Premium
Structural and biophysical analysis of UCH37: ubiquitin recognition and binding to ADRM1 (952.3)
Author(s) -
Morrow Marie,
Kim MyungIl,
Paul Lake,
Ronau Judith,
Das Chittaranjan
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.952.3
Subject(s) - deubiquitinating enzyme , ubiquitin , proteasome , biochemistry , mutant , isothermal titration calorimetry , plasma protein binding , ubiquitin ligase , allosteric regulation , chemistry , protein subunit , binding site , microbiology and biotechnology , biology , biophysics , enzyme , gene
Ubiquitin C‐terminal hydrolase 37 (UCH37) is a deubiquitinating enzyme capable of dismantling Lys48‐linked polyubiquitin chains when associated with the 26S proteasome. Upon binding to the proteasomal subunit ADRM1, UCH37 is activated for cleavage of the distal monomer in a chain, an exo‐specific activity. Prior to binding with ADRM1, it is thought that UCH37 is inhibited by its own C‐terminal UCHL5‐like domain (ULD). Our interests lie in UCH37’s mechanism of activation by ADRM1 as well as its exo‐specificity. Previously we have reported the crystal structure of T. spiralis UCH37 bound to ubiquitin vinyl methyl ester, which revealed that the ULD domain interacts with ubiquitin’s Lys48, an event we believe accounts for its exo‐specificity. In order to further examine specificity and activation, we have generated a series of ULD mutants of UCH37. To probe exo‐specificity, we are purifying the 19S regulatory particle of the proteasome from rabbit skeletal muscle, will reconstitute each ULD mutant within the 19S, and then test their cleavage with an asymmetric triubiquitin substrate. When mutated, ULD residues involved in ubiquitin recognition may lose exo‐specificity against the asymmetric substrate. Additionally, we have analyzed UCH37 and ADRM1’s biophysical interactions through isothermal titration calorimetry, finding that the two bind with 1:1 stoichiometry and a dissociation constant of 22 nM. Due to this tight binding and our structural data, we hypothesize that ADRM1’s activation of UCH37 may not be due to an enhancement of UCH37’s turnover rate, but rather due to an increase in ubiquitin’s k off rate; that is, the ULD merely slows product dissociation. We hope to characterize this phenomenon through surface plasmon resonance studies of ubiquitin binding to ULD mutants in the presence of ADRM1. These findings should not only provide a better understanding of UCH37’s activation and exo‐specificity, but will further probe the role of deubiquitination at the proteasome. Grant Funding Source : Supported by National Institutes of Health Grant 1R01RR026273 (C.D.)