Gurkan Guntas

Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface

Robust generation of IgG bispecific antibodies has been a long-standing challenge. Existing methods require extensive engineering of each individual antibody, discovery of common light chains, or complex and laborious biochemical processing. Here we combine computational and rational design approaches with experimental structural validation to generate antibody heavy and light chains with orthogonal Fab interfaces. Parental monoclonal antibodies incorporating these interfaces, when simultaneously co-expressed, assemble into bispecific IgG with improved heavy chain–light chain pairing. Bispecific IgGs generated with this approach exhibit pharmacokinetic and other desirable properties of native IgG, but bind target antigens monovalently. As such, these bispecific reagents may be useful in many biotechnological applications.

Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins

Significance Photoactivatable proteins are powerful tools for studying biological processes. Light-induced dimers are especially useful because they can be turned on and off with high spatial and temporal resolution in living systems, allowing for control of protein localization and activity. Here, we develop and apply methods for identifying mutations that improve the effectiveness of a light-induced dimer. The engineered switch is modular, can be used in most organisms, has more than 50-fold change in binding affinity upon light stimulation, and can be used to initiate signaling pathways in a specific region of a cell. The discovery of light-inducible protein–protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.

Engineering a protein–protein interface using a computationally designed library

Computational algorithms for protein design can sample large regions of sequence space, but suffer from undersampling of conformational space and energy function inaccuracies. Experimental screening of combinatorial protein libraries avoids the need for accurate energy functions, but has limited access to vast amounts of sequence space. Here, we test if these two traditionally alternative, but potentially complementary approaches can be combined to design a variant of the ubiquitin-ligase E6AP that will bind to a nonnatural partner, the NEDD8-conjugating enzyme Ubc12. Three E6AP libraries were constructed: (i) a naive library in which all 20 amino acids were allowed at every position on the target-binding surface of E6AP (13 positions), (ii) a semidirected library that varied the same residue positions as in the naive library but disallowed mutations computationally predicted to destabilize E6AP, and (iii) a directed library that used docking and sequence optimization simulations to identify mutations predicted to be favorable for binding Ubc12. Both of the directed libraries showed > 30-fold enrichment over the naive library after the first round of screening with a split-dihydrofolate reductase complementation assay and produced multiple tight binders (Kd < 100 nM) after four rounds of selection. Four rounds of selection with the naive library failed to produce any binders with Kd’s lower than 50 μM. These results indicate that protein design simulations can be used to create directed libraries that are enriched in tight binders and that in some cases it is sufficient to computationally screen for well-folded sequences without explicit binding calculations.

Circular Permutation in the Ω-Loop of TEM-1 β-Lactamase Results in Improved Activity and Altered Substrate Specificity

Generating diverse protein libraries that contain improved variants at a sufficiently high frequency is critical for improving the properties of proteins using directed evolution. Many studies have illustrated how random mutagenesis, cassette mutagenesis, DNA shuffling and similar approaches are effective diversity generating methods for directed evolution. Very few studies have explored random circular permutation, the intramolecular relocation of the N- and C-termini of a protein, as a diversity-generating step for directed evolution. We subjected a library of random circular permutations of TEM-1 β-lactamase to selections on increasing concentrations of a variety of β-lactam antibiotics including cefotaxime. We identified two circularly permuted variants that conferred elevated resistance to cefotaxime but decreased resistance to other antibiotics. These variants were circularly permuted in the Ω-loop proximal to the active site. Remarkably, one variant was circularly permuted such that the key catalytic residue Glu166 was located at the N-terminus of the mature protein.

Protein Switches Identified From Diverse Insertion Libraries Created Using S1 Nuclease Digestion of Supercoiled-Form Plasmid DNA

We demonstrate that S1 nuclease converts supercoiled plasmid DNA to unit‐length, linear dsDNA through the creation of a single, double‐stranded break in a plasmid molecule. These double‐stranded breaks occur not only in the origin of replication near inverted repeats but also at a wide variety of locations throughout the plasmid. S1 nuclease exhibits this activity under conditions typically employed for the nucleases single‐stranded nuclease activity. Thus, S1 nuclease digestion of plasmid DNA, unlike analogous digestion with DNaseI, effectively halts after the first double‐stranded break. This property makes easier the construction of large domain insertion libraries in which the goal is to insert linear DNA at a variety of locations throughout a plasmid. We used this property to create a library in which a circularly permuted TEM1 β‐lactamase gene was inserted throughout a plasmid containing the gene encoding Escherichia coli ribose binding protein. Gene fusions that encode allosteric switch proteins in which ribose modulates β‐lactamase catalytic activity were isolated from this library using a combination of a genetic selection and a screen. Biotechnol. Bioeng. 2011;108: 2535–2543.

Combined computational design of a zinc-binding site and a protein-protein interaction: one open zinc coordination site was not a robust hotspot for de novo ubiquitin binding.

We computationally designed a de novo protein–protein interaction between wild‐type ubiquitin and a redesigned scaffold. Our strategy was to incorporate zinc at the designed interface to promote affinity and orientation specificity. A large set of monomeric scaffold surfaces were computationally engineered with three‐residue zinc coordination sites, and the ubiquitin residue H68 was docked to the open coordination site to complete a tetrahedral zinc site. This single coordination bond was intended as a hotspot and polar interaction for ubiquitin binding, and surrounding residues on the scaffold were optimized primarily as hydrophobic residues using a rotamer‐based sequence design protocol in Rosetta. From thousands of independent design simulations, four sequences were selected for experimental characterization. The best performing design, called Spelter, binds tightly to zinc (Kd < 10 nM) and binds ubiquitin with a Kd of 20 µM in the presence of zinc and 68 µM in the absence of zinc. Mutagenesis studies and nuclear magnetic resonance chemical shift perturbation experiments indicate that Spelter interacts with H68 and the target surface on ubiquitin; however, H68 does not form a hotspot as intended. Instead, mutation of H68 to alanine results in tighter binding. Although a 3/1 zinc coordination arrangement at an interface cannot be ruled out as a means to improve affinity, our study led us to conclude that 2/2 coordination arrangements or multiple‐zinc designs are more likely to promote high‐affinity protein interactions. Proteins 2013; 81:1245–1255.

Engineering a genetically encoded competitive inhibitor of the KEAP1–NRF2 interaction via structure-based design and phage display

In its basal state, KEAP1 binds the transcription factor NRF2 (Kd = 5 nM) and promotes its degradation by ubiquitylation. Changes in the redox environment lead to modification of key cysteines within KEAP1, resulting in NRF2 protein accumulation and the transcription of genes important for restoring the cellular redox state. Using phage display and a computational loop grafting protocol, we engineered a monobody (R1) that is a potent competitive inhibitor of the KEAP1-NRF2 interaction. R1 bound to KEAP1 with a Kd of 300 pM and in human cells freed NRF2 from KEAP1 resulting in activation of the NRF2 promoter. Unlike cysteine-reactive small molecules that lack protein specificity, R1 is a genetically encoded, reversible inhibitor designed specifically for KEAP1. R1 should prove useful for studying the role of the KEAP1-NRF2 interaction in several disease states. The structure-based phage display strategy employed here is a general approach for engineering high-affinity binders that compete with naturally occurring interactions.

Redesigning the NEDD8 pathway with a bacterial genetic screen for ubiquitin-like molecule transfer.

Pathways of ubiquitin-like (UBL) molecule transfer regulate a myriad of cellular cascades. Here, we report a high-throughput assay that correlates catalytic human NEDD8 transfer to bacterial survival. The assay was utilized to screen mutant NEDD8 and NAE (NEDD8-activating enzyme) libraries to engineer a more stable NEDD8 and redesign the NEDD8-NAE interface. This approach will be useful in understanding the specificities underlying UBL pathways.