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 NEDD8-activating enzyme (NAE) 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

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

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

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

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

MIC of beta-lactam antibiotics conferred by BLA and variants of BLA.

a<p>at 37°C.</p>b<p>at 22°C.</p

Figure 2

<p>Sites of circular permutation BLA based on DNA sequencing of (A) 25 randomly selected members from the naïve library, (B) 20 randomly selected members capable of growing on plates containing 16 µg/ml ampicillin, and (C) 10 randomly selected members capable of growing on plates containing 250 µg/ml ampicillin. The first amino acid in the mature wild-type BLA is 24 (since the signal sequence of amino acids 1–23 is removed) and the last amino acid is 286. The linker joining the N- and C-termini has the amino acid sequence DKS.</p

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 (K(d) = 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 K(d) 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 NEDD8-activating enzyme (NAE) 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