| Summary: | Repeat-in-Toxin (RTX) represents a broad family of protein produced by Gram-negative bacteria. RTX protein consists of RTX parallel β-roll motif repeat
structure. Previously, AMS8 lipase from Antarctic Pseudomonas fluorescens
strain AMS8 was classified as RTX lipase. The previous study has been
reported the Ca2+ ions play a role in the formation of RTX parallel β-roll motif
repeat structure and involve in the folding and stabilization of many RTX
protein. However, the contributions of Ca2+ ions towards the folding and
stabilization of AMS8 lipase have not been understood. It is hypothesized that
the Ca2+ ions induce the formation of RTX parallel β-roll motif repeat structure
and involve in the folding and stabilization of AMS8 lipase. Thus, this research
aimed to examine the influence of Ca2+ ion towards the activity, folding and
stabilization of AMS8 lipase through the in-silico approach and various
biophysical characterizations.
AMS8 lipase contains six Ca2+ ions (Ca1, Ca2, Ca3, Ca4, Ca5 and Ca6) and
RTX parallel β-roll motif repeat structures. In-silico studies were done to
analyze the structural conformational changes of the AMS8 lipase structure
using molecular docking and molecular dynamics (MD) simulation. As a result,
metal ion docking analysis gives high binding energy, especially for Ca4 and
Ca5. To further analyze the function of each Ca2+ ions, MD simulation was
performed. The removal of Ca3, Ca4 and Ca5 caused the AMS8 lipase
structure to become unstable and unfolded. These suggested that Ca3, Ca4
and Ca5 were involved in the stabilization and folding of the RTX parallel β-roll
motif repeat structure.AMS8 lipase activity was increased in the presence of CaCl2, where the
optimum CaCl2 concentration was detected at 80 mM. To further confirm the
contribution of Ca2+ ion, various biophysical characterizations using circular
dichroism (CD), fourier-transform infrared (FTIR), intrinsic and extrinsic
fluorescence, dynamic light scattering (DLS) and isothermal titration calorimetry
(ITC) were performed. The far-UV CD and FTIR analyses suggested that the
secondary structure content was improved with the addition of CaCl2. Intrinsic
and extrinsic fluorescence analysis showed that the presence of CaCl2
increased protein folding and compactness. DLS analysis suggested the AMS8
lipase became aggregated at a high concentration of CaCl2. The binding
constant (Kd) value from the ITC analysis proved that the Ca2+ ion was tightly
bound to the AMS8 lipase.
In conclusion, in-silico approach and various biophysical characterizations
revealed that Ca2+ ions play essential roles in the activity, folding and stability
of the AMS8 lipase. Furthermore, Ca2+ ions also induced the folding of the RTX
parallel β-roll motif repeat structure and played a crucial role in the folding and
stabilization purposes of the whole AMS8 lipase structure.
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