Stochastic Gate Dynamics Regulate the Catalytic Activity of Ubiquitination Enzymes

Abstract

Initiation of the DNA damage and innate immune responses is dependent upon the flow of chemical information through coupled protein-protein interaction networks and driven by the synthesis and recognition of Lys 63 linked polyubiquitin (polyUb) chains on adaptor proteins. The central chemical step in Lys 63-linked protein ubiquitination involves the reaction of a specific lysine on a target protein with Ub that is covalently attached as a thioester conjugate to the Ub conjugating enzyme (E2) Ubc13. The active site cysteine of Ubc13, and E2 enzymes in general, is buttressed by a flexible loop. The role of loop dynamics in catalysis was investigated by mutating the central and hinge residues to glycine. The loop dynamics were experimentally characterized through measurement of enzyme kinetics, main chain NMR relaxation, X-ray crystallographic studies, and in vivo studies in yeast. The experimental data were complemented by analysis of MD simulations of the dynamics and kinetics for the loop motion. The results show that fast pico- to nanosecond time scale active site loop fluctuations play a crucial role in regulating the catalytic activity of Ubc13 by functioning as a stochastic active site gate, which is characterized by precisely balanced rates of opening and closing. In vivo functional complementation assays in yeast demonstrate that defects within this regulatory mechanism can have profound biological consequences, given that Ubc13 is the only E2 dedicated to synthesizing Lys 63-linked polyUb chains.

Covalent Inhibition of Ubc13 Affects Ubiquitin Signaling and Reveals Active Site Elements Important for Targeting

“…Flexibility of this loop is further suggested by the observation that the loop adopts still other conformations in complex with E3 ligases and other regulatory proteins (Supporting Information Figure 12). Indeed, we have recently shown the dynamics of this loop to be important for the catalytic activity of Ubc13 35 . This is consistent with previous suggestions that E3 ligases may activate Ubc13 and other E2s by driving conformational change within the active site that propagates from the site of E3 binding 8, 9, 36 .…”

Conformational Dynamics and Allostery in E2:E3 Interactions Drive Ubiquitination: gp78 and Ube2g2

“…Multiple molecular dynamics studies of SCF-E3s indicate the role of functional dynamics in the E3s (Liu and Nussinov, 2009, 2013). For E2s UbcH5c and Ubc13, the ps-ns dynamics of the β4α2 loop near the active site were correlated to its function (Pruneda et al, 2012; Pruneda et al, 2011; Rout et al, 2014). However, the dynamics in these enzymes upon interaction with their reaction partners has not been previously explored, and no studies have addressed the μs-ms timescale, which can be relevant to the recognition of binding partners and may correlate with the Ub transfer step.…”

“…Solution NMR studies of the picosecond-nanosecond (ps-ns) dynamics have been studied in four E2s: Ubc13, UbcH5b, UbcH5c and Ube2g2 (Houben et al, 2004; Ju et al, 2010; Pruneda et al, 2011; Rout et al, 2014; Soss et al, 2013). In UbcH5b and Ubc13, the β4α2 loop lacks the acidic extension found in Ube2g2 and Cdc34.…”

“…In UbcH5b and Ubc13, the β4α2 loop lacks the acidic extension found in Ube2g2 and Cdc34. Nevertheless, the ps-ns dynamics of the shorter β4α2 loop were shown to have an effect on the catalytic activity of Ubc13 (Rout et al, 2014). Although individual residues in the UbcH5b β4α2 loop were found to be crucial for its catalytic activity, the possible role of dynamics has not been revealed as yet (Houben et al, 2004).…”

Tracking individual membrane proteins and their biochemistry: The power of direct observation

“…A stochastic model can be used to describe the operation of molecular machines, which links allosteric motions and chemical reactions to changes in free energy. (Elber and Karplus, 1987;Jülicher and Bruinsma, 1998;Keller and Bustamante, 2014;Kurzynski and Chelminiak, 2014;Lazaridis and Karplus, 1999;Tsai, 2008) Such descriptions include the following phenomena: the closing of ligand binding clefts, (McCammon et al, 1976) rotations of subdomains, (De Groot et al, 1998;Noji et al, 1997) twisting of subunits, binding and unbinding of ligands, (Ghanouni et al, 2001;Lau and Roux, 2007;Ma et al, 2000;Ravindranathan et al, 2005) the metabolism of substrates (enzymes), (Rout et al, 2014) and the movement of proteins through lipid domains. (Wang et al, 2014) Each machine has a small number of key conformational degrees of freedom linked to these motions and chemical reactions, which result in the observable functional states (discrete states) of the protein.…”