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.
