Date of Award

Summer 8-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

First Advisor

Innokentiy Maslennikov

Second Advisor

Elshahawi Sherif

Third Advisor

Kaur Kamaljit

Fourth Advisor

Dintzner Madeline

Abstract

Ubiquitin signaling plays a central role in orchestrating cellular processes and regulating dynamic molecular interactions. Consequently, dissecting the mechanisms that govern diverse biological pathways relies heavily on our ability to interpret the complexity of ubiquitin modifications. A persistent challenge in the field is the limited understanding of how cells interpret heterotypic ubiquitin polymers, mixed or branched chains that encode multilayered regulatory information beyond that of homotypic linkages.

To address this challenge, in this study we engineered a chimeric polypeptide probe by fusing ubiquitin-interacting motif(UIM) domains from two distinct ubiquitin-binding proteins and optimizing the linker that connects the UIMs. Our initial construct, RA20, exhibited selective recognition of heterotypic tri-ubiquitin chains with K48 and K63 linkages, both in mixed and in branched architectures with comparable affinity. Notably, its specificity for mixed chains depended on the sequential arrangement of linkages, underscoring the importance of chain topology.

Through a combination of mutational analyses, surface plasmon resonance (SPR), NMR spectroscopy, and structural modeling, we identified key residues mediating RA20’s interaction with the distal K48-linked ubiquitin within tri-ubiquitin assemblies. Docking studies performed using HADDOCK further revealed structural constraints that differentiate RA20’s binding modes for mixed versus branched chains.

Guided by these insights, we rationally designed an improved variant, △4RA20, featuring a shortened inter-UIM linker. This optimized probe displays increased affinity (KD ≈ 1.4 μM) for K48-K63 branched tri-ubiquitin chains and exhibits a roughly tenfold enhancement in selectivity comparing to the mixed K48-K63 tri-ubiquitin chains. Collectively, △4RA20 not only offers an effective tool for decoding the molecular recognition principles of heterotypic ubiquitin architectures but also establishes a strategic foundation for designing next-generation probes tailored to interrogate diverse polyubiquitin topologies.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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