Metal-Directed Design of Supramolecular Protein Assemblies

Abstract

Owing to their central roles in cellular signaling, construction, and biochemistry, protein-protein interactions (PPIs) and protein self-assembly have become a major focus of molecular design and synthetic biology. In order to circumvent the complexity of constructing extensive non-covalent interfaces, which are typically involved in natural PPIs and protein self-assembly, we have developed two design strategies, Metal-Directed Protein Self-Assembly (MDPSA) and Metal-Templated Interface Redesign (MeTIR). These strategies, inspired by both the proposed evolutionary roles of metals and their prevalence in natural PPIs, take advantage of the favorable properties of metal coordination (bonding strength, directionality, and reversibility) to guide protein self-assembly with minimal design and engineering. Using a small, monomeric protein (cytochrome cb562) as a model building block, we employed MDPSA and MeTIR to create a diverse array of functional supramolecular architectures which range from structurally tunable oligomers to metalloprotein complexes that can properly self-assemble in living cells into novel metalloenzymes. The design principles and strategies outlined herein should be readily applicable to other protein systems with the goal of creating new PPIs and protein assemblies with structures and functions not yet produced by natural evolution.

Metal‐dependent assembly of a protein nano‐cage

“…There has been extensive interest in creating metal‐dependent protein assemblies that may have uses as sensors or responsive biomaterials . The experiments we describe here demonstrate the potential of using metal‐binding coiled coils to mediate protein assembly, a concept that could be extended to other environmentally responsive coiled coil designs.…”

“…Inspired by Nature, various strategies have been developed to control the assembly of extended protein-based nanomaterials that form filaments, 30,31 2D lattices, 32,33 and 3D crystals. [34][35][36] Assembly has been controlled by, for example, the judicious use of protein-protein disulfide linkages, 33,37 the use of bifunctional small molecules 38 to connect together protein subunits and the design of "split" metal ion-binding sites 30,31,33,36,39,40 that span two protein subunits. Natural protein cages such as ferritin have been reengineered to undergo environmentally responsive assembly and disassembly; for example, a Cu(II)-responsive ferritin cage was designed by introducing His residues at the inter-subunit interface of ferritin, 41 whereas a pH-responsive ferritin cage was engineered by introducing a pH-responsive amphipathic helix into the inter-subunit interface.…”

Constructing protein polyhedra via orthogonal chemical interactions

“…X-ray diffraction data were collected at 100 K at either the Advanced Light Source (ALS) beamline BL 8.3.1 (using 1.12-Å radiation for BMC3 and 1.33-Å radiation for BMC4) or at the Stanford Synchrotron Radiation Lightsource (SSRL) beamlines 9-2 (using 0.98-Å radiation for BMC2) and 12-2 (using 0.98-Å radiation for BMC1). Data integration was performed using the XDS Program Package, truncated at CC 1/2 > 0.5 35 Datasets of the same structure recorded at different wavelengths were scaled to the highest resolution dataset with XSCALE 36 , 37 . Phaser-MR 38 was employed to carry out molecular replacement with search models based on the CMFC1 monomer (PDB ID: 3M4B) containing the expected side chain mutations (generated in Pymol 39 ) but lacking HA.…”

Hierarchical design of artificial proteins and complexes toward synthetic structural biology

“…In particular, design strategies of metal-directed protein self-assembly (MDPSA) and metal-templated interface redesign (MeTIR) were described (Fig. 1f) (Salgado et al 2010a, b;Bailey et al 2016). Using these strategies, a building block protein was designed and engineered to form homodimers bearing interfacial Zn- (Lai et al 2014).…”