Cryo Electron Tomography and Reaction-Diffusion Simulations Reveal a Molecular and Evolutionary Basis for Charged Archaeal Surface Layer Proteins

Abstract

Surface layers (S-layers) are 2D, proteinaceous lattices that form the outermost cell envelope component of many microbes. S-layers, which exhibit exceptional sequence diversity, are found in nearly all archaea and numerous bacteria. Despite their variation, S-layer proteins display several unifying characteristics such as their ability to form crystalline sheets punctuated with nano-scale pores, and their propensity for charged amino acids. However, the precise role of these charged functional groups and how they relate to cellular function is unknown. Here, we offer a rationale for charged S-layer proteins in the context of the structural evolution of S-layers. We have chosen ammonia-oxidizing archaea (AOA) as a model system for S-layer and used the cryo electron tomographic reconstruction of the AOA to develop a 2D electro-diffusion reaction computational framework to simulate diffusion and consumption of the charged solute ammonium. The AOA create energy directly from electrons evolved during ammonia oxidation by ammonia monooxygenase (AMO). While the specific location of the archaeal AMO active site is unknown, a bacterial homologue indicates a location underneath the S-layer in pseudo periplasmic space (PPS). Our simulations suggest that charged S-layers and nanopores expedite diffusion of charged solutes into the PPS when the electro-diffusion-reaction system is driven away from equilibrium, replenishing reacted NH_4^+ in PPS. By contrast, a neutral S-layer would inhibit diffusion of charged molecules, while removing the S-layer altogether dramatically reduced ammonium concentration throughout the PPS. Strikingly, analysis of annotated S-layer amino acid sequences from all known archaeal clades indicated a clear dearth of sequences in the neutral regime. Our simulations suggest that charged S-layers and nanopores impart a potential fitness advantage. Thus, S-layer charge may have emerged by convergent evolution to enhance metabolic function in diverse ecosystems.