CRL3 and CRL4 are functionally diverse ubiquitination complexes that regulate fundamental cell processes from human DNA damage repair to plant pathogen immunity

Thesis (Ph.D.)--University of Washington, 2017Ubiquitination is a mechanism used by eukaryotes to precisely alter the level, functionality, and location of specific target proteins through the post-translational attachment of one or more ubiquitin tags. This attachment proceeds through a three-step enzymatic process involving ubiquitin activating proteins, conjugating proteins, and ligases. Of these three steps, the final ubiquitin ligation reaction to the target substrate shows the most variability due to the vast number of different substrates targeted by the cell. These ligase complexes are broadly divided into three families, with the Cullin-RING ligases (CRLs) comprising the largest physical structures. CRLs can be further subdivided into different complexes based on the specific Cullin backbone (1-5, 7) used to bridge the complex. Among the many roles these complexes play, CRL3 has been implicated in the oxidative stress response in animals (Nguyen et al., 2004) as well as hormone signaling perception in plants (Lechner et al., 2011). CRL4 has been shown to be involved in a variety of cellular processes such as DNA damage repair and cell cycle regulation (Jackson and Xiong, 2009; Scrima et al., 2011). CRLs perform these functions by employing a modular architecture whereby individual substrate receptors are recruited to the complex to provide specificity for an array of possible targets. The large variety of target substrates these complexes ubiquitinate presents the technical challenge of identifying them in the context of their individual pathways. It also points to the need for a cell to be able to precisely regulate these processes. Here we solve the crystal structure of DDB1, an adaptor component of CRL4, in complex with an N-terminal minimal binding fragment of DDA1. DDA1 has previously been shown to regulate CRL4 activity (Pick et al., 2007; Gao et al., 2017). We hypothesize this regulation is due to conformational control of the flexible BPB domain of DDB1, which normally confers a large degree of freedom to the alignment of the bound substrate with the incoming ubiquitin tag. We also show that CRL3 is involved in perception of the plant hormone salicylic acid (SA) via the hormone receptor NPR4, which binds to radiolabeled SA over a size-exclusion chromatography column. Finally, we show that NPR4 can be crystallized. We have generated a variety of monoclonal antibodies that bind to NPR4 to improve crystal packing and diffraction resolution. Taken together, these studies highlight the important role that CRLs play in eukaryotic biology as well as the importance of efficient regulatory regimes to fine-tune their activity