wrabl
    • , Jeremy A. Anderson
    • , Jing Li
    •  & Vincent J. Hilser
  2. T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA

    • Harry G. Saavedra
    • , James O. Wrabl
    • , Jeremy A. Anderson
    • , Jing Li
    •  & Vincent J. Hilser


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H.G.S. designed research, performed experiments, analysed data, interpreted results, discussed research, and wrote the manuscript. J.O.W. analysed data, interpreted results, discussed research, and wrote the manuscript. J.A.A. performed experiments, analysed data, interpreted results, and discussed research. J. L. designed research and discussed research. V.J.H. designed research, analysed data, interpreted results, discussed research, and wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Vincent J. Hilser.

Extended data figures and tables

  1. Extended Data Fig. 1 Kinases of known structure that may exhibit similar open/close architecture as E. coli adenylate kinase.

    ad, In each panel, the left cartoon represents a putative ‘lid open’ apo-structure, and the right panel represents a putative ‘lid closed’ holo-structure. Protein chains are rainbow-coloured from blue (N terminus) to red (C terminus). Note that in b and c there is crystallographic evidence of disorder (magenta) in the conformationally changing ‘lid’ domain, a situation very similar to that seen for the low-population locally unfolded state in E. coli adenylate kinase, given in a for comparison. a, E. coli adenylate kinase: PDB accessions 4AKE (left) and 1AKE (right). b, Mycobacterium tuberculosis adenylyl sulfate kinase: PDB accessions 4RFV (left) and 4BZX (right). c, Helicobacter pylori shikimate kinase: PDB accessions 1ZUH (left) and 1ZUI (right). d, Sulfolobus tokodaii hexokinase: PDB accessions 2E2N (left) and 2E2O (right).

  2. Extended Data Fig. 2 Other enzymes of known structure that may exhibit similar open/close architecture as E. coli adenylate kinase.

    ad, In each panel, the left cartoon represents a putative ‘lid open’ apo-structure, and the right panel represents a putative ‘lid closed’ holo-structure. Protein chains are rainbow-coloured from blue (N terminus) to red (C terminus). Note that in ac there is crystallographic evidence of disorder (magenta) in the conformationally changing ‘lid’ domain, a situation very similar to that seen for the low-population locally unfolded state in E. coli adenylate kinase. a, E. coli 2-glycinamide ribonucleotide transformylase: PDB accessions 1CDD (left) and 1CDE (right). b, l,d-carboxypeptidase: PDB accessions 4JID (left) and 4OX5 (right). c, Thermus thermophilus ribosomal protein L11 methyltransferase PrmA: PDB accessions 2NXC (left) and 2NXE (right). d, Lactobacillus casei dihydrofolate reductase: PDB accessions 1l7o (left) and 2HQP (right).

  3. Extended Data Fig. 3 Comparison of wild-type adenylate kinase HSQC spectrum with mutants A55G and V142G.

    a, b, Overlay HSQC spectra at 19 °C for A55G (blue, a) and V142G (red, b); in both panels an identical wild-type spectrum is shown in black. Peak dispersion in all spectra is consistent with folded protein and also is not inconsistent with a similar ground state structure shared among all three proteins. Individual resonances for both mutants exhibited minimal shifts from the wild type, and thus generally permitted transference of assignments from the wild type.

  4. Extended Data Fig. 4 DSC control experiments.

    a, Test of the two-state model using wild-type adenylate kinase. Wild-type thermal denaturation is not consistent with a two-state process, as data simulated under the two-state assumption do not agree with experiment, and calorimetric to van’t Hoff enthalpy ratio is substantially greater than 1. Results represent n = 1 independent experiments. b, Reversibility test. Wild-type adenylate kinase exhibited approximately 80% of the original calorimetric area upon re-heating. Results represent n = 1 independent experiments. c, High temperature test. Wild-type adenylate kinase demonstrates complete reversibility when extreme high temperature is avoided. Results represent n = 1 independent experiments. d, Calorimetric heat capacity (ΔCp) of adenylate kinase LID variants. Dependence of enthalpy on melting temperature for wild type, V135G and V142G results in ΔCp,LID of 0.7 ± 0.1 kcal mol−1 K−1 (mean ± s.d.). This value is reasonably consistent with energetics determined from accessible surface areas. Results represent mean ± s.d. of n = 3 independent experiments.

  5. Extended Data Fig. 5 Modelled domain stabilities and ensemble probabilities of adenylate kinase variants.

    The DSC data for wild type, LID and AMPbd mutants were each fit to three-state transitions. The fitted parameters correspond to population profiles that differ dramatically between the LID and AMPbd mutants (Fig. 2b and c, respectively). As determined previously from circular dichroism and ITC15, the locally unfolded intermediate (LU), which is 5% populated at physiological temperature for wild-type adenylate kinase (37 °C), is increased to approximately 40% in the LID mutants (Fig. 2b). In contrast to the LID, mutations to the AMPbd do not stabilize the intermediate. Instead, the unfolded (U) state is stabilized, accounting for the decrease in the apparent Tm of the main peak, with no change in the temperature of onset of the intermediate (Fig. 2c). ae, Representative domain stabilities calculated from DSC experiments, see Extended Data Table 1. ΔGtotal = ΔGCA + ΔGLID; in which ‘CA’ denotes ‘CORE-AMPbd’. Mutations other than those in the LID domain have a small effect on the stability of adenylate kinase. a, Wild type. b, A37G AMPbd mutant. c, A55G AMPbd mutant. d, V135G LID mutant. e, V142G LID mutant. fj, Ensemble probability calculations were based on values in Extended Data Table 1. LID mutations V135G and V142G clearly modulate the ensemble by reducing the population of fully folded state and increasing population of unfolded LID domain. f, Wild type. g, A37G AMPbd mutant. h, A55G AMPbd mutant. i, V135G LID mutant. j, V142G LID mutant.

  6. Extended Data Fig. 6 Representative ITC data.

    All measurements were obtained at 37 °C, fitting parameters are indicated in each panel. Results represent n = 1 independent experiments. a, Wild type. b, A55G AMPbd mutant. c, V135G LID mutant.

  7. Extended Data Fig 7 Examples of holo-enzymes’ degree of ligand burial.

    ac, In each panel, atoms are shown as van der Waals’ spheres. Dark grey indicates protein atoms and yellow indicates ligand. The left side of each panel shows the protein and ligand together, and the right side shows ligand alone. a, ‘Little’ ligand surface area is buried in the complex of deoxyhypusine synthase and nicotinamide adenine-dinucleotide inhibitor, PDB accession 1RLZ. b, ‘Partial’ ligand surface area is buried in the complex of glyceraldehyde-3-phosphate dehydrogenase, nicotinamide adenine-dinucleotide cofactor, and glyceraldehyde-3-phosphate substrate, PDB accession 1NQA. c, ‘Mostly’ ligand surface area is buried in the complex of chorismate-pyruvate lyase and p-hydroxybenzoic acid product, PDB accession 1TT8.

  8. Extended Data Table 1 Summary of thermodynamic parameters obtained from DSC

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