Ashish, | bioRxiv Science

bioRxiv2023

Assessment of structural behaviour of new L-asparaginase and SAXS data-based evidence for catalytic activity in its monomeric form

The present study reports the structural and functional characterization of a new glutaminase-free recombinant L-asparaginase (PrASNase) from Pseudomonas resinovorans IGS-131. PrASNase showed substrate specificity to L-asparagine, and its kinetic parameters, Km, Vmax, and kcat were 9.49x10-3 M, 25.13 IUmL-1min-1, and 3.01x103 s-1, respectively. The CD spectra showed that PrASNase consists of 30.9% -helix and 69.1% other structures in its native form. FTIR was used for the functional characterization, and molecular docking predicted that the substrate interacts with serine, alanine, and glutamine in the binding pocket of PrASNase. Different from known asparaginases, structural characterization by small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) unambiguously revealed PrASNase to exist as a monomer in solution at low temperatures and oligomerized to a higher state with temperature rise. Through SAXS studies and enzyme assay, PrASNase was found to be mostly monomer and catalytically active at 37{degrees}C. Furthermore, this glutaminase-free PrASNase showed killing effects against WIL2-S and TF-1.28 cells with IC50 of 7.4 {micro}g.mL-1 and 5.6 {micro}g.mL-1, respectively. This is probably the first report with significant findings of fully active L-asparaginase in monomeric form using SAXS and AUC and demonstrates the potential of PrASNase in inhibiting cancerous cells, making it a potential therapeutic candidate. HIGHLIGHTSO_LIA new L-asparaginase (PrASNase) was structurally and functionally characterized. C_LIO_LISAXS revealed PrASNase is functionally active in monomeric form and oligomerizes with temperature rise. C_LIO_LIMonomeric PrASNase showed an IC50 value of 7.4 and 5.6 {micro}g mL-1 against WIL2-S and TF-1.28 cells. C_LIO_LICytotoxicity of PrASNase against leukemic cell lines showed its potential as a biotherapeutic. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/522448v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@ece494org.highwire.dtl.DTLVardef@92cdbeorg.highwire.dtl.DTLVardef@19a2borg.highwire.dtl.DTLVardef@1308c70_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2017

Atypical domain communication and domain functions of a Hsp110 chaperone

Hsp110s are well recognized nucleotide exchange factors (NEFs) of Hsp70s, in addition they are implicated in various aspects of cellular proteostasis as discrete chaperones with yet enigmatic molecular mechanism. Stark similarity in domain organization and structure between Hsp110s and Hsp70s, is easily discernible although the nature of domain communication and domain functions of Hsp110s are still puzzling. Here, we report atypical domain communication of yeast Hsp110, Sse1 using single molecule FRET, small angle X-ray scattering measurements (SAXS) and Molecular Dynamic simulations. Our data show that Sse1 lacks typical domain movements as exhibited by Hsp70s, albeit it undergoes unique structural alteration upon nucleotide and substrate binding. Hsp70-like domain-movements can be artificially salvaged in chimeric constructs of Hsp110-Hsp70 although such salvaging proves detrimental for the NEF activity of the protein. Furthermore, we show that substrate binding domain (SBD) of Hsp110, chaperones self, as well as foreign nucleotide binding domains (NBD). Interestingly, the substrate binding specificity of Hsp110 is largely determined by its NBD rather than SBD, the latter being the foremost substrate binding region for Hsp70s.

biochemistry↗