A minimal sequence code for switching protein structure and function

Abstract

We present here a structural and mechanistic description of how a protein changes its fold and function, mutation by mutation. Our approach was to create 2 proteins that (i) are stably folded into 2 different folds, (ii) have 2 different functions, and (iii) are very similar in sequence. In this simplified sequence space we explore the mutational path from one fold to another. We show that an IgG-binding, 4beta+alpha fold can be transformed into an albumin-binding, 3-alpha fold via a mutational pathway in which neither function nor native structure is completely lost. The stabilities of all mutants along the pathway are evaluated, key high-resolution structures are determined by NMR, and an explanation of the switching mechanism is provided. We show that the conformational switch from 4beta+alpha to 3-alpha structure can occur via a single amino acid substitution. On one side of the switch point, the 4beta+alpha fold is >90% populated (pH 7.2, 20 degrees C). A single mutation switches the conformation to the 3-alpha fold, which is >90% populated (pH 7.2, 20 degrees C). We further show that a bifunctional protein exists at the switch point with affinity for both IgG and albumin.

Fig. 1.

G_A77 in complex with HSA (from 1TFO.pdb) (36) and G_B77 in complex in IgG (from 1fcc.pdb) (37). The side chains of amino acids at the 13 positions of nonidentity are depicted as yellow sticks.

Fig. 2.

Sequence alignment for 3-α G_A proteins (Top) and 4β+α G_B proteins (Bottom) described in the text. Secondary structure regions for G_A95 and G_B95 are shown at the top and bottom of the alignment, respectively. The 13 nonidentities between G_A77 and G_B77 are shown in cyan, the 7 nonidentities between G_A88 and G_B88 in green, the 4 nonidentities between G_A91 and G_B91 in blue, the 3 nonidentities between G_A95 and G_B95 yellow, and the single amino acid difference between G_A98 and G_B98 at residue 45 in red.

Fig. 3.

¹H, ¹⁵N HSQC spectra. Main chain amide assignments are shown for G_A98 (black) and G_B98 (red). Of the 56 aa in these 2 proteins, 55 are identical but have different chemical environments reflecting the 2 different folds. Amide proton signals for side chains are connected by the horizontal lines.

Fig. 4.

Cartoon depiction of backbone topology of G_A95 and G_B95. Residues 1–8 are blue, 9–23 are green, 24–37 are red, 38–52 are yellow, and 53–56 are cyan.

Fig. 5.

Ensemble of 20 NMR structures for (A) G_A95 (residues 7–52) and (B) G_B95 (residues 1–56). Backbone RMSDs are ≈0.5 Å for both structures. Full structure statistics are in Table S2. Ordered core residues are shown in cyan, whereas the 3 amino acid differences between G_A95 and G_B95 are highlighted in red. Note that L20 is ordered in the hydrophobic core of G_A95, whereas I30 and L45 are more disordered and solvent accessible. Conversely, F30 and Y45 contribute significantly to stabilization of the core in G_B95.

Fig. 6.

Switching mechanism. Alternative conformations of the N-terminal (orange) and C-terminal (blue) amino acids in the 3-α and 4β+α folds. The critical switch amino acid occurs at position 45 (red). Also depicted are the hydrophobic packing of the N- and C-terminal amino acids in the core of the 4β+α fold.