Heat induces end to end repetitive association in P. furiosus l-asparaginase which enables its thermophilic property

Abstract

It remains undeciphered how thermophilic enzymes display enhanced stability at elevated temperatures. Taking l-asparaginase from P. furiosus (PfA) as an example, we combined scattering shapes deduced from small-angle X-ray scattering (SAXS) data at increased temperatures with symmetry mates from crystallographic structures to find that heating caused end-to-end association. The small contact point of self-binding appeared to be enabled by a terminal short β-strand in N-terminal domain, Leu179-Val-Val-Asn182 (LVVN). Interestingly, deletion of this strand led to a defunct enzyme, whereas suplementation of the peptide LVVN to the defunct enzyme restored structural frameworkwith mesophile-type functionality. Crystal structure of the peptide-bound defunct enzyme showed that one peptide ispresent in the same coordinates as in original enzyme, explaining gain-of lost function. A second peptide was seen bound to the protein at a different location suggesting its possible role in substrate-free molecular-association. Overall, we show that the heating induced self-assembly of native shapes of PfA led to an apparent super-stable assembly.

Plasmodium falciparum Kelch13 and its artemisinin‐resistant mutants assemble as hexamers in solution: a SAXS data‐driven modelling study

“…Using the shape information in the SAXS data profiles, and a combination of manual curation coupled with molecular modelling, we generated molecular models for the TrK13 proteins at dummy residue and atomic scale. It is pertinent to mention that acquired scattering information was desmeared using incident beam profile for the experiments, as done before for several systems [46][47][48][49][50][51][52][53][54][55]. This allowed the scattering data collected using line collimation to be integrated in a manner that it represents scattering arising from the point collimation.…”

“…These I 0 values were compared with those from the standard protein samples of lysozyme (in 40 mM NaOAc with 150 mM NaCl, pH 3.8) and gelsolin (in 1 mM Tris. HCl with 40 mM NaCl pH 8 and 1 mM EGTA) [47,60].…”

“…During the review of this manuscript, a query was raised about the desmearing using slit beam profile and the SAXSQUANT program used in this study. We iterate that our method has been validated in previously published studies using standard proteins [46][47][48][49][50][51][53][54][55]87], and provided intensity profiles which could suitably be used for other analysis. To compare the desmearing by PRIMUS/GNOM with SAXSQUANT, we read-in the slit beam profile and attempted distance distribution profile calculation in GNOM plugin.…”

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

“…It also indicates that the active site of the protein may get occluded with an increase in temperature, and thus, in states or temperatures forming higher-order association than dimer, the total activity of the protein may drastically reduce. This “side-ways” association profile is in contrast to the temperature-induced end-to-end association of L-asparaginase from P. furiosus (PfLAsp) when heated [46]. The differential relevance of this contrasting behaviour to the shape-function of this enzyme is that while PrASNase displayed diminished activity with heating, while thermophile PfLAsp showed enhanced activity at elevated temperatures than ambient or physiological ones.…”

“…SAXS analysis showed the protein to be mostly monomeric till 37°C and catalytically active at that temperature under enzyme assay conditions. As discussed above, the side-ways association of this enzyme upon heating is in sharp contrast with the end-to-end spiral order of association for thermophilic P. furiosus L-asparaginase [46]. In the latter, the dimeric association is the building block of the higher-order association that disassembles upon heating withdrawal.…”