AbstractSurface 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 C. crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multi-step assembly pathway. This pathway involves two domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2D crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using time-resolved electron cryo-microscopy (Cryo-EM) reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the two 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.Significance StatementMany microbes assemble a crystalline protein layer on their outer surface as an additional barrier and communication platform between the cell and its environment. Surface layer proteins efficiently crystallize to continuously coat the cell and this trait has been utilized to design functional macromolecular nanomaterials. Here, we report that rapid crystallization of a bacterial surface layer protein occurs through a multi-step pathway involving a crystalline intermediate. Upon calcium-binding, sequential changes occur in the structure and arrangement of the protein, which are captured by time-resolved small angle x-ray scattering and transmission electron cryo-microscopy. We demonstrate that a specific domain is responsible for enhancing the rate of self-assembly, unveiling possible evolutionary mechanisms to enhance the kinetics of 2D protein crystallization in vivo.
Monte Carlo Study of the Molecular Mechanisms of S-Layer Protein Self-Assembly