Origins of allostery in vertebrate hemoglobin evolution.

Allostery-regulation of a protein's activity by binding to an effector-is an essential functional feature of many proteins. Its structural basis is complex: a protein must bind effector at one site-an interaction that typically involves many complementary residues-and this binding event must change the protein's conformation at the distant active site. How allosteric proteins evolved this architecture is unknown because there are no cases in which the historical mechanisms by which allostery was acquired from nonallosteric precursors have been identified. Vertebrate hemoglobin (Hb) is a tetrameric protein whose oxygen affinity is reduced by binding organic phosphates. We used ancestral protein reconstruction, biochemical experiments, and in silico studies of protein structure and dynamics to identify the changes in protein sequence and structure that caused allosteric Hb to evolve from its non-allosteric dimeric precursor. We found that just two historical substitutions were sufficient to confer positive allostery on the ancestor-one that caused the dimer to tetramerize, and another that created an effector binding site in the tetramer's central cavity. Two additional substitutions changed the effector's position and conferred the modern form of negative allostery. This short evolutionary path to allostery was possible because most of the key requirements for allostery already existed as by-products of the protein's structure: the tertiary transition between oxygenated and deoxygenated conformations is an ancient and intrinsic property of the globin fold. It affects the surface patch that ultimately mediated tetramer assembly, so as soon as tetramerization evolved because of one substitution on that surface, tertiary heterogeneity propagated to large-scale quaternary heterogeneity. The other substitution(s) conferred effector binding at a site within the tetramer that is preferentially accessible in the deoxygenated state. One of the most complex and essential protein phenomena therefore evolved via simple and intelligible mechanisms.