Smoothing a rugged protein folding landscape by sequence-based redesign

Abstract

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α₁-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.

Figure 1. Conserpin conforms to the serpin fold and has superior biophysical properties compared with α1-AT.

(A) Cartoon representation of the 2.4 Å X-ray crystal structure of native conserpin, identifying the breach and shutter regions, the A, B and C sheets (colored in red, green and yellow respectively), and the RCL stumps (magenta). (B) Structural alignment of conserpin (grey) with α1-AT (PDB: 3NE4; spectrum, blue to red). Root mean square deviation (RMSD) = 0.91 Å across 296 backbone Cα atoms. Chemical refolding of (C) α1-AT and (D) conserpin shows that conserpin can refold to a monomer. Chromatograms from a Superdex 75 10/300 size exclusion column are shown. Final protein concentrations loaded onto column were 2 μM. Samples were unfolded in 5 M GuHCl and then diluted out to 0.5 M GuHCl (dotted line). Control samples of native protein are shown as the solid black line. (E) Intrinsic fluorescence equilibrium unfolding (red dots) and refolding (blue diamonds) curves of conserpin coincide, demonstrating reversible folding. (F) CD spectral scans of conserpin before (solid blue line) and after (dashed red line) heating to 110 °C. Variable temperature thermal melts of (G) α1-AT and (H) conserpin as measured by CD at 222 nm. (I) Conserpin shows a significant reduction of intermediate formation during bis-ANS fluorescent equilibrium unfolding of α1-AT (blue circles) and conserpin (green triangles). (J) Kinetic unfolding and refolding experiments. The plot shows the [GuHCl]-dependence of the natural logarithm of the rate constants for unfolding and refolding of conserpin (chevron plot). Two discernable refolding rates are observed (red squares, fast rate; black circles, slower folding rate). The positive slope in each refolding arm suggests the presence of intermediate species that have to partially unfold to reach the native state.

Figure 2. Structural analysis reveals alterations of the electrostatic surface and stabilization of the D-helix in conserpin.

(A) The electrostatic potential surface of conserpin and α1-AT models (blue =  +ve, red = −ve), in the same orientation as Fig. 1A (front) and a 180° rotation reveals an overall increase in positive charge on the back face of conserpin. (B) The introduced salt bridge in hD of conserpin with residues Q105R₇₉ and E376₃₄₆. There is no comparable interaction present in α1-AT. Inset shows the shortened D-helix in conserpin. (C) H-bonding between A-1 of the extended N-terminus and D65 of hD, as seen in the conserpin crystal structure. (D) Persistent hydrogen bonding between Q-4, G-3 and A-1 of the extended N-terminus and E63 and D65 of hD in conserpin as seen in MD simulation.

Figure 3. The electrostatic network of the breach region is extended in conserpin.

(A) A-sheet salt bridge interactions (dashed lines) in the crystal structures of conserpin (carbon atoms in grey) and α1-AT (carbon atoms in wheat; PDB: 3NE4). (B) A simulation snapshot taken at 500 ns, showing A-sheet salt bridge interactions as described above. The modeled RCL of conserpin is colored magenta.

Figure 4. W160 stabilizes hF in conserpin.

(A) A structural overlay of hF in conserpin (grey) and α1-AT (wheat), highlighting the positions of Y160W₁₃₂, Y187A₁₅₉ and G115A₈₈. (B) Solvent inaccessible cavities (red blobs) surrounding hF of conserpin and α1-AT. Y160W₁₃₂ reduces cavity volumes from 233.8 to 120.9 ų. (C) MD simulation frames (every 50 ns), highlighting the dynamic differences of W132 in conserpin and Y160 in α1-AT.

Figure 5. Structural analysis of the B/C barrel in conserpin (grey) and α1-AT (wheat).

(A) Stabilizing hydrophobic mutations surrounding F275W₂₄₇. (B) Remodeling of the inner barrel surrounding W238K₂₁₀.

Figure 6. Configurational frustration analysis for conserpin and α1-AT.

Minimal, neutral and highly frustrated contacts are represented in green, gray and red respectively. Calculations were performed with different electrostatic strengths by varying the electrostatic constant (k). According to ref , larger k values are related to stronger effects of the Debye–Hückel term.