Graduation Year

2024

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Molecular Biosciences

Major Professor

Gary Wayne Daughdrill, Ph.D.

Committee Member

Huzefa Dungrawala, Ph.D.

Committee Member

Ioannis Gelis, Ph.D.

Committee Member

Jiandong Chen, Ph.D.

Keywords

Allovalency, Intrinsically Disordered Proteins, MDMX, Short Linear Motifs, WF motif, WW motif

Abstract

Regulation of protein binding through autoinhibition commonly occurs via interactions involving intrinsically disordered regions (IDRs). These intramolecular interactions can directly or allosterically inhibit intermolecular protein or DNA binding, regulate enzymatic activity, and control the assembly of large macromolecular complexes. Autoinhibitory interactions mediated by protein disorder are inherently transient, making their identification and characterization challenging. In this work, we explore the structural and functional diversity of disorder-mediated autoinhibition for a variety of biological mechanisms, with a focus on the role of multivalency and effective concentration. We also discuss the evolution of disordered motifs that participate in autoinhibition using examples where sequence conservation varies from high to low. In some cases, identifiable motifs that are essential for autoinhibition remain intact within a rapidly evolving sequence, over long evolutionary distances. We discuss some recent examples of autoinhibition regulated by IDRs and describe how multivalency and effective concentration are key features. We also examine methods used to identify and characterize autoinhibitory IDRs and how new tools like AlphaFold2 will facilitate the discovery of IDR-mediated protein autoinhibition.Autoinhibition of p53 binding to MDMX requires two short-linear motifs (SLiMs) containing adjacent tryptophan (WW) and tryptophan-phenylalanine (WF) residues. NMR spectroscopy was used to show the WW and WF motifs directly compete for the p53 binding site on MDMX and circular dichroism spectroscopy was used to show the WW motif becomes helical when it is bound to the p53 binding domain (p53BD) of MDMX. Binding studies using isothermal titration calorimetry showed the WW motif is a stronger inhibitor of p53 binding than the WF motif when they are both tethered to p53BD by the natural disordered linker. We also investigated how the WW and WF motifs interact with the DNA binding domain (DBD) of p53. Both motifs bind independently to similar sites on DBD that overlap the DNA binding site. Taken together our work defines a model for complex formation between MDMX and p53 where a pair of disordered SLiMs bind overlapping sites on both proteins.

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