Complexity in the Membrane: The Fluc family of Fluoride Channels and Small Multidrug Resistance Family of Transporters as Models for Understanding Membrane Protein Structural and Functional Evolution
Macdonald, Christian
2021
Abstract
The cellular membrane comprises the barrier between self and environment, the development of which laid the foundation for the individual organism. The membranes of organisms today are packed full of molecular machines which mediate communication, metabolism, and interaction between the homeostatic internal environment and the uncontrolled environments they find themselves in. Understanding the molecular bases for their function, as well as the evolutionary origins of their roles, is an important task for understanding modern microbial biology. This thesis examines two families of membrane proteins in their functional and evolutionary context. The first, the Fluc family of fluoride channels, is an exquisitely selective ion channel which confers resistance to environmental Fluoride via electrodiffusive transport. The structure of this family has been shaped by gene duplication, as phylogenetic analysis reveals. This reveals that duplication occurs often but that, once duplicated, reversion is rare. Additionally, functional redundancy leads to one (of two) pores becoming non-functional. Gene duplication has a key role in protein evolution, and in membrane transporters in particular has been involved in most transporters that are known today. The Flucs are an appealing model system for studying this, as they exist in several evolutionary states, and their function depends on asymmetric interactions between dimers which can be used to probe drift after duplication. This interaction can be harnessed to develop an experimental system for studying the fitness effects of gene duplication. Several models exist which propose mechanisms for the retention of gene duplicates before adaptive or non-adaptive forces fix them, but any experimental test has been difficult to obtain. The asymmetric interactions of a key motif in the Flucs allows constructs which differentiate between unduplicated, duplicated, and heterodimeric (fixed duplicated) evolutionary states of the same protein to be created. Deep mutational scanning of these constructs and comparison between the contexts then allows insight into the natural trajectories duplicates took in the Flucs. Small Multidrug Resistance family of prokaryotic proton-coupled transporters has long been thought to be mainly involved in antibiotic resistance. A more careful analysis reveals that the major role of these is in fact guanidinium efflux. Using a combination of phylogenetics and in vitro assays, a variety of homologs are shown to be specific and well-coupled electrogenic guanidinium/proton antiporters. To understand the relationship between subtypes, a novel electrophysiological technique, solid-supported membrane electrophysiology, was used to study the comparative substrate capacities of the guanidinium-exporting subtype (called Gdxs) and the multispecific drug-exporting subtype (called Qacs). Surprisingly, a chemical region of shared recognition between the subtypes was discovered: aromatic or hydrophobic singly-substituted guanidiniums are transported by both subtypes. To understand the structural basis for this, the first high-resolution crystal structure of an SMR was solved. Both subtypes share aromatic binding pockets, but loss of important H-bonds in the Qac subtype likely introduces polyspecificity. These combined results reveal how structure, function, and topology interact to shape the evolution of membrane proteins, and provide important insights and tools for understanding how extant membrane machinery arose.Deep Blue DOI
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membrane protein electrophysiology biochemistry
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