Intersubunit Coupling Enables Fast CO2-Fixation by Reductive Carboxylases.

Enoyl-CoA carboxylases/reductases (ECRs) are some of the most efficient CO2-fixing enzymes described to date. However, the molecular mechanisms underlying the extraordinary catalytic activity of ECRs on the level of the protein assembly remain elusive. Here we used a combination of ambient-temperature X-ray free electron laser (XFEL) and cryogenic synchrotron experiments to study the structural organization of the ECR from Kitasatospora setae. The K. setae ECR is a homotetramer that differentiates into a pair of dimers of open- and closed-form subunits in the catalytically active state. Using molecular dynamics simulations and structure-based mutagenesis, we show that catalysis is synchronized in the K. setae ECR across the pair of dimers. This conformational coupling of catalytic domains is conferred by individual amino acids to achieve high CO2-fixation rates. Our results provide unprecedented insights into the dynamic organization and synchronized inter- and intrasubunit communications of this remarkably efficient CO2-fixing enzyme during catalysis.

A bacterial surface layer protein exploits multistep crystallization for rapid self-assembly.

Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the Caulobacter crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multistep assembly pathway. This pathway involves 2 domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2-dimensional (2D) crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using a time course of electron cryo-microscopy (Cryo-EM) imaging reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the 2 domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials.

Transport Properties of Nanoporous, Chemically Forced Biological Lattices.

Permselective nanochannels are ubiquitous in biological systems, controlling ion transport and maintaining a potential difference across a cell surface. Surface layers (S-layers) are proteinaceous, generally charged lattices punctuated with nanoscale pores that form the outermost cell envelope component of virtually all archaea and many bacteria. Ammonia oxidizing archaea (AOA) obtain their energy exclusively from oxidizing ammonia directly below the S-layer lattice, but how the charged surfaces and nanochannels affect availability of NH4+ at the reaction site is unknown. Here, we examine the electrochemical properties of negatively charged S-layers for asymmetrically forced ion transport governed by Michaelis-Menten kinetics at ultralow concentrations. Our 3-dimensional electrodiffusion reaction simulations revealed that a negatively charged S-layer can invert the potential across the nanochannel to favor chemically forced NH4+ transport, analogous to polarity switching in nanofluidic field-effect transistors. Polarity switching was not observed when only the interior of the nanochannels was charged. We found that S-layer charge, nanochannel geometry, and enzymatic turnover rate are finely tuned to elevate NH4+ concentration at the active site, potentially enabling AOA to occupy nutrient-poor ecological niches. Strikingly, and in contrast to voltage-biased systems, magnitudes of the co- and counterion currents in the charged nanochannels were nearly equal and amplified disproportionally to the NH4+ current. Our simulations suggest that engineered arrays of crystalline proteinaceous membranes could find unique applications in industrial energy conversion or separation processes.

Nutrient transport suggests an evolutionary basis for charged archaeal surface layer proteins.

Surface layers (S-layers) are two-dimensional, proteinaceous, porous lattices that form the outermost cell envelope component of virtually all archaea and many bacteria. Despite exceptional sequence diversity, S-layer proteins (SLPs) share important characteristics such as their ability to form crystalline sheets punctuated with nano-scale pores, and their propensity for charged amino acids, leading to acidic or basic isoelectric points. However, the precise function of S-layers, or the role of charged SLPs and how they relate to cellular metabolism is unknown. Nano-scale lattices affect the diffusion behavior of low-concentration solutes, even if they are significantly smaller than the pore size. Here, we offer a rationale for charged S-layer proteins in the context of the structural evolution of S-layers. Using the ammonia-oxidizing archaea (AOA) as a model for S-layer geometry, and a 2D electrodiffusion reaction computational framework to simulate diffusion and consumption of the charged solute ammonium (NH4+), we find that the characteristic length scales of nanoporous S-layers elevate the concentration of NH4+ in the pseudo-periplasmic space. Our simulations suggest an evolutionary, mechanistic basis for S-layer charge and shed light on the unique ability of some AOA to oxidize ammonia in environments with nanomolar NH4+ availability, with broad implications for comparisons of ecologically distinct populations.

Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer.

Surface layers (S-layers) are paracrystalline, proteinaceous structures found in most archaea and many bacteria. Often the outermost cell envelope component, S-layers serve diverse functions including aiding pathogenicity and protecting against predators. We report that the S-layer of Caulobacter crescentus exhibits calcium-mediated structural plasticity, switching irreversibly between an amorphous aggregate state and the crystalline state. This finding invalidates the common assumption that S-layers serve only as static wall-like structures. In vitro, the Caulobacter S-layer protein, RsaA, enters the aggregate state at physiological temperatures and low divalent calcium ion concentrations. At higher concentrations, calcium ions stabilize monomeric RsaA, which can then transition to the two-dimensional crystalline state. Caulobacter requires micromolar concentrations of calcium for normal growth and development. Without an S-layer, Caulobacter is even more sensitive to changes in environmental calcium concentration. Therefore, this structurally dynamic S-layer responds to environmental conditions as an ion sensor and protects Caulobacter from calcium deficiency stress, a unique mechanism of bacterial adaptation. These findings provide a biochemical and physiological basis for RsaA's calcium-binding behavior, which extends far beyond calcium's commonly accepted role in aiding S-layer biogenesis or oligomerization and demonstrates a connection to cellular fitness.

Subwavelength localization of near fields in coupled metallic spheres for single-emitter polarization analysis.

We numerically demonstrate selective near-field localization determined by the polarization state of a single emitter coupled to a plasmonic nanocluster. Seven gold nanospheres are carefully arranged such that up to 10 polarization states of the single emitter, including linear, circular, and elliptical polarizations, can be distinguished via the distinct field localization in four gaps. The ability to transform polarization states into field spatial localizations may find application in single emitter polarization analysis.