![]() ![]() ![]() Thus, substrate-free Cezanne-1 is more prone to adopt a catalytically noncompetent conformation. Considering a Cys194-His358 distance of 0.4 nm and below as indicative of catalytic competency, the probability to find substrate-free Cezanne-1 in a catalytically competent low energy state is 0.25 in a neutral state and 0.51 in a zwitterionic charge state. Apparently, residue Glu157 does not directly promote the formation of a catalytically competent active site. It can be seen that the close positioning and correct orientation of His358 with respect to Cys194 occurs independently of the Glu157 The free energy barriers between the states are 1.5 kcal/mol ( Cez 0 apo) and 2 kcal/mol ( Cez +/- apo), and several transitions can be observed within 3 μs of cumulative MD sampling (see the bottom panel in Figure S2). His358 distances are occurring at 0.35 and 0.55 nm for Cez 0 apo and at 0.31 and 0.57 nm for Cez +/- apo ( Figure 1B and C).This information cannot be obtained from protein crystallographic studies alone but is important when exploring Cezanne-1 as a drug target and aiming at selectively inhibiting a particular state of function. The release of the cleaved ubiquitin monomers is sequential and initiated by dissociation of the proximal ubiquitin, reconstitution of the inactive state of Cezanne-1, and then release of the distal ubiquitin. Although this residue is not actively involved in iso-peptide bond cleavage, it is catalytically relevant in terms of correct substrate positioning close to the catalytic center. Substrate binding, however, shifts the equilibrium toward the catalytically active state by means of an electrostatic stabilization of the substrate by a glutamate residue. Even in absence of diubiquitin, there is an equilibrium between the inactive and active states of the catalytic dyad. ![]() The MD simulations show that substrate binding is not necessary for the activation of Cezanne-1. Our MD simulations, covering a total of 18.3 μs for Cezanne-1 in different states along the deubiquitinylating process, reveal the dynamics of conformational changes of this OTU during its enzymatic performance. Activity-based probes of cysteine proteases lead to non-native protein–protein contacts, which need to be resolved in order to be able to issue statements about physiological states and substrate binding. The process of activation and catalytic turnover of DUB Cezanne-1 is a multistage cycle with several critical dynamic transitions that cannot be characterized based on protein structures alone. The subsequent release of the distal ubiquitin fully reconstitutes the inactive substrate-free state of Cezanne-1. After cleavage of the scissile bond, sequential dissociation of first the proximal ubiquitin induces the inactivation of Cezanne-1. The subsequent binding of the proximal ubiquitin shifts the equilibrium toward the catalytically competent state of the dyad, thereby promoting proteolysis of the iso-peptide bond. The catalytically competent substrate-free Cezanne-1 promotes distal ubiquitin substrate access to the catalytic center. Our results show that ubiquitin-free Cezanne-1 dynamically shuttles between catalytically competent and incompetent states which suggests that its activation is independent of substrate binding. Conformational changes of Cezanne-1, during its process of activation and proteolytic activity, are investigated using all-atom molecular dynamics (MD) simulations of the ubiquitin-free, diubiquitin-bound, and monoubiquitin-bound Cezanne-1 DUB for a total of ∼18 μs. Those, however, may lead to structures that are non-native. Activity-based probes are used to isolate intermediate states upon forming covalent bonds with the DUB active site. Crystal structures of Cezanne-1 in different states provide important insight regarding the complex formation and global changes during the catalytic cycle but are lacking details of dynamics and control of activation. The DUB Cezanne-1 catalyzes the cleavage of the iso-peptide bond of Lys11-linked polyubiquitin chains with high selectivity. Deubiquitinylating enzymes (DUBs) regulate the deubiquitinylation process of post-translationally modified proteins and thus control protein signaling in various cellular processes. ![]()
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