Evolution and molecular basis of a novel allosteric property of crocodilian hemoglobin
Understanding the evolution of novel protein functions requires data on the mechanistic effects of causative mutations and the extent of coupling between the gain of new function and loss of ancestral function. Here, we use ancestral protein resurrection and directed mutagenesis to dissect the molecular basis of a novel mode of allosteric regulation in crocodilian hemoglobin. We discovered that regulation of Hb-O2 affinity via allosteric binding of bicarbonate ions (a biochemical adaptation unique to crocodilians) and the concomitant loss of allosteric regulation by ATP-binding are attributable to non-overlapping sets of substitutions. Gain of bicarbonate-sensitivity involved direct effects of few replacements at key sites in combination with indirect effects of numerous replacements at structurally disparate sites. Due to the context-dependence of causative substitutions, the unique allosteric properties of crocodilian hemoglobin cannot be easily transplanted into divergent homologs of other species. Significance StatementThe extraordinary breath-hold diving capacity of crocodilians is partly attributable to a unique mode of allosterically regulating hemoglobin-oxygenation in circulating red blood cells. We investigated the origin and mechanistic basis of this novel biochemical adaptation by performing directed mutagenesis experiments on resurrected ancestral crocodilian hemoglobins. Our results revealed that evolved changes in allosteric regulation involved the direct effect of few amino acid substitutions at key sites in combination with indirect effects of numerous other substitutions at structurally disparate sites. Such indirect interaction effects suggest that the evolution of novel protein functions may often depend on neutral mutations that produce no adaptive benefit when they first arise, but which contribute to a permissive background for subsequent function-altering mutations at other sites.