Biophysical and structural characterization of the thermostable WD40 domain of a prokaryotic protein, Thermomonospora curvata PkwA

Abstract

WD40 proteins belong to a big protein family with members identified in every eukaryotic proteome. However, WD40 proteins were only reported in a few prokaryotic proteomes. Using WDSP (http://wu.scbb.pkusz.edu.cn/wdsp/), a prediction tool, we identified thousands of prokaryotic WD40 proteins, among which few proteins have been biochemically characterized. As shown in our previous bioinformatics study, a large proportion of prokaryotic WD40 proteins have higher intramolecular sequence identity among repeats and more hydrogen networks, which may indicate better stability than eukaryotic WD40s. Here we report our biophysical and structural study on the WD40 domain of PkwA from Thermomonospora curvata (referred as tPkwA-C). We demonstrated that the stability of thermophilic tPkwA-C correlated to ionic strength and tPkwA-C exhibited fully reversible unfolding under different denaturing conditions. Therefore, the folding kinetics was also studied through stopped-flow circular dichroism spectra. The crystal structure of tPkwA-C was further resolved and shed light on the key factors that stabilize its beta-propeller structure. Like other WD40 proteins, DHSW tetrad has a significant impact on the stability of tPkwA-C. Considering its unique features, we proposed that tPkwA-C should be a great structural template for protein engineering to study key residues involved in protein-protein interaction of a WD40 protein.

Protein Thermodynamic Destabilization in the Assessment of Pathogenicity of a Variant of Uncertain Significance in Cardiac Myosin Binding Protein C

“…To study this scenario, recombinant WT and I603M C4 domains were produced (Figure 2, C) and analysed by Circular Dichroism (CD) in the far-UV (200-250 nm, informative about secondary structure) and near-UV (250-350 nm, resulting from tertiary structure) at 25°C (Figure 3 A, B). In agreement with the homology modelling prediction, the far-UV spectrum of C4 WT showed a minimum of ellipticity at 215 nm, characteristic of β-sheet structure 50,51 . C4 WT and I603M spectra showed similar CD spectra, suggesting that the mutation did not have major impact in the fold of the protein at 25ºC (Figure 3, A, B).…”

Thermodynamic destabilization informs pathogenicity assessment of a variant of uncertain significance in cardiac myosin binding protein C

“…To study this scenario, recombinant WT and I603M C4 domains were produced ( Figure 2, C ) and analysed by Circular Dichroism (CD) in the far-UV (200-250 nm, informative about secondary structure) and near-UV (250-350 nm, resulting from tertiary structure) at 25°C ( Figure 3 A, B ). In agreement with the homology modelling prediction, the far-UV spectrum of C4 WT showed a minimum of ellipticity at 215 nm, characteristic of β-sheet structure 50,51 . C4 WT and I603M spectra showed similar CD spectra, suggesting that the mutation did not have major impact in the fold of the protein at 25°C ( Figure 3, A, B ).…”

Effects of SDS on the activity and conformation of protein tyrosine phosphatase from thermus thermophilus HB27

“…Although our knowledge on the structure and cellular function of PTPase is still increasing, little is known how the activity of PTPase from thermus thermophiles HB27 is determined by its conformation. Denaturant is usually used to characterize the activity and conformation of an enzyme [13][14][15][16][17][18] . Urea is known to break non-covalent interactions.…”