The rational design and construction of a cuboidal iron-sulfur protein

Abstract

Rational protein design is an emerging approach for testing general theories of protein chemistry through the creation of new structures and functions. Here we present the first successful introduction by rational design of a [Fe4S4] cuboidal cluster into the hydrophobic core of Escherichia coli thioredoxin, a protein normally devoid of metal centers. Cuboidal [Fe4S4] is one of the stable forms of self-assembled iron-sulfur clusters that are thought to represent some of the earliest evolved biological redox centers. [Fe4S4] clusters have been recruited for use in a variety of proteins whose functions are central to many of the major biochemical processes ranging from simple soluble electron-transfer agents, to membrane-bound components of electron-transfer chains, to electron reservoirs in complex metalloenzymes such as nitrogenase. By situating an [Fe4S4] cluster into a protein environment not previously adapted by evolution we can explore the factors by which their activity is modulated by the protein matrix.

Figure 1

Ribbon and space-filling representations of the designed cuboidal iron–sulfur protein Trx-Fe₄S₄. Iron is cyan, sulfur yellow. (A) Ribbon diagram showing the location of all mutations used in the construction of Trx-Fe₄S₄ and the region of the Trx host protein implicated in phage assembly. The cuboidal [Fe₄S₄] cluster binding residues (Leu-24-Cys, Leu-42-Cys, Val-55-Cys, and Leu-99-Cys) are buried between the central β-sheet and two α-helices. Cys-32-Ser and Cys-35-Ser mutations were made to remove the native Trx disulfide bond, thereby eliminating any interaction from cysteine residues that are not part of the designed site. An isosteric Asp-26-Leu mutation was also introduced to improve the global stability of the protein; the Asp-26-Ala mutant of Trx is stabilized by 3 kcal compared with the wild-type protein (27, 28). The residues that are believed to be important for protein–protein interactions in Trx function (–, 75, 76, 91–93) are colored red (29). (B) Space-filling representation of residues 24, 42, 55, and 99 in native E. coli Trx (Left) and Trx-Fe₄S₄ (Right). Incorporation of the [Fe₄S₄(Cys)₄]ⁿ⁻ design moiety represents a conservative isovolume exchange; there is only a negligible decrease in the volumes occupied by amino acid side chains in the interior of the designed iron–sulfur protein relative to the host.

Figure 2

Optical spectrum of apo Trx-Fe₄S₄ (trace A), holo Trx-Fe₄S₄ (trace B) in 15 mM Mops/100 mM NaCl (pH 7.4) at 5°C, and the synthetic cluster [Fe₄S₄(S-EtOH)₄](Me₄N)₂ (trace C) in 10 mM Ches/10 mM βME (pH 8.5). holo Trx-Fe₄S₄ contains 3.9 ± 0.2 mol of iron and 3.9 ± 0.2 mol acid labile sulfide per Trx protein; λ_max = 280 nm, ɛ_M,280 = 36,000 λ_max = 413 nm, ɛ_M,413 = 16,100. (Inset) CD spectra of apo Trx-Fe₄S₄ (upper trace) and Trx (lower trace) in 50 mM potassium phosphate (pH 7.0) at 25°C. The upper trace has been offset slightly to clarify the close superposition of the two curves.

Figure 3

Stability of the free synthetic cluster [Fe₄S₄(S-EtOH)₄](Me₄N)₂ (□) in the presence of low levels (0.4 mM) of stabilizing exogenous βME and reconstituted holo Trx-Fe₄S₄ (▪) in the absence of stabilizing exogenous βME. Solution conditions: 10 mM Mops/100 mM NaCl (pH 7.4) at 5°C.

Figure 4

EPR spectrum of holo Trx-Fe₄S₄ in 15 mM Mops/100 mM NaCl (pH 7.4), protein concentration 70 ± 5 μM. (Trace B) holo Trx-Fe₄S₄ oxidized with 10 equivalents of K₃[Fe(CN)₆]. (Trace A) Untreated (resting state) holo Trx-Fe₄S₄. EPR spectra collected on a Varian E-line EPR spectrometer operating at 9.24 GHz, 1.5 mW observe power, 10 G modulation amplitude, 25 K.