How proteins evolved the ability to form complexes made up of multiple subunits—and what functions this new structure enabled in early multimeric proteins—isn’t well understood. To better understand how such changes might have happened, Joseph W. Thornton of the University of Chicago and coworkers used ancestral protein reconstruction to trace the evolutionary pathway of the oxygen-transporting protein hemoglobin (Nature 2020, DOI: 10.1038/s41586-020-2292-y).
Modern hemoglobin forms a heterotetramer with two α subunits and two β subunits. The researchers used computational methods to infer the ancestral amino acid sequences of each of those subunits, as well as the more ancient precursor from which both α and β likely originated. They engineered bacteria to produce the proteins and characterized them with mass spectrometry and size-exclusion chromatography. When expressed together, the α and β subunits formed hemoglobin-like tetramers, but their ancient precursor formed only dimers.
By introducing mutations that happened during hemoglobin’s history into the reconstructed ancestral protein sequences, and having the bacteria produce them, the researchers found that just two changes at an interface between the subunits were sufficient to cause tetramers to form. They also found that the formation of tetramers causes cooperative binding, in which the binding of oxygen increases the hemoglobin’s affinity for additional oxygen molecules.
The researchers suggest that the ease of evolving structural complexity and cooperativity may not be limited to hemoglobin. The team plans to use this method to study whether other proteins also evolved multimeric structures and cooperative binding with relatively few mutations.