Abstract
The metzincins are a clan of metallopeptidases consisting of families that share a series of structural elements. Among them is the Met-turn, a tight 1,4-turn found directly below the zinc-binding site, which is structurally and spatially conserved and invariantly shows a methionine at position 3 in all metzincins identified. The reason for this conservation has been a matter of debate since its discovery. We have studied this structural element in Methanosarcina acetivorans ulilysin, the structural prototype of the pappalysin family, by generating 10 mutants that replaced methionine with proteogenic amino acids. We compared recombinant overexpression yields, autolytic and tryptic activation, proteolytic activity, thermal stability, and three-dimensional structure with those of the wild type. All forms were soluble and could be purified, although with varying yields, and three variants underwent autolysis, could be activated by trypsin, and displayed significant proteolytic activity. All variants were analyzed for the thermal stability of their zymogens. None of the mutants analyzed proved more stable or active than the wild type. Both bulky and small side chains, as well as hydrophilic ones, showed diminished thermal stability. Two mutants, leucine and cysteine, crystallized and showed three-dimensional structures that were indistinguishable from the wild type. These studies reveal that the Met-turn acts as a plug that snugly inserts laterally into a core structure created by the protein segment engaged in zinc binding and thus contributes to its structural integrity, which is indispensable for function. Replacement of the methionine with residues that deviate in size, side-chain conformation, and chemical properties impairs the plug-core interaction and prejudices molecular stability and activity.
Highlights
Evolutionary conservation of structural elements in proteins usually results from stringent steric requirements for function
The strict conservation of the Met-turn and its methionine among metzincin structures has intrigued structural biologists since its discovery, and it contributed to the name of this metallopeptidase clan [2]
We found an inverse correlation between function/stability and deviation from the optimal size as granted by methionine
Summary
Site-directed Mutagenesis—Plasmids based on pET28a, which were modified to encode for both M. acetivorans proulilysin wild-type and variant C269A [16, 17], were used as a template to mutate Met290 to alanine, leucine, valine, phenylalanine, glycine, serine, cysteine, aspartate, lysine, and histidine (M290X mutants). Only mature mutants M290L (autolytically activated) and M290C (trypsin-treated) rendered crystals suitable for x-ray analysis. Both variants (at 10 mg/ml and 5.8 mg/ml, respectively, in 20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 5 mM CaCl2) crystallized at 20 °C in, respectively, 0.2 M CaCl2, 0.1 M Hepes, pH 7, 20% (w/v) PEG 6000; and 0.2 M CaCl2, 0.1 M Hepes, pH 8, 22% (w/v) PEG 6000 as precipitating agent. The final model of ulilysin M290C contained residues 63–321 for each of the two molecules (chains A and B) in the asymmetric unit plus an Arg-Val dipeptide in the active-site cleft of molecule B. The final co-ordinates have been deposited with the Protein Data Bank at the RCSB (PDB access code 3LUM for M290L ulilysin and 3LUN for M290C ulilysin)
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