An active site loop mutant of a zinc metallo-deubiquitinase suggests the importance of loop dynamics to catalysis
AMSH is a conserved zinc metalloprotease that functions with endosomal sorting complexes required for transport (ESCRT) machinery in order to down regulate and degrade cell-surface receptors. It has been shown to have high specificity toward Lys63-linked polyubiquitin chains, a signal used for receptor down regulation by ESCRT machinery. It has previously been found that Sst2, an AMSH orthologue from fission yeast, contains a flexible "loop" near the proximal ubiquitin binding site, composed of residues L402 and F403. The different mobility of the side chains of these residues contribute to the opening and closing conformation of the catalytic cleft which allow access to the active-site and may contribute to linkage specificity and catalysis. More specifically, F403 has been shown to make contacts with the isopeptide bond of substrate diubiquitin during cleavage, as well as have the potential to make interactions with residues D315 and T316 across from the mobile loop. To probe role of dynamics in this area, the conserved residue F403 was mutated to a bulkier yet still hydrophobic tryptophan residue with the thought that it would slow loop mobility by having the potential to make increased contacts. An additional mutant was made in which F403 was mutated to alanine to create a more flexible mutant incapable of making these contacts for comparison. Crystallographic structures were obtained of the F403W mutant alone and bound to monoubiquitin, revealing little change in overall structure compared to that of wild type, as well as little change in the alignment with the isopeptide bond of diubiquitin through aid of molecular modeling. Kinetic measurement of the tryptophan mutant showed a decreased value in Kcat by about 10-fold from that of wild type (0.17s-1 vs. 1.5s-1 respectively), while the KM was relatively unchanged. This indicated that while catalytic activity of the mutant was significantly impaired, substrate binding affinity was not affected. To show that this decrease in catalytic activity was not due to a structural change, circular dichroism and thermal denaturation were done on the mutant. Results indicate minimal structural changes due to the tryptophan mutation, as supported by crystallographic results. Isothermal titration calorimetry (ITC) data was obtained for the tryptophan mutant binding to monoubiquitin, showing that substrate (or product) binding of the tryptophan mutant (10.2 + 0.3 μM) is relatively unchanged when compared to that of wild type (3.6 + 0.3 μM). Finally, cleavage of chain types K11 and K48 were done and compared to K63 by the mutant was measured, as well as the cleavage ability of several surrounding loop residue mutants. Results indicate that the tryptophan mutant allows better cleavage of other chain types than WT due to dynamics and an increased flexibility in loop, as well as hint to the importance of other key residues in the protein that may impact protein stability and function.
Das, Purdue University.
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