Journal: ACS Omega

Article Title: Conformation-Specific Design: Engineering Extracellular Signal-Regulated Kinase 2 Variants with Bias toward Active or Inactive States

doi: 10.1021/acsomega.6c01185

Figure Lengend Snippet: CSDesign applied to ERK2. The CSDesign computational approach shown is used to identify sequences predicted to most likely fold into the active Holo conformation of ERK2 while biased against folding into the alternative inactive Apo structure. The reciprocal function was also applied to predict sequences most likely to remain in the Apo inactivated state and biased against the active Holo conformation. The activation-loop is designated by color in the magnified boxes.

Article Snippet: All four ERK2 constructs were synthesized and cloned by TWIST Bioscience (San Francisco, CA) into the pJEx411c expression vector containing a kanamycin resistance marker.

Techniques: Activation Assay

Journal: ACS Omega

Article Title: Conformation-Specific Design: Engineering Extracellular Signal-Regulated Kinase 2 Variants with Bias toward Active or Inactive States

doi: 10.1021/acsomega.6c01185

Figure Lengend Snippet: Design and functional characterization of ERK2 variants. (A) Table of ERK2 wild-type sequence and select variants indicating their nomenclature, design objective, and purification success. (B) Kinase activity assay (ADP-Glo) of computationally designed ERK2 constructs. Kinase activity is reported as a percent conversion of ATP to ADP over the course of the assay. The control represents samples lacking ERK2. All reactions were performed in triplicate; error bars represent the mean ± SEM.

Article Snippet: All four ERK2 constructs were synthesized and cloned by TWIST Bioscience (San Francisco, CA) into the pJEx411c expression vector containing a kanamycin resistance marker.

Techniques: Functional Assay, Sequencing, Purification, Kinase Assay, Construct, Activity Assay, Control

Journal: ACS Omega

Article Title: Conformation-Specific Design: Engineering Extracellular Signal-Regulated Kinase 2 Variants with Bias toward Active or Inactive States

doi: 10.1021/acsomega.6c01185

Figure Lengend Snippet: Structural comparison of CSD103 and CSD104 with wild-type ERK2. (A) Superposition of CSD103 (cyan) with wild-type ERK2 (magenta; PDB ID: 2ERK ). The activation-loop (A-loop) is highlighted in blue for CSD103 and in orange for ERK2, illustrating local conformational differences. (B) Superposition of CSD104 (blue) with wild-type ERK2 (magenta). The A-loop is shown in green for CSD104 and orange for ERK2, highlighting the loop similarity. (C) Left panel: The A-loop of the AF3 model of CSD104 shows a potential disulfide bridge between the A-loop and α-loop 3, along with hydrophobic interactions involving Val138, Phe164, Leu176, and Tyr179. This covalent linkage may help stabilize the active conformation, consistent with the observed innate kinase activity of CSD104. ATP (purple sticks) and the coordinated Mg 2+ ions (purple spheres) were added manually for visualization. Right panel: The crystal structure of wild-type active ERK2 (PDB ID: 2ERK ) highlights the A-loop conformation, where Thr183 stabilizes the loop via ionic interactions with Arg146 and Arg170 (shown as sticks). ATP (purple sticks) and the coordinated Mg 2+ ions (purple spheres) were added manually for visualization and are not present in the original 2ERK structure.

Article Snippet: All four ERK2 constructs were synthesized and cloned by TWIST Bioscience (San Francisco, CA) into the pJEx411c expression vector containing a kanamycin resistance marker.

Techniques: Comparison, Activation Assay, Activity Assay