Preorganization of molecular binding sites in designed diiron proteins

Abstract

De novo protein design provides an attractive approach to critically test the features that are required for metalloprotein structure and function. Previously we designed and crystallographically characterized an idealized dimeric model for the four-helix bundle class of diiron and dimanganese proteins [Dueferri 1 (DF1)]. Although the protein bound metal ions in the expected manner, access to its active site was blocked by large bulky hydrophobic residues. Subsequently, a substrate-access channel was introduced proximal to the metal-binding center, resulting in a protein with properties more closely resembling those of natural enzymes. Here we delineate the energetic and structural consequences associated with the introduction of these binding sites. To determine the extent to which the binding site was preorganized in the absence of metal ions, the apo structure of DF1 in solution was solved by NMR and compared with the crystal structure of the di-Zn(II) derivative. The overall fold of the apo protein was highly similar to that of the di-Zn(II) derivative, although there was a rotation of one of the helices. We also examined the thermodynamic consequences associated with building a small molecule-binding site within the protein. The protein exists in an equilibrium between folded dimers and unfolded monomers. DF1 is a highly stable protein (K(diss) = 0.001 fM), but the dissociation constant increases to 0.6 nM (deltadeltaG = 5.4 kcalmol monomer) as the active-site cavity is increased to accommodate small molecules.

Fig 1.

(a) The structure of di-Zn(II) DF1: the Leu-13 and Leu-13′ residues block substrate access to the active site. The backbone trace of residues 7–15 and 33–42 is shown, along with Glu-10, Leu-13, Glu-36, and His-39 side chains. (b) The active site of L13A DF1. The decreased steric bulk of Leu-13 and Leu-13′ results in the formation of an active-site pocket, which accommodates a DMSO molecule, the oxygen atom of which bridges between the Mn(II) ions.

Fig 2.

Gdn denaturation curves of DF1, L13A, and L13G. The ellipticity at 222 nm was monitored as a function of the concentration of added denaturant in 10 mM phosphate buffer (pH 5.5). The smooth curves are generated by globally fitting the free energy of dimerization (ΔG_U) in the absence of Gdn, m (δΔG_U/δ[Gdn]) and the baseline parameters to the data as described in Supporting Text. The identity and concentrations of the proteins are as follows: ○, L13G-DF1 3.76 μM; □, L13G-DF1 16 μM; ▪, L13A-DF1 0.9 μM; •, L13A-DF1 12 μM; ♦, DF1 0.78 μM; ▵, DF1 3.7 μM.

Fig 3.

(a) One-dimensional NMR spectrum of DF1 [H₂O/DMSO 90:10 (vol/vol), pH 4.0] at 600 MHz and 298 K. (b) Total correlation spectroscopy in the NH-aliphatic region. Of 47 possible NH–^αCH cross peaks, 45 are observed. (c) NOESY spectrum (120-ms mixing time) in the NH–NH region.

Fig 4.

Stereoview of the superposition of the best 14 minimized structures for apo-DF1.

Fig 5.

Superposition of the di-Zn(II) and apo structures of DF1. The backbone trace of the di-Zn(II) structure is in green, and the side chains are plotted with Corey–Pauling–Koltun colors (C, green; N, blue; O, red). The backbone of apo-DF1 is in yellow, the Glu side chain is in red, and the His residue is in blue. (Right) Helices 1 and 1′, which superpose well between the structures. (Left) A much poorer superposition of helices 2 and 2′ arises from a rotation of the helices about their axes, which increases the exposure of the His and Glu side chains.