title: Modulation of the FKBP51 structure by protein engineering and isolation of conformation-locking antibodies and affibodies
Creators: name: Lerma Romero, Jorge Alberto
Publisher: name: UNSPECIFIED
Year: 2024

description: Proteins are biomolecules with an intrinsic flexibility that enables them to carry out their functions by interacting with other proteins, substrates, cells, and many other molecules. The structural flexibility of a protein influences ligand-protein binding, and for some therapeutic targets, this feature can limit the access to its binding site. This presents a constraint for the development of novel molecules to inhibit or activate a target protein.
The FK506 binding protein 51 (FKBP51) is a member of the immunophilin family, and it is linked to several psychiatric and stress-related disorders, among many other reported diseases. Various members of the FKBP family possess an FK1 domain with a highly conserved active site (or orthosteric site). This is not only specific for FKBPs, but it is common to find largely conserved orthosteric sites across protein families. Due to small differences in the amino acid sequence of FKBP51 compared to other members of its family, this protein exhibits higher flexibility than other FKBPs, which translates into a higher number of possible conformers. One of these reported conformers forms a transient binding pocket that can potentially accommodate FKBP51-selective ligands without interacting with other members of this pro-tein family. This doctoral thesis focuses on the development of research tools to shift the conformational ensemble of FKBP51 from the native low-energy state (closed conformation) to a conformer with a sta-bilized transient binding pocket that can bind conformation-specific ligands.
The first part of this work focuses on a protein engineering and screening strategy that was used to successfully identify FKBP51 variants with a favored open conformation of a transient binding pocket. To that end, the amino acids of the FKBP51 FK1 domain were systematically modified by random and Site Saturation Mutagenesis (SSM) to spot FKBP51 muteins that change the distribution of the protein to an open conformation, favoring the binding of two reported conformation-specific ligands. Firstly, random mutagenesis was used to mutate the FK1 domain-encoding gene of FKBP51. With this genetic material, a Yeast Surface Display (YSD) library presenting a pool of mutagenesis variants was created and screened via high throughput Fluorescence-Activated Cell Sorting (FACS). From this first library, a specific region of the protein that destabilizes the FKBP51 binding pocket was identified. Subsequently, an SSM of the identified region spanning glycine-64 to serine-69 was applied, and a second YSD library was created and screened via FACS. From this SSM library, key residues for the destabilization of the FKBP51 binding pocket were identified. Three muteins with enhanced affinity to conformation-specific ligands (G64S, F67E, and D68Y) were characterized by fluorescence polarization and crystallography, and demonstrated to facilitate a cryptic site formation which enables the binding of the open confor-mation ligands.
In subsequent experiments, these three improved FKBP51 variants were used as a starting point to create libraries with multiple mutations to further optimize the affinity to open conformation ligands. Addi-tional SSM libraries based on combinatorial and iterative mutagenesis yielded over 20 FKBP51 variants with two or more mutations that improved ligand binding. Moreover, variants with a mutated position K121 confirmed the importance of this loop region which was not identified in the initial experiments. From all four different libraries, over 30 unique FKBP51 muteins with one or multiple exchanged amino acids were found.
FKBP51 is one of the many proteins with a reported transient binding pocket. Cryptic sites in a protein represent a unique opportunity to design high-affinity ligands that bind selectively to the target protein without interacting with structurally similar proteins and consequently, avoid off-target effects. Our pro-tein engineering-based research used FKBP51 as a model to test the possibilities and potential benefits of protein engineering methods coupled with a cell display system such as yeast surface display (YSD) and FACS to identify variants with enhanced binding to open conformation ligands. These methods were described in detail and are transferable to other proteins that, similarly to FKBP51, exhibit a transient binding pocket. This step-wise methodology can be used to systematically modify a target protein to shift a population from a native state with a low-populated conformer with a cryptic site, to a population with an open conformation that permits the binding of selective ligands.
The second part of this study focused on the use of chicken-derived single-chain variable fragments (scFvs) as conformation-locking molecules to stabilize the open conformation of FKBP51 and facilitate the screening of novel selective inhibitors. An allosteric effector, in this case an scFv, would be able to redistribute the protein conformational ensembles, optimally favoring a low-populated conformation found in the unbound state.
Because of the large evolutionary distance between humans and avians, chickens have a stronger im-mune response to human proteins than conventional murine immunizations to develop antibodies. For this reason, a Gallus gallus specimen was immunized with the FK1 domain of FKBP51. From the splenic RNA of the immunized animal, a pool of scFvs was synthesized and used to create a YSD library. This library was then screened in different campaigns to obtain either high-affinity binders, conformation locking, or blocking scFvs.
While no blocking scFvs were found, the conformation-locking screening campaign generated six differ-ent scFvs, and one of them showed potential allosteric effects on FKBP51 which influenced the binding of canonical and conformation-selective ligands. Multiple fluorescence polarization (FP) and biolayer interferometry (BLI) assays were able to demonstrate the effect of the isolated clone T32, as a conformation-locking scFv. The allosteric effects of T32 were orthogonally assessed by Hydrogen/Deu-terium exchange Mass Spectrometry (HDX-MS), which revealed the binding epitope on FKBP51 and a clear conformational change in the region of the binding pocket.
Moreover, the same goal was pursued by transferring the search of high-affinity binders and allosteric effectors to the screening of affibody molecules. These small molecules are derived from the Z-domain of Staphylococcus aureus. An affibody consists of a 3-helical structure where selected amino acids on two of their helices are randomized to create a synthetic library that acts as an antibody-mimicking molecule able to deliver high-affinity binders for many target proteins. An Escherichia coli display library express-ing affibodies was screened with the FK1 domain of FKBP51. Six screening rounds by magnetic-activated cell sorting (MACS) and FACS yielded four affibodies which bind FKBP51 and one of them with potential effects on ligand intake.
Either by the use of FKBP51 muteins or interactions with an scFv, methods for the stabilization of the transient binding pocket of FKBP51 represent an invaluable set of research tools to characterize the molecular dynamics of the binding pocket and provide an alternative to find new FKBP51 selective lig-ands on a fragment screening basis. Moreover, both scFvs and affibodies as FKBP51 binders have the potential to be conjugated to detection reagents and make them effective resources for the biochemical characterization of FKBP51 selectively. The results presented in this thesis yielded biomolecules with potential applications in the research and characterization of FKBP51 and the search for high affinity and selective ligands that bind to the cryptic pocket of FKBP51.