CONSPECTUSProteins are Nature's premier building blocks for constructing sophisticated nanoscale architectures that carry out complex tasks and chemical transformations. It is estimated that 70-80% of all proteins are permanently oligomeric, that is, they are composed of multiple proteins that are held together in precise spatial organization through non-covalent interactions. While it is of great fundamental interest to understand the physicochemical basis of protein self-assembly, the mastery of proteinprotein interactions (PPIs) would also allow access to novel biomaterials using Nature's favorite and most versatile building block. With this possibility in mind, we have developed a new approach, Metal Directed Protein Self-Assembly (MDPSA), which utilizes the strength, directionality and selectivity of metal-ligand interactions to control PPIs.At its core, MDPSA is inspired by supramolecular coordination chemistry which exploits metal coordination for the self-assembly of small molecules into discrete, more-or-less predictable higherorder structures. Proteins, however, are not exactly small molecules or simple metal ligands: they feature extensive, heterogeneous surfaces that can interact with each other and with metal ions in unpredictable ways. We will start this Account by first describing the challenges of using entire proteins as molecular building blocks. This will be followed by our work on a model protein (cytochrome cb 562 ) to both highlight and overcome those challenges toward establishing some ground rules for MDPSA.Proteins are also Nature's metal ligands of choice. In MDPSA, once metal ions guide proteins into forming large assemblies, they are by definition embedded within extensive interfaces formed between protein surfaces. These complex surfaces make an inorganic chemist's life somewhat difficult, yet they also provide a wide platform to modulate the metal coordination environment through distant, non-covalent interactions -exactly as natural metalloproteins and enzymes do. We will describe our computational and experimental efforts on restructuring the non-covalent interaction network formed between proteins surrounding the interfacial metal centers. This approach of metal templating followed by the redesign of protein interfaces (Metal-Templated Interface Redesign, MeTIR) not only provides a route to engineer de novo PPIs and novel metal coordination environments, but also carries possible parallels to the evolution of metalloproteins.Correspondence to: F. AKIF TEZCAN. To circumvent the challenge of ruling over numerous weak bonds, we envisioned that if protein surfaces are appropriately modified, PPIs could be controlled through metal coordination. Using metal coordination to guide PPIs offers several advantages. First, metal-ligand bonds are stronger than the non-covalent bonds that make up protein interfaces, obviating the need to engineer large surfaces to produce favorable protein-protein docking. Second, metal-ligand bonds are highly directional, thus the stereochemical preference an...
Protein-protein interactions (PPI's) are central to nearly all processes within cells, whether they are formed transiently in dynamic networks or permanently in macromolecular assemblies. There has been considerable progress towards our understanding of how proteins recognize their partners and how the energetics of their interactions are tuned. 1 Nevertheless, the ability to predict or interfere with natural PPI's or engineer new ones remains a great challenge, owing to the fact that protein-protein docking processes are guided by the superposition of many weak, non-covalent bonds that spread over large and often flexible surfaces. Our goal is to utilize the strength, directionality and selectivity of metal-ligand interactions to control PPI's, thereby achieving specificity and affinity without requiring extensive binding surfaces. We describe here the Zn-mediated construction of a 16-helix architecture comprising four copies of cytochrome cb 562 (cyt cb 562 ), a 4-helix
Metal coordination is a key structural and functional component of a large fraction of proteins. Given this dual role we considered the possibility that metal coordination may have played a templating role in the early evolution of protein folds and complexes. We describe here a rational design approach, Metal Templated Interface Redesign (MeTIR), that mimics the time course of a hypothetical evolutionary pathway for the formation of stable protein assemblies through an initial metal coordination event. Using a folded monomeric protein, cytochrome cb 562 , as a building block we show that its non-self-associating surface can be made self-associating through a minimal number of mutations that enable Zn coordination. The protein interfaces in the resulting Zn-directed, D 2 -symmetrical tetramer are subsequently redesigned, yielding unique protein architectures that self-assemble in the presence or absence of metals. Aside from its evolutionary implications, MeTIR provides a route to engineer de novo protein interfaces and metal coordination environments that can be tuned through the extensive noncovalent bonding interactions in these interfaces.evolution | metal coordination | protein self-assembly | protein-protein interactions | templating
The enzyme ␣-galactosidase (␣-GAL, also known as ␣-GAL A; E.C. 3.2.
Protein-protein interactions (PPIs) are the most diverse of biological self-assembly processes, central to the construction and mechanical integrity of biological machinery on one hand, and cellular dynamics and communication on the other. While rational design and control of PPIs would provide access to novel protein assemblies 1 as well as the manipulation of cellular processes, 2, 3 such efforts are hampered by the fact that PPIs comprise an extensive set of noncovalent bonds distributed over large and typically non-contiguous molecular surfaces. 4 We have taken on the challenge of controlling PPIs using an inorganic chemical approach. Work by several groups has shown that small organic building blocks with acceptor groups can self-assemble into discrete superstructures and frameworks through metal coordination. 5 We envisioned that the self-assembly of proteins can likewise be controlled by metal binding, with the added complications that a) protein surfaces are replete with polar side chains capable of coordinating metals, and b) the interactions between individual proteins in contrast to those between organic building blocks may not be negligible. Our previous studies have shown that metal coordination can be largely localized onto multidentate metal binding motifs inserted on surface helices that can outcompete other potential binding sites; one such construct (His 4 -cb 562 ; hereafter referred to as MBPC-1, Figure 1) was found to self-assemble into a discrete superstructure upon Zn coordination. 6 In this study, we show that secondary interactions between proteins, i.e. those not involving metal coordination, can play a significant role in tuning the oligomerization/aggregation behavior of a protein.MBPC-1 was engineered with two di-His motifs (59/63 and 73/77) located near each terminus of a single α-helix (Helix 3, spanning residues 56-80) in the parent protein cytochrome cb 562 , 7 with the idea that such an arrangement would yield a closed superstructure upon metal coordination. Indeed, at appropriate Zn and protein concentrations, MBPC-1 formed a tetrameric assembly (Zn 4 :MBPC-1 4 ). 6 The crystal structure of Zn 4 :MBPC-1 4 revealed a unique quaternary architecture, in which two V-shaped dimers wedged into one another, held together by four Zn ions with identical His 3 (63/73/77) Asp 1 (74) coordination environments (Figure 2a). The key to this supramolecular arrangement was the Asp74 residue located within the His73/77 clamp: Zn coordination by Asp74 rather than His59 allowed the V-shaped dimers, and ultimately, the observed tetramer to be formed ( Figure S1). Hydrodynamic measurements showed that MBPC-1 did not have a tendency to self-associate in the absence of metals, which indicated that metal binding was the driving force for oligomerization. At the same time, the tetramer was found to have an extensive PPI surface area of nearly 5000 Å 2 , raising the possibility that secondary interactions between proteins may still influence the formation of Supporting Information Available: Material...
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