Engineering of a Small Protein Scaffold To Recognize Sulfotyrosine with High Specificity.

Protein tyrosine O-sulfation is an essential post-translational modification required for effective biological processes such as hemostasis, inflammatory response, and visual phototransduction. Because of its unstable nature under mass spectrometry conditions and residing on low-abundance cell surface proteins, sulfated tyrosine (sulfotyrosine) residues are difficult to detect or analyze. Enrichment of sulfotyrosine-containing proteins (sulfoproteins) from complex biological samples are typically required before analysis. In this work, we seek to engineer the phosphotyrosine binding pocket of a Src Homology 2 (SH2) domain to act as an antisulfotyrosine antibody mimic. Using tailored selection schemes, several SH2 mutants are identified with high affinity and specificity to sulfotyrosine. Further molecular docking simulations highlight potential mechanisms supporting observed characteristics of these SH2 mutants. Utilities of the evolved SH2 mutants were demonstrated by the detection and enrichment of sulfoproteins.

Functional genetic encoding of sulfotyrosine in mammalian cells.

Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

Engineering of a sulfotyrosine-recognizing small protein scaffold for the study of protein tyrosine O-sulfation.

Protein tyrosine O-sulfation is considered as one of the most common types of posttranslational modification of tyrosine in nature. The introduction of a negatively charged sulfate group plays crucial roles in extracellular biomolecular interactions that dictate various cellular processes, including cell adhesion, leukocyte trafficking, hormone activities, and immune responses. Despite substantial advances in our knowledge about protein tyrosine O-sulfation in recent years, our understanding of its biological significance is still in its infancy. This is largely hindered by a chronic lack of suitable biochemical tools. We seek to meet this challenge by engineering a small protein scaffold that can recognize sulfated tyrosine (sulfotyrosine) residues with high affinity. In this chapter, we describe the directed evolution of a Src Homology 2 (SH2) domain to recognize sulfotyrosine. In the first part, the design strategy for the phage display of SH2 variants is discussed. In the second part, the techniques required for phage propagation and selection are described. The evolved SH2 variants are characterized and validated in vitro through fluorescence polarization assays. Finally, the evolved SH2 domain mutants are applied to the visualization of sulfated proteins on the cell surface.

A high throughput approach for the generation of orthogonally interacting protein pairs.

In contrast to the nearly error-free self-assembly of protein architectures in nature, artificial assembly of protein complexes with pre-defined structure and function in vitro is still challenging. To mimic nature's strategy to construct pre-defined three-dimensional protein architectures, highly specific protein-protein interacting pairs are needed. Here we report an effort to create an orthogonally interacting protein pair from its parental pair using a bacteria-based in vivo directed evolution strategy. This high throughput approach features a combination of a negative and a positive selection. The newly developed negative selection from this work was used to remove any protein mutants that retain effective interaction with their parents. The positive selection was used to identify mutant pairs that can engage in effective mutual interaction. By using the cohesin-dockerin protein pair that is responsible for the self-assembly of cellulosome as a model system, we demonstrated that a protein pair that is orthogonal to its parent pair could be readily generated using our strategy. This approach could open new avenues to a wide range of protein-based assembly, such as biocatalysis or nanomaterials, with pre-determined architecture and potentially novel functions and properties.

Deciphering molecular details in the assembly of alpha-type carboxysome.

Bacterial microcompartments (BMCs) are promising natural protein structures for applications that require the segregation of certain metabolic functions or molecular species in a defined microenvironment. To understand how endogenous cargos are packaged inside the protein shell is key for using BMCs as nano-scale reactors or delivery vesicles. In this report, we studied the encapsulation of RuBisCO into the α-type carboxysome from Halothiobacillus neapolitan. Our experimental data revealed that the CsoS2 scaffold proteins engage RuBisCO enzyme through an interaction with the small subunit (CbbS). In addition, the N domain of the large subunit (CbbL) of RuBisCO interacts with all shell proteins that can form the hexamers. The binding affinity between the N domain of CbbL and one of the major shell proteins, CsoS1C, is within the submicromolar range. The absence of the N domain also prevented the encapsulation of the rest of the RuBisCO subunits. Our findings complete the picture of how RuBisCOs are encapsulated into the α-type carboxysome and provide insights for future studies and engineering of carboxysome as a protein shell.