Journal: bioRxiv

Article Title: Helical ultrastructure of the oncogenic metalloprotease meprin α in complex with a small molecule hydroxamate inhibitor

doi: 10.1101/2022.03.13.484121

Figure Lengend Snippet: a. Periodic arrangement of the active site and prodomain are highlighted as yellow in the surface representation of an idealised meprin α helical segment. b. Example curve representing a series of first-order rates (determined by fitting the linear region of fluorescence versus time) at a given meprin concentration. The kcat/Km is determined from this graph for several meprin concentrations and in triplicate c. First order rate constants (k cat /K m ) of meprin α and variants for small fluorogenic peptide cleavage. Oligomeric state does not appear to affect rate of cleavage of small molecule substrate. d. Inhibitory constant (IC 50 ) of meprin α inhibitors. Determined by fitting and normalising the linear region of fluorescence versus time, in the presence of varying amount of inhibitor at a fixed meprin α concentration. e. Globular proteinaceous inhibitor, murine fetuin-B, and small molecule compound 10d were unaffected by oligomeric state. f. Proteolytic stability of meprin α and variants against trypsin and plasmin. Meprin α oligomers were more stable compared to lower-stoichiometric variants. g. Meprin α thermal stability measured by nanoDSF. Meprin α possess superior thermal stability compared to variants that lack either the disulphide bridge (most drastic) or helical interface interactions. All measurements are statistically significant to all others (p<0.0001, ****) unless otherwise indicated.

Article Snippet: His-tagged recombinant human fetuin-B (11834-H08H) was purchased from SinoBiological.

Techniques: Fluorescence, Concentration Assay, Nano Differential Scanning Fluorimetry

Journal: bioRxiv

Article Title: Helical ultrastructure of the oncogenic metalloprotease meprin α in complex with a small molecule hydroxamate inhibitor

doi: 10.1101/2022.03.13.484121

Figure Lengend Snippet: a. Rigid body fit of murine fetuin-B/meprin β crystal structure (PDB 7AUW) to helical structure of meprin α. Arrangement of fetuin-B reveal monomers may pack into a slanted intercalated state that is not significantly prohibited by steric clashes. b. View of a tetramer of fetuin-B based on meprin α docking reveals potential interactions to form a higher-order inhibitory filamentous complex are possible. c. Side view of single fetuin-B dimer forms a “horseshoe” where putative interactions between inter-subunit fetuin-B domains may occur shown in (d), and (e). d, e. Models are not refined, rigid body fitting results in some minor clashes. f. The predicted oligomeric interface corresponds to an evolutionarily conserved interface revealed by ConSurf analysis. g. Side and top views of the cryo-EM reconstruction of human fetuin-B (red) in complex with meprin α (grey, black) at 3.7 Å resolution. Fetuin-B is observed to pack intimately within the meprin α active groove and intercalate as a secondary helix.

Article Snippet: His-tagged recombinant human fetuin-B (11834-H08H) was purchased from SinoBiological.

Techniques: Cryo-EM Sample Prep