Most proteins associate into multimeric complexes with specific architectures1,2, which often have functional properties such as cooperative ligand binding or allosteric regulation3. No detailed knowledge is available about how any multimer and its functions arose during evolution. Here we use ancestral protein reconstruction and biophysical assays to elucidate the origins of vertebrate haemoglobin, a heterotetramer of paralogous α- and β-subunits that mediates respiratory oxygen transport and exchange by cooperatively binding oxygen with moderate affinity. We show that modern haemoglobin evolved from an ancient monomer and characterize the historical ‘missing link’ through which the modern tetramer evolved—a noncooperative homodimer with high oxygen affinity that existed before the gene duplication that generated distinct α- and β-subunits. Reintroducing just two post-duplication historical substitutions into the ancestral protein is sufficient to cause strong tetramerization by creating favourable contacts with more ancient residues on the opposing subunit. These surface substitutions markedly reduce oxygen affinity and even confer cooperativity, because an ancient linkage between the oxygen binding site and the multimerization interface was already an intrinsic feature of the protein’s structure. Our findings establish that evolution can produce new complex molecular structures and functions via simple genetic mechanisms that recruit existing biophysical features into higher-level architectures.
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Reconstructed ancestral sequences have been deposited in GenBank (IDs MT079112, MT079113, MT079114, MT079115). Alignment and inferred phylogeny, raw mass spectra, oxygen-binding data, and homology model coordinates have been deposited at https://doi.org/10.5061/dryad.w0vt4b8mx. HDX-MS data are available at https://doi.org/10.5287/bodleian:5zRrdMB7E.
Scripts for analysis for the HDX permutation analysis and identification of contacts between subunits in modelled structures have been deposited at https://github.com/JoeThorntonLab/Hemoglobin-evolution.
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We thank C. Natarajan for technical advice and the Hb co-expression plasmid and members of the Thornton laboratory for technical advice and comments on the manuscript. Supported by NIH R01-GM131128 and R01-GM121931 (J.W.T.), NIH R01-HL087216 and NSF OIA-1736249 (J.F.S.), NIH T32-GM007197 (C.R.C.-R.), a Chicago Fellowship (G.K.A.H.), BBSRC BB/L017067/1 and Waters Corp. (J.L.P.B.).
The authors declare no competing interests.
Peer review information Nature thanks Nobuhiko Tokuriki and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Phylogeny of Hb and related globins. Node supports are shown as approximate likelihood ratio statistic59,60. The number of sequences in each group is shown in parentheses. Ancestral sequences reconstructed in this study are shown as coloured circles. Arrow, branch swap that differentiates this phylogeny from the unconstrained ML phylogeny, which requires additional gene gains and losses. The tree is rooted on neuroglobin and globin X, paralogues that duplicated before the divergence of deuterostomes and protostomes61. Inset, pairwise sequence identities among extant (human, Hsa) and reconstructed ancestral globins. b, Distribution across sites of the posterior probabilities (PP) of maximum a posteriori states for reconstructed ancestral proteins. c, Thermal stability of ancestral globins. Points, fraction of secondary structure lost as temperature increases in Ancα/β (purple), Ancα + Ancβ (blue) and AncMH (black), measured by circular dichroism spectroscopy at 222 nm, relative to signal at 23 °C. Tm and its s.e. were estimated by nonlinear regression; the best-fit curve (lines) are shown. Each point is the mean of four measurements. d, Native mass spectra of globin Y from elephant shark (top, Callorhinchus milii) and African clawed frog (bottom, X. laevis) at 30 μM. Charge states of haem-bound monomer shown. Asterisk, cleavage products. Spectra were collected once. e, Sequence alignment of reconstructed ancestral globins. Dots, states identical to Ancα/β; yellow, IF2 sites; orange, IF1 sites; h, sites 4 Å away from the haem; a, sites that link the haem-coordinated proximal histidine (H95) to IF2. f, Statistical test of cooperativity of oxygen binding for ancestral proteins and mutants. An F-test was used to compare the fit of a model in which the Hill coefficient (n) is a free parameter to a null model with no cooperativity (n = 1). Computed P value and degrees of freedom (df) are shown. N, number of concentrations measured. *P < 0.05. Data were pooled across replicate experiments for nonlinear regression.
a, Homology model of Ancα + Ancβ (template 1A3N) showing haem (tan spheres). Blue cartoon, Ancβ subunits; red, Ancα. Helices and interfaces are labelled. Green, proximal histidine. b, SEC and multiangle light scattering of Ancα/β (90 μM) and Ancα + Ancβ (60 μM). Black, relative refractive index; red, estimated molar mass. Dashed lines, Ancα/β; solid lines, Ancα+Ancβ. Dashed horizontal lines, expected mass for dimers and tetramers. c, SEC of human Hb (dashed) and Ancα + Ancβ (solid) at 100 μM. Top inset, SDS–PAGE of these complexes, with bands corresponding to α- and β-subunits. Bottom inset, masses estimated by denaturing MS of Ancα + Ancβ, compared to expected masses based on primary sequence. d, SEC of Ancα/β across a series of concentrations. Dashed vertical lines, elution peak volumes of human haemoglobin tetramer and myoglobin monomer. e, Tandem MS of the heterotetrameric peak in the Ancα + Ancβ nMS (indicated in Fig. 1b). Ejected monomer and trimer charge series and the subunits they contain are shown. Pink, Ancα; blue, Ancβ. f, nMS of Ancα + Ancβ and Ancα/β at 4 μM and 100 μM. Charge series and fitted stoichiometries are indicated. *Unhaemed apo form. g, Monomer–dimer association by Ancα/β. Abundances of monomers and dimers were characterized using nMS across a range of concentrations. Circles, fraction of all subunits that were assembled into dimers as a function of the concentration of subunits in all states. Nonlinear regression (line) was used to estimate the dissociation constant (Kd, with s.e.). h, SEC of Ancα/β at high concentrations (purple and grey lines). Black curves show SEC traces of human Hb and myoglobin for comparison. i, nMS of human Hb at 50 μM. j, SEC of AncMH (cyan) at a high concentration. SEC traces of human Hb and myoglobin (black) are shown for reference. Dashed line, Ancα/β dimer elution peak volume (see f). k, Alternative estimation of affinity of dimer–tetramer association by nMS. For human Hb (green) and Ancα/β14 + Ancα (orange), the fraction of heterodimers incorporated into heterotetramers includes both haem-deficient and holo-heterodimers. For Ancα + Ancβ (red), caesium iodide adduct was included. Compare to Figs. 1d and 3d. Kd values (with s.e.) were estimated by nonlinear regression (lines). All concentrations are expressed in terms of monomer. All nMS and SEC experiments were performed once at each concentration.
a, SEC of Ancα at 75 μM. b, nMS spectrum (top, at 20 μM) and SEC–MALS (bottom) of Ancβ. Blue, UV absorption; red, molar mass estimated by light scattering. c, Colorimetric haemoglobin concentration assay. Absorbance spectra before (black) and after (red) adding 150 μl Triton/NaOH reagent to 50 μl purified Ancα/β. In the presence of reagent, globins absorb at 400 nm. d, SEC of crude cell lysate after expression of AncMH (purple) and Ancα/β (black). Dashed lines, expected elution volumes for monomer (human myoglobin) and dimer (Ancα/β). e, Colorimetric haemoglobin concentration assay on collected SEC fractions of crude lysate containing AncMH (purple) and Ancα/β (black). f, nMS of His-tagged AncMH at 70 μM, with monomer charge series indicated. *Cleavage product. Green, apo. Fractional occupancy of the monomeric form is shown. All experiments were performed once.
Extended Data Fig. 4 Biochemical inferences about ancestral Hbs are robust to uncertainty in sequence reconstructions.
a–e, Maximum parsimony inferences of ancestral stoichiometry and interface losses or gains based on the distribution of stoichiometries among extant globins. a, Hbs in all extant lineages of jawed vertebrates are heterotetramers, supporting the inference that AncHb was heterotetrameric. Stoichiometries from representative species’ Hbs are shown with PDB IDs. b–e, Each panel shows a hypothetical set of ancestral stoichiometries, plotted on the phylogeny of extant Hb subunits and closely related globins, with the minimal number of changes required by each scenario. b, The most parsimonious reconstruction is that Ancα/β was a homodimer and AncMH was a monomer. c, For Ancα/β to have been a tetramer, early gain and subsequent loss of IF2 in Hbα would be required. d, For Ancα/β to have been a monomer, IF1 would have to have been independently gained in Hbα and Hbβ. e, For AncMH to have been a dimer, IF1 would have to have been lost in lineages leading to the monomers myoglobin (Mb) and globin E (GbE)12,13. The dimeric globins most closely related to Hb—agnathan ‘haemoglobin’ (aHb) and cyotoglobin (Cyg)—use interfaces that are structurally distinct from those in Hb15,16, indicating independent acquisition. f–j, Alternative reconstructions of Ancα/β are biochemically similar to the ML reconstruction. f, Alternative ancestral versions of Ancα/β were constructed, each containing the the ML state at every unambiguously reconstructed site and the second most likely state at all ambiguously reconstructed sites, using different thresholds of ambiguity. For each alternative reconstruction, the table shows the threshold posterior probability (PP) used to define an ambiguous site, as well as the fold-difference in total PP of the entire sequence and the number of sites that differ from the ML reconstruction. g, SEC at 75 μM of ML reconstruction of Ancα/β and AltAll reconstructions, which contain all plausible alternative states with PP above a threshold. Dashed lines show elution peak volumes for the dimeric ML α/β and monomeric human myoglobin. Constructs that elute between the expected volumes for dimer and monomer indicate dimers that partially dissociate during the run. None tetramerize; all form predominantly dimers, except AltAll(PP >0.2), which is ~62,000 times less probable than ML, which is mostly monomeric. UV traces were collected once for each construct. h, Oxygen binding curves of Ancα/β-AltAll(0.25), the dimeric AltAll with the lowest PP, with and without 2× IHP. Dissociation constant (P50, with s.e.) estimated by nonlinear regression is shown. Lack of cooperativity is indicated by the Hill coefficient (n50 = ~1.0). Oxygen binding at each concentration was measured once. i, Alternate globin phylogeny that is more parsimonious than the ML topology with respect to gene duplications and synteny but has a lower likelihood given the sequence data. A version of Ancα/β (Ancα/β-AltPhy) was reconstructed on this phylogeny. j, SEC of Ancα/β-AltPhy. Dashed lines show expected elution volumes for various stoichiometric forms.
a–c, Deuterium uptake measurements across time for three peptides. Left vertical axis, raw deuterium incorporation; right vertical axis, deuterium incorporation divided by the total number of exchangeable amide hydrogens per peptide. Uptake curves for four concentrations of mutants IF1rev and P127R are shown. Each point shows mean ± s.e. of three replicate measurements. d–f, Raw MS spectra for the peptides shown in a–c, respectively, at 0.67 μM (red, at which the protein is monomeric), and 75 μM (purple, at which it is entirely dimeric: see Extended Data Fig. 2). The traces are slightly offset to allow visualization. One replicate at each incubation time is shown. g, Amino acids 99 to 111 contact IF1 (orange) or IF2 (yellow). The homology model of one chain of Ancα/β (cartoon and sticks) was aligned to the α-subunit of human Hb (PDB 1A3N); β-subunits are shown as surfaces. h, Normalized deuterium uptake difference (mean ± s.e. from three replicates), defined as the uptake difference between monomer and dimer divided by the uptake of the monomer, observed for peptides containing amino acids 99–111. Grey N-terminal residues do not contribute to uptake. Amino acid sequences are aligned and labelled (orange dots, IF1; yellow dots, IF2).
Extended Data Fig. 6 Statistical analysis of HDX-MS results for peptides containing interface residues.
a, Residues in human Hb (PDB 1A3N) that bury at least 50% of their surface area in either IF1 (orange) or IF2 (yellow) are shown as spheres. Red and pink, α-subunits; blue, β-subunits. b, Homology models of Ancα/β dimer across IF1 (left) and IF2 (right). Two subunits of Ancα/β were computationally docked using HADDOCK using the α1/β1 interface (IF1, left) or α1/β2 interface (IF2, right) of human Hb (1A3N) as a template. c, Coverage of peptides produced by trypsinization of Ancα/β, assessed by MS. Orange and yellow, sites that bury surface area at IF1 and IF2 in the modelled dimeric structures, respectively. d, Classification of trypsin-produced peptides that contribute to IF1 or IF2. Each circle represents one peptide, plotted by average surface area per residue buried at each interface (total buried area divided by total number of residues). Dashed lines, cutoffs to classify peptides as contributing to IF1 (orange) or IF2 (yellow). e, f, Correlation between change in deuterium uptake and burial of surface area at IF1 or IF2. Each point is one of 47 peptides, plotted according to the normalized difference in deuterium uptake between concentrations at which monomer or dimer predominates (0.67 or 75 μM, normalized by uptake at 75 μM) and average buried surface area at IF1 or IF2. r, Pearson correlation coefficient. g, Permutation test to evaluate the difference in deuterium uptake at two time points by peptides containing IF1 versus all other peptides (orange), or IF2 versus all other peptides (yellow). To avoid non-independence, the experimental data were reduced to a set of nonoverlapping peptides by sampling without replacement. Peptides were categorized by whether they contained residues at IF1, IF2, or neither; peptides that contributed to both IFs were excluded. For each interface, the mean uptake by peptides contributing to the interface was calculated, as was the mean uptake by peptides not in that category, and the difference in means was recorded. Peptide assignment to categories was then randomized, and the difference in mean uptake recorded; this permutation process was repeated until all possible randomized assignment schemes for those peptides had been sampled once. P value, fraction of permuted assignment schemes with a difference in mean uptake between categories greater than or equal to that from the true scheme. This process was repeated for 1,000 nonoverlapping peptide sets; the histogram shows the frequency of P values across these sets. Dashed line, P = 0.05.
a, b, Peptides with residues contributing to IF1 (a) or IF2 (b) that have the largest relative uptake difference upon dimerization are shown as purple tubes. Sticks, side chains predicted to contact the other subunit (orange surface, IF1; yellow surface, IF2). Side chains are coloured orange (IF1) or yellow (IF2) if they were substituted between AncMH and Ancα/β; purple, unchanged in that interval; green, site for targeted mutation P127; blue, Q40. Circled numbers show the rank of each peptide among all peptides for the normalized difference in deuterium uptake between monomer and dimer conditions. Homology models of the Ancα/β dimer using half-tetramers of human Hb (1A3N) are shown. In a, the dimer is modelled using the α1/β1 subunits; in b, it is modelled on the α1/β2 subunits. c, d, nMS of interface mutants Q40R (at IF2) and P127R (at IF1) and for mutants IF1rev and IF2rev, in which interface residues in Ancα/β were reverted to their states in AncMH. All assays at 20 μM. Stoichiometries and charge states are labelled. Unhaemed peak series due to haem ejection during nMS is labelled. Spectra were collected once.
a, Deuterium uptake difference between monomer (0.67 μM) and dimer (75 μM) at each time point was normalized by the length of each peptide. Peptides were categorized by the interface to which they contribute, as in Fig. 2c. *Interface peptide sets that show significantly increased uptake upon dilution when compared to peptides outside of that interface, as determined by a permutation test (see Extended Data Fig. 6). Each point shows the mean ± s.e. from three replicates. b, Permutation test to evaluate the difference in deuterium uptake at 60 min by peptides at each interface, when uptake difference per peptide is normalized by length (as described in Extended Data Fig. 6g). Orange, peptides with IF1-containing residues versus those with no IF1 residues. Yellow, IF2-containing peptides versus those with no IF2 residues. Dashed line, P = 0.05. c, d, Average deuterium uptake difference per residue (c) and uptake difference normalized by dimer uptake (d) for peptides at different time points. Orange, IF1 sites; yellow, IF2 sites. Each rectangle shows the position of the peptide in the linear sequence and its uptake (mean of three replicates).
a, b, SEC of mutants at IF2 (Q40R and IF2rev, which reverts all substitutions that occurred between AncMH and Ancα/β at IF2 sites) and at IF1 (P127R and IF1rev) at 100 μM. Dashed line, elution peak volume for Ancα/β. c, Circular dichroism spectra for P127R and Ancα/β, showing comparable helical structure. d, SEC from IF1 mutant V119A at 64 μM, compared to Ancα/β. e, nMS of Ancα/β, P127R and IF1rev at 10 μM. Stoichiometries and charges are shown. For a–d, nMS and SEC experiments were performed once per concentration. f, Normalized deuterium uptake by IF1-containing peptide 106–111 in HDX-MS of Ancα/β (75 μM) and mutants P127R (2 μM) and IF1rev (2 μM). Mean ± s.e. of three replicates. g, h, Difference between deuterium uptake by each peptide in Ancα/β and uptake by the same peptide in IF1 mutants P127R (g) and IF1rev (h), both at 2 μM, normalized by uptake in Ancα/β. Peptides are classified by interface category. Mean ± s.e. of three replicates. *Peptide sets that have significantly increased relative uptake (by permutation test, see Extended Data Fig. 6) compared to all other peptides (peptides containing both IF1 and IF2 residues excluded).
a, c, SEC of Ancα/β containing sets of historical substitutions, when coexpressed and purified with Ancα. Dashed lines, elution volumes of known stoichiometries (4-mer, Ancα + Ancβ; 2-mer, Ancα/β; monomer, human myoglobin). Pie charts, relative proportions of α (pink) and α/β mutant (purple) subunits in fractions corresponding to each peak, as determined by high-resolution MS (Extended Data Fig. 11). b, nMS of tetrameric fraction in a at 20 μM (monomer concentration). *Apparent impurity. Together, a and b show that tetramers formed by coexpression of Ancα/β4 + Ancα incorporate virtually no α-subunits. Occupancy from this experiment is shown in Fig. 3b. d, f, nMS of unfractionated purified protein complexes of Ancα/β5 + α and Ancα/β14 + α at 20 μM. Charge series, stoichiometries indicated. Red arrows, peaks isolated for further characterization by tandem MS (Extended Data Fig. 11). e, Homology model of Ancα/β14 + α using Human Hb (1A3N) as template. Yellow and cyan sticks, Ancβ-lineage substitutions on IF2; orange sticks, Ancβ substitutions on IF1; yellow surface, αIF2; orange surface, αIF1; green, five β substitutions close to the interfaces included in Ancα/β14 + α. g, nMS of Ancα/β2 across concentrations. Charge series and stoichiometries indicated. h, Similarity between interfaces in Ancα/β14 + Ancα homology model and X-ray crystal structure of Human Hb. Venn diagrams show sites buried at IF1 and IF2 in one or both structures. Small circle, number of shared interface sites with identical amino acid state. i, Hydrogen-bond contacts at interfaces in Ancα/β14 + α homology model are also found in X-ray crystal structures of extant haemoglobins. Residue pairs hydrogen-bonded in Ancα/β14 + α IF2 (yellow) and IF1 (orange) are listed; +also present in crystal structure; *interactions discussed in the main text. PDB identifiers are shown. j, Oxygen equilibrium curves of Ancα/β14 + α, Ancα/β4, Ancα/β2. All experiments were performed once per concentration. Lines, best-fit curves by nonlinear regression.
Extended Data Fig. 11 Stoichiometric characterization of Ancα/β containing historical substitutions.
a, SEC of Ancα/β5. Circles show stoichiometry associated with each peak’s elution volume. b, High-resolution accuracy mass spectrometry (HRA-MS) of Ancα/β5 + α. Purple circles, peaks associated with Ancα/β5; pink, Ancα. c, HRA-MS of tetramer-containing SEC fraction of Ancα/β4 + Ancα. d, HRA-MS of monomer-containing SEC fraction of Ancα/β4 + Ancα. *922 m/z calibration reference standard. e, HRA-MS of Ancα/β9 + Ancα. f, nMS of tetramer-containing SEC fraction of Ancα/β4 + Ancα (Fig. 3a, b). Black circle, most abundant peak used for tandem MS. g, Tandem MS of isolated most-abundant peak in f, showing trimer-containing peaks. Charge states and number of haems (h) in the 8+ peak are indicated. h, Monomer-containing (M) peaks. i–k, nMS (i) and tandem MS (j, k) of Ancα/β14 + Ancα (Fig. 3f) as in f–h. l–n, nMS and tandem MS of Ancα/β5 + Ancα (Fig. 3c, d) as in f–h. Black dots in n mark charge species produced by cleavage of Ancα/β5. All experiments were performed once.
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Pillai, A.S., Chandler, S.A., Liu, Y. et al. Origin of complexity in haemoglobin evolution. Nature 581, 480–485 (2020). https://doi.org/10.1038/s41586-020-2292-y
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