Computational design of a protein-based enzyme inhibitor

Abstract

While there has been considerable progress in designing protein-protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding.

Keywords: BSA; FACS; HA; HEL; MW; PAK1; PBS; PDB; Protein Data Bank; Rosetta molecular modeling program; SEC; VNAR; bovine serum albumin; fluorescence-activated cell sorting; hemagglutinin; hen egg lysozyme; hot spot; molecular weight; p21-activated kinase 1; phosphate-buffered saline; protein engineering and design; protein–protein interactions; size-exclusion chromatography; variable new antigen receptor.

Fig. 1

A HEL-binding protein built using a dock-and-design strategy makes interactions inconsistent with the computational model. (a) Outline of the computational design strategy. Residues within the HEL active site were selected to be part of the target interaction surface (i). A curated set of 865 protein structures (referred to as scaffolds) were individually docked to the target site using PatchDock (ii). Docked configurations were refined using ROSETTA, followed by sequence design of the scaffold protein within 8 Å of the interface to minimize the assembly's energy (iii). Designed protein-HEL complexes were filtered by interface metrics. (b) Designed proteins were expressed on the yeast surface with a C-terminal myc epitope tag detected with a FITC-conjugated antibody (x-axis). Biotinylated HEL (1 μM) was premixed with phycoerythrin (PE)-conjugated streptavidin (0.5 μM) to form an oligomeric/avid complex, which was incubated with the yeast cells to detect surface interactions (y-axis) by flow cytometry. Shown are yeast display data for HEL-binding design DnvLB16. An identical analysis with negative control proteins (biotinylated IgG and biotinylated Mycobacterium tuberculosis acyl-carrier protein, MycoACP) failed to show interactions with DnvLB16. (c) Designed residues on DnvLB16 were mutated back to their original amino acid identities in the starting scaffold, and binding to avid HEL was tested as above. (d) DnvLB16-M40R;W41H;H42S with improved expression and solubility (Supplementary Fig. S1) was evolved. The yeast display library consisted of 1 × 10⁶ transformants with 0 to ~5 amino acid substitutions per DnvLB16 clone, and was sorted for 3 rounds. For each round, cells were stained with 50 nM monomeric HEL followed by PE-streptavidin after washing off unbound ligand. Plasmids from improved HEL-binding mutants were isolated and the DnvLB16 gene sequenced. Shown is a region of the sequence alignment for isolated clones (designated cl.X). (e) The designed HEL-interaction site is shown with a black dashed line on a surface representation of DnvLB16. Positions of reversion mutations that maintained binding (blue) or lost binding (red) with HEL are colored. Positions identified as important for high affinity binding by directed evolution are colored orange.

Fig. 2

Construction of a HEL-binding protein by transplanting hot spot residues and computationally designing the surrounding interface. (a) Schematic of the design process. Hot spot residues R100 and Y101 (magenta sticks) were taken from shark VNAR (cyan cartoon) bound to HEL (green cartoon) (i). Rotamers for the disembodied hot spot residues compatible with the binding geometry are enumerated (ii). Then, protein scaffolds are docked against the target surface using PatchDock and ROSETTA with a modified energy function that biases towards backbone overlap between scaffold and hot spots. Inverse hot spot rotamers are placed sequentially on the scaffold backbone (iii) and the surrounding surface of the scaffold in contact with HEL is redesigned with ROSETTA to minimize the total energy (iv). Designs are filtered by multiple criteria to assess interface quality. (b) Binding of HtsptLB12 to HEL assessed by yeast display and flow cytometry using protocol described in Figure 1B legend. HtsptLB12 binds HEL but not IgG or MycoACP. (c) The grafted hot spot residues, R45 and Y46 of HtsptLB12, were mutated back to their original identities in the scaffold protein, or to charged glutamates. Binding of the mutants to HEL was assessed as in panel B.

Fig. 3

Directed evolution of HtsptLB12 reveals affinity-enhancing mutations at the interface periphery. (a) HtsptLB12 was diversified by error-prone PCR, a yeast display library sorted and affinity enhancing mutations S50A and K52M identified (Supplementary Figure S4). Shown at top is the mutated region in the computational model, with HEL in green and HtsptLB12 in orange. S50 and K52 are highlighted with cyan spheres. Below are the apparent dissociation constants of targeted HtsptLB12 mutants, determined by titrating monomeric HEL and measuring binding signals to HtsptLB12-expressing yeast (n = 3-4, excluding the K_D for parental HtsptLB12, which was determined from a single titration series due to limited reagent). ND, not determined due to too low affinity. (b) A second round of directed evolution was applied to a library of HtsptLB12.v1 mutants. Three mutations improved affinity (S19Y, Q22R and K67X, where X is any of several amino acids), shown at left as cyan spheres on the modeled structure of HtsptLB12-HEL (orange and green, respectively). To the right are the apparent dissociation constants of HtsptLB12.v1 mutants determined by yeast display (n = 4-5).

Fig. 4

HtsptLB12 has correct unbound backbone structure and interacts with HEL at the designed surface. (a) The crystal structure of HtsptLB12.v1 (two copies A, blue, and B, purple, in the asymmetric unit) is superposed with the computational model of unbound HtsptLB12 (orange). (b) Zoomed in region of the modeled HtsptLB12 (orange) and HEL (green) complex encompassing the hot spot residues. The crystal structure of unbound HtsptLB12.v1 (crystal chains A and B are blue and purple) is overlaid with HtsptLB12 in the computational model. The hot spot residue conformations are very close to the design model; this is particularly notable for the conformationally flexible arginine of chain A. (c) HEL (14 kD, 20 nmol, grey line) elutes as a higher MW complex (black line) from a size exclusion chromatography column when mixed with purified HtsptLB12.v1 (20 kD, 20 nmol, orange line). A MW standard, bovine carbonic anhydrase (CA, 29 kD, 20 nmol), is shown as a blue line for comparison. HEL elutes anomalously from dextran-based gel filtration resins with an apparent MW of 7 kD. Results are representative of two repeats. (d) 4-Fluorophenylalanine was incorporated in to HtsptLB12.v2 for ¹⁹F NMR studies. Shown is the computational model of HtsptLB12 (orange cartoon) bound to HEL (grey surface), with the nine phenylalanines of HtsptLB12 as red sticks and the fluorine-substituted para-positions as red spheres. The three phenylalanines of HtsptLB12 in or near the interface are labeled. (e) ¹⁹F NMR spectra of fluorophenylalanine-substituted HtsptLB12.v2 (100 μM), titrated with HEL (0 to 200 μM). The titration spectra are overlaid to highlight the appreciable differences in the intensities and frequencies of ¹⁹F resonances, assigned to F17 and F40, both of which are in or near the HEL binding site. The increase in the ¹⁹F resonance intensity of F125 is attributed to degeneracy of this resonance with one or both of F17 and F40 in the complex.

Fig. 5

A binding fitness landscape indicates that interface residues surrounding the grafted hot spots are critical for activity. (a) Fifty-three surface positions of HtsptLB12.v2 were chosen for single site-saturation mutagenesis. Mutants were selected by yeast display and one round of FACS after incubation with 4 nM HEL. The number of transformants in the yeast library was 1.5 × 10⁶, sufficient for the 1,696 unique DNA sequences. Cells falling within the top 1.5% of events measured by binding signal relative to protein expression were collected, plasmid DNA harvested and sequenced. The log₂ enrichment ratio for each amino acid substitution is plotted from -3.5 (i.e. depleted, orange) to +3.5 (i.e. enriched, blue). Residues within 10 Å of the designed interface are in red text. *, stop codon. (b-d) Regions of the computationally designed interface discussed in main text. HEL is green and HtsptLB12 is orange.

Fig. 6

The designed binding surface is conserved during in vitro evolution. Sequence conservation measured by Shannon entropy is mapped on to the surface of HtsptLB12. Entropy color scale is from ≤ 3.2 (blue) to 4.3 (red).

Fig. 7

HtsptLB12 inhibits lysozyme activity. (a) A HtsptLB12.v2 variant library was constructed combining multiple affinity-enhancing mutations. The yeast display library (containing 2 × 10⁶ transformants) was sorted for four rounds with increasing stringency: 2 nM HEL incubation in round 1, 0.5 nM HEL in round 2, and 0.2 nM HEL for rounds 3-4. Twenty clones from the final enriched population were sequenced, and the proportion of sequences with a particular amino acid at each of the diversified positions is tabulated (middle column). The most abundant clone sequence, representing 65% of the final enriched population, is shown at right. (b) Yeast display titration curves. HtsptLB12 variants were expressed on the yeast surface, and cells were incubated with monomeric HEL at the indicated concentrations. HEL binding signal is detected by flow cytometry in the FL2 fluorescence channel. (c) Cell wall hydrolysis of a M. lysodeikticus suspension by 500 nM HEL is inhibited by HtsptLB12 variants.