The evolution of novel protein functions and protein families is a fundamental question within both evolutionary biology and biochemistry. While many gene families follow predictable patterns of molecular tinkering, many protein families exist with completely novel functions now essential. The guanylate kinase protein interaction domain (GKPID) of the membrane associated guanylate kinases (MAGUK) represents a model system for the study of protein evolution in which a protein scaffolding domain has evolved from a nucleotide kinase ancestor. Here we elucidate the ancient mechanisms by which these new functions evolved by combining ancestral protein reconstruction with in vitro and cell-biological molecular experiments. We found that the GKPID's capacity to serve as a mitotic spindle-orienting scaffold evolved by duplication and divergence of an ancient guanylate kinase enzyme before the divergence of animals and choanoflagellates. Re-introducing a single historical substitution into the ancestral guanylate kinase is sufficient to abolish the ancestral enzyme activity, confer the derived scaffolding function, and establish the capacity to mediate spindle orientation in cultured cells. This substitution appears to have revealed a latent protein-binding site, rather than constructing a novel interaction interface, apparently by altering the dynamics or conformational occupancy of a hinge region that determines whether the binding site is exposed or hidden. Three further substitutions also conveyed a measure of ligand specificity to phosphorylated Pins, which is necessary in metazoan spindle orientation pathways. These findings show how a small number of simple, ancient genetic changes caused the evolution of novel molecular functions crucial for the evolution of complex animals and laid the groundwork for an entirely new family of metazoan scaffolding proteins. This dissertation contains previously unpublished, co-authored material.

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