Metal-directed protein self-assembly

Abstract

Proteins are nature's premier building blocks for constructing sophisticated nanoscale architectures that carry out complex tasks and chemical transformations. Some 70%-80% of all proteins are thought to be permanently oligomeric; that is, they are composed of multiple proteins that are held together in precise spatial organization through noncovalent interactions. Although it is of great fundamental interest to understand the physicochemical basis of protein self-assembly, the mastery of protein-protein interactions (PPIs) would also allow access to novel biomaterials with nature's favorite and most versatile building block. In this Account, we describe a new approach we have developed with this possibility in mind, 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 higher order 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 begin by first describing the challenges of using entire proteins as molecular building blocks. We follow with an examination of our work on a model protein (cytochrome cb(562)), highlighting challenges toward establishing ground rules for MDPSA as well as progress in overcoming these challenges. 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, noncovalent interactions, exactly as natural metalloproteins and enzymes do. We describe our computational and experimental efforts toward restructuring the noncovalent 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 suggests possible parallels with the evolution of metalloproteins.

Figure 1

Cyt cb₅₆₂ and representative organic building blocks used for constructing supramolecular coordination complexes and MOFs. Surface residues with coordinating ability are shown as sticks.

Figure 2

(a) Antiparallel arrangement of cyt cb₅₆₂ molecules in the crystal lattice along their Helix3’s (magenta). (b) Model of MBPC-1, where key residues involved in metal binding and secondary interactions are depicted as sticks.

Figure 3

(a) Molecular mass distributions for MBPC-1 and equimolar Zn(II) (except where noted) determined by SV measurements, and proposed Zn-induced supramolecular geometries corresponding to dimeric and tetrameric species. (b) Sideview of Zn₄:MBPC-1₄. (c) Topview of Zn₄:MBPC-1₄, highlighting the coordination environment of interfacial Zn ions.

Figure 4

Crystal structures of (a) Ni₂:MBPC-1₃ and (b) Cu₂:MBPC-1₂ viewed from the side and the top.

Figure 5

(a) Model for MBP-Phen1, highlighting potential metal binding sites on Helix3. (b) Crystal structure of Ni₃:MBP-Phen1₃. (c) Surface representation of Ni₃:MBP-Phen1₃, showing the burial of the Phen group under the 50’s loop. (d) Ni coordination environment in Ni₃:MBP-Phen1₃. The H-bond between the P53 carbonyl and the PhenC59 amide nitrogen is indicated with a red dashed line.

Figure 6

(a) Cylindrical representations of Zn₄:MBPC-1₄ and Zn₄:MBPC-2₄ Helix3’s and sidechains involved in Zn coordination viewed from the side. N- and C-termini of Helix3’s in each assembly are labeled accordingly. (b) Closeup of the interfacial Zn coordination environment.

Figure 7

(top) Interfacial H-bonding interactions in Zn₄:MBPC-1₄ and Zn₄:MBPC-2₄. (bottom) Molecular weight distributions of MBPC-1 and MBPC-2 species as determined by SV measurements. All samples contain 600 μM protein and 600 μM Zn, with the exception of R34A-MBPC-1, which contains 300 μM Zn.

Figure 8

Terraced energy landscapes for MDPSA in the absence (a) or presence (b) of specific protein-protein interactions. The funnels shown apply to the Zn-driven oligomerization of MBPC-1 (a) and RIDC-1 (b). ICN denotes “interprotein coordination number” for interfacial metal ions. The asterisk denotes the preferred conformation, which is a D₂-symmetrical assembly shown in the Inset.

Figure 9

Cartoon outline for MeTIR. 1. Protein/peptide with a non-self-associating surface; 2. 1 modified with metal coordinating groups; 3. Initial Metal1-templated protein complex with non-complementary interfaces; 4. Metal1-templated protein complex with optimized, complementary interfaces; 5. Protein with a self-associating surface; 6. Metal-independent protein complex biased towards Metal1 binding; 7. Protein complex with distorted Metal2 coordination.

Figure 10

Three pairs of interfaces (i1, i2, i3) formed within the Zn₄:MBPC-1₄ tetramer; Zn-coordination environment in each interface is listed below.

Figure 11

Detailed view of the crystalographically-characterized sidechain conformations in i1 and i2 before (left) and after (right) redesign (Zn – large red spheres; water molecules – small red spheres.)

Figure 12

(a) From left to right: Sedimentation coefficient distributions for MBPC-1, RIDC-1 and RIDC-2 in the presence of equimolar Zn(II). (b) Backbone superposition of Zn₄:MBPC-1₄ (green), Zn₄:RIDC-1₄ (blue), and Zn₄:RIDC-2₄ (red).

Figure 13

Side and topviews of the RIDC-1₂ crystal structure, along with the closeup of one of the two symmetrical interaction zones in the dimer interface detailing the interfacial contacts. An ordered water molecule is highlighted as a red sphere.

Figure 14

(a) Influence of Zn-templated interfacial mutations in i1 on the conformations of Cu-mediated dimeric assemblies. (b) Backbone superposition of Cu₂:RIDC-1₂ (grey) and a dimeric half of Zn₄:RIDC-1₄ (orange) that contains i1. (c) Cu coordination environment in Cu₂:RIDC-1₂, highlighting the open coordination sites occupied by two water molecules. The Glu81 sidechain from a crystallographic symmetry-related dimer is also shown.