The mechanism used by the ubiquitin-conjugating enzyme, Ubc13, to catalyze ubiquitination is probed with three computational techniques: Born-Oppenheimer molecular dynamics, single point quantum mechanics/molecular mechanics energies, and classical molecular dynamics. These simulations support a long-held hypothesis and show that Ubc13-catalyzed ubiquitination uses a stepwise, nucleophilic attack mechanism. Furthermore, they show that the first step-the formation of a tetrahedral, zwitterionic intermediate-is rate limiting. However, these simulations contradict another popular hypothesis that supposes that the negative charge on the intermediate is stabilized by a highly conserved asparagine (Asn79 in Ubc13). Instead, calculated reaction profiles of the N79A mutant illustrate how charge stabilization actually increases the barrier to product formation. Finally, an alternate role for Asn79 is suggested by simulations of wild-type, N79A, N79D, and H77A Ubc13: it stabilizes the motion of the electrophile prior to the reaction, positioning it for nucleophilic attack.
We present classical molecular dynamics (MD), Born-Oppenheimer molecular dynamics (BOMD), and hybrid quantum mechanics/molecular mechanics (QM/MM) data. MD was performed using the GPU accelerated pmemd module of the AMBER14MD package. BOMD was performed using CP2K version 2.6. The reaction rates in BOMD were accelerated using the Metadynamics method. QM/MM was performed using ONIOM in the Gaussian09 suite of programs. Relevant input files for BOMD and QM/MM are available.
with familial autism spectrum disorder while loss of function results in the Angelman syndrome neurological disorder. BIOGRID recognizes 174 E6APinteractors but only a few have been validated as substrates for ubiquitination. Recently, phosphorylation on E6APT485, an autism related residue, affected RPN10/S5A substrate ubiquitination in cells. The current aim included kinetic analysis of E6AP-catalyzed conjugation of target proteins to provide new insights into the mechanism of substrate ubiquitination. E6AP-dependent Lys 48 -polyubiquitin chain assembly in the absence of substrate requires two functionally distinct UbcH7~ubiquitin binding sites on the ligase surface and oligomerization. Here, the E6AP ubiquitin-function was analyzed in the presence of RNP10/S5A (regulatory subunit of proteasome 26S) and PRDX1 (antioxidant enzyme). Rates of substrate ubiquitin adduct formation were analyzed under E6AP rate-limiting conditions. Adducts of PRDX1-Ub1 or RPN10/S5A-Ub1 showed K m values of 851 mM and 0.250.06 mM, respectively; while the k cat values were 0.3 s À1 , comparable to 0.5 s À1 observed for polyubiquitin chain assembly without substrate and indicating that the ligase cannot distinguish the lysine nucleophile in Lys 48 -ubiquitin and Lys-target protein ubiquitination. Analysis of pH-dependent E6AP ligase function inferred a pK a of~8.4, either in the absence or presence of PRDX1. Removal of the first 250 N-terminal residues reduced ubiquitination of both substrates supporting the presence of previously unrecognized substrate binding domains in this region. A T485D mutation mimicking E6AP phosphorylation, or a D212A mutation, an Angelman syndrome mutation, abrogated substrate ubiquitination, although they retained the polyubiquitin chain assembly function. The results provide new insights of the E6AP ubiquitination mechanism in the presence of target proteins that might explain the deleterious effect of some mutations associated with Angelman syndrome.
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