Mapping synthetic binding proteins epitopes on diverse protein targets by protein structure prediction and protein-protein docking.

Synthetic binding proteins (SBPs) are a class of artificial proteins engineered from privileged protein scaffolds, which can form highly specific molecular recognition interfaces with a variety of targets. Due to the characteristics of small size, high stability, and good tissue permeability, SBPs have important applications in biomedical research, disease diagnosis and treatment. However, knowledge of SBPs epitopes on the structures of target proteins is still limited, which hinder the development of novel SBPs. In this study, based on the currently available information of SBPs and their targets, 96 pairs of interacting proteins referring to 96 representative SBPs and 80 different targets, were systemically investigated using the state-of-the-art computational modeling techniques including AlphaFold2 protein structure prediction and Rosetta protein-protein docking. As a result, 71 out of the 96 pairs were successfully docked, of which 18, 33, and 20 pairs were defined as models with high, medium, and acceptable quality, respectively. In addition, the interface information was analyzed to decipher the interaction types driven SBPs and targets recognition. Overall, this work not only provides important structural information for understanding the mechanism of action of other SBPs with same protein scaffold, but also for aiding the rational protein engineering and to design of novel SBPs with biomedical applications.

Biosensors Show the Pharmacokinetics of S-Ketamine in the Endoplasmic Reticulum.

The target for the "rapid" (<24 h) antidepressant effects of S-ketamine is unknown, vitiating programs to rationally develop more effective rapid antidepressants. To describe a drug's target, one must first understand the compartments entered by the drug, at all levels-the organ, the cell, and the organelle. We have, therefore, developed molecular tools to measure the subcellular, organellar pharmacokinetics of S-ketamine. The tools are genetically encoded intensity-based S-ketamine-sensing fluorescent reporters, iSKetSnFR1 and iSKetSnFR2. In solution, these biosensors respond to S-ketamine with a sensitivity, S-slope = delta(F/F0)/(delta[S-ketamine]) of 0.23 and 1.9/µM, respectively. The iSKetSnFR2 construct allows measurements at <0.3 µM S-ketamine. The iSKetSnFR1 and iSKetSnFR2 biosensors display >100-fold selectivity over other ligands tested, including R-ketamine. We targeted each of the sensors to either the plasma membrane (PM) or the endoplasmic reticulum (ER). Measurements on these biosensors expressed in Neuro2a cells and in human dopaminergic neurons differentiated from induced pluripotent stem cells (iPSCs) show that S-ketamine enters the ER within a few seconds after appearing in the external solution near the PM, then leaves as rapidly after S-ketamine is removed from the extracellular solution. In cells, S-slopes for the ER and PM-targeted sensors differ by <2-fold, indicating that the ER [S-ketamine] is less than 2-fold different from the extracellular [S-ketamine]. Organelles represent potential compartments for the engagement of S-ketamine with its antidepressant target, and potential S-ketamine targets include organellar ion channels, receptors, and transporters.

Computational design of a protein-based enzyme inhibitor.

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.