DOI

10.17077/etd.8lgx-dt8x

Document Type

Dissertation

Date of Degree

Spring 2019

Access Restrictions

Access restricted until 07/29/2021

Degree Name

PhD (Doctor of Philosophy)

Degree In

Biochemistry

First Advisor

Fuentes, Ernesto J.

Second Advisor

Spies, Maria

First Committee Member

Washington, M. Todd

Second Committee Member

Schnieders, Michael J.

Third Committee Member

Musselman, Catherine A.

Fourth Committee Member

Smith, Richard J.

Abstract

PSD-95/Dlg/ZO-1 (PDZ) domain - PDZ binding motif (PBM) interactions have been one of the most well studied protein-protein interaction systems through biochemical, biophysical and high-throughput screening (HTS) strategies. This has allowed us to understand the mechanism of individual PDZ-PBM interactions and the re-engineering of PBMs to bind tighter or to gain or lose certain specificity. However, there are several thousand native PDZ domains whose biological ligands remain unknown. Because of the low sequence identity among PDZ domain homologues, promiscuous binding profiles (defined as a PDZ domain that can accommodate a set of PBMs or a PBM that can be recognized by many PDZ domains), and context-dependent interaction mechanism, we have an inadequate understanding of the general molecular mechanisms that determine the PDZ-PBM specificity. Therefore, predicting PDZ specificity has been elusive. In addition, no de novo PBM ligand or artificial non-native PDZ domain have been successfully designed. This reflects the general challenges in understanding the general principles of PDZ-ligand interactions, namely that they are context-dependent, exhibit weak binding affinity, narrow binding energy range, and larger interaction surface than other protein-ligand interactions. Together, PDZ domains make good model systems to investigate the fundamental principles of protein-protein interactions with a wide spectrum of biomedical implications.

My studies suggest that understanding PBM specificity with the set of structural positions forming the binding pocket can connect sequence, structure and function of a PDZ domain in a general context. They also suggest that this way of understanding the specificity will shed light on prediction and engineering of specificity rationally. Structural analysis on most of the available PDZ domain structures was established to support the principle (Chapter I). The principle was tested against two different types of PBM; C-terminal PBM (Chapter II) and internal PBM (Chapter III), and shown to support better understanding and design of PDZ domain specificity. We further applied the principle to design de novo PDZ domains, and the preliminary data hints that it is optimistic to engineer PDZ domain specificity (Appendix A and B).

Keywords

Artificial Protein Design, Domain Specificity, PDZ Domain, Protein Engineering, Protein-protein Interaction

Pages

xv, 191 pages

Bibliography

Includes bibliographical references (pages 174-191).

Copyright

Copyright © 2019 Young Joo Sun

Available for download on Thursday, July 29, 2021

Included in

Biochemistry Commons

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