Functional Evolution of Solute-Binding Proteins

Abstract

Solute-binding proteins (SBPs) comprise an abundant and adaptable superfamily of extracytoplasmic solute receptors involved in nutrient uptake and chemotaxis, and constitute an important component of the nutrient-scavenging arsenal in bacteria. The SBP superfamily exemplifies the power of evolution to generate functional diversity by tinkering with an existing protein fold; SBPs have evolved to recognise a wide variety of solutes with high affinity and specificity, and have also been co-opted into roles in signal transduction, transcriptional regulation and catalysis. However, the historical sequence-structure-function relationships that explain how this functional diversity could have evolved are not well understood. This thesis describes the use of ancestral protein reconstruction, a technique that leverages phylogenetic information to enable experimental characterisation of extinct proteins, to investigate two case studies of functional evolution in the SBP superfamily: the evolution of new binding specificities in the amino acid-binding protein (AABP) family, and the emergence of the enzyme cyclohexadienyl dehydratase (CDT) from a non-catalytic ancestor that belonged to the SBP superfamily. The evolution of binding specificity in the AABP family was explored by reconstruction and functional characterisation of ancestral AABPs that predated the divergence of modern AABP subfamilies. The binding specificities of these ancestral proteins were comparable with modern AABPs, contradicting the prevailing view that ancient proteins had lower specificity than modern proteins. X-ray crystallography and isothermal titration calorimetry experiments showed that specialised glutamine-binding proteins originated from ancestral arginine-binding proteins that bound glutamine promiscuously, and that the promiscuous binding of glutamine was enabled by multi-scale conformational plasticity, water-mediated hydrogen bonding interactions and co option of an alternative low energy conformational sub-state productive for glutamine binding. This promiscuous binding mode was enthalpically favourable and entropically unfavourable; evolution of high-affinity glutamine-binding proteins was achieved by reduction of this entropic penalty to binding. CDT catalyses the decarboxylative aromatisation of prephenate and arogenate; these reactions are involved in phenylalanine biosynthesis. Because CDT is closely related to non-catalytic SBPs, this enzyme provides a useful model system for understanding the emergence of catalytic activity de novo. The evolution of CDT from a SBP was investigated by functional characterisation of reconstructed ancestors and extant homologues of the enzyme, which showed that CDT evolved from cationic amino acid-binding proteins. Directed evolution, X-ray crystallography and molecular dynamics simulations were used to determine the genetic, structural and dynamic bases for this functional transition. These experiments showed how individual substitutions contributed to activation of the ancestral SBP scaffold for decarboxylative aromatisation of cyclohexadienols by remodelling, functionalisation and refinement of the active site. These case studies of functional evolution in the SBP superfamily provide insight into two important evolutionary processes: the evolution of protein-ligand interactions with high affinity and specificity by adaptive improvement of promiscuous interactions, and the de novo evolution of enzymes from non-catalytic ancestors. Show more