Papers in the Biological Sciences


Date of this Version



Nature 581 (28 May 2020), pp. 480–485.

doi: 10.1038/s41586-020-2292-y


Copyright © 2020 Arvind S. Pillai, Shane A. Chandler, Yang Liu, Anthony V. Signore, Carlos R. Cortez-Romero, Justin L. P. Benesch, Arthur Laganowsky, Jay F. Storz, Georg K. A. Hochberg, and Joseph W. Thornton. Published by Springer Nature Limited. Used by permission.


Most proteins associate into multimeric complexes with specific architectures, which often have functional properties such as cooperative ligand binding or allosteric regulation. 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 hemoglobin, 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 hemoglobin 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 favorable 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.

The interfaces that hold molecular complexes together typically involve sterically tight, electrostatically complementary interactions among many amino acids. Similarly, allostery and cooperativity usually depend on numerous residues that connect surfaces to active sites. The acquisition of such complicated machinery would seem to require elaborate evolutionary pathways. The classical explanation of this process, by analogy to the evolution of morphological complexity, is that multimerization conferred or enhanced beneficial functions, allowing selection to drive the many substitutions required to build and optimize new interfaces.

Whether this account accurately describes the evolution of any natural molecular complex requires a detailed reconstruction of the historical steps by which it evolved. Hemoglobin (Hb) is a useful model for this purpose, because the structural mechanisms that mediate its multimeric assembly, cooperative oxygen binding, and allosteric regulation are well established. Moreover, its subunits descend by duplication and divergence from the same ancestral proteins, so their history can be reconstructed in a single analysis. Despite considerable speculation, virtually nothing is known about the evolutionary origin of Hb’s heterotetrameric architecture and the functions that depend on it.

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