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Insights to the Assembly of a Functionally Active Leptospiral ClpP1P2 Protease Complex along with Its ATPase Chaperone ClpX
journal contribution
posted on 2019-07-31, 08:14 authored by Anusua Dhara, Md Saddam Hussain, Debika Datta, Manish KumarLeptospira interrogans genome is
predicted to encode multiple
isoforms of caseinolytic proteases (ClpP1 and ClpP2). The ClpP proteins
with the aid of its ATPase chaperone are known to be involved in establishing
cellular proteostasis and have emerged as a target for developing
new antibiotics. We report the molecular characterization of recombinant
ClpP1 (rClpP1) and rClpP2 of Leptospira along with its ATPase chaperone rClpX. The two isoforms of rClpPs
when coupled together in an equivalent concentration exhibit optimum
activity on small fluorogenic peptide substrates, whereas the pure
rClpP isoforms are enzymatically inactive. Isothermal titration calorimetry
analysis suggests that the two rClpP isoforms bind each other moderately
in a 1:1 stoichiometry with a dissociation constant of 2.02 ±
0.1 μM at 37 °C and is thermodynamically favored. Size
exclusion chromatography fractionates the majority of pure rClpP1
at ≥308 kDa (14–21-mer) and the pure rClpP2 at 308 kDa
(tetradecamer), whereas the functionally active rClpP isoform mixture
fractionates as a tetradecamer. The distinct and unprecedented oligomeric
form of rClpP1 was also evident through native-gel and dynamic light
scattering. Moreover, the rClpP isoform mixture formed after the site-directed
mutation of either or both the isoforms at one of the catalytic triad
residues (Ser 98/97 to Ala 98/97) resulted in the complete loss of
protease activity. The rClpP isoform mixture gets stimulated to degrade
the casein substrate in the presence of rClpX and in an energy-dependent
manner. On the contrary, pure rClpP1 or the rClpP2 isoform in association
with rClpX are incapable of forming operative protease. The reported
finding suggests that in Leptospira, the enzymatic activity of the rClpP protease complex in the presence
or absence of cochaperone is performed solely by the tetradecamer
structure which is hypothesized to be composed of 2-stacked ClpP heptameric
rings, wherein each ring is a homo-oligomer of ClpP1 and ClpP2 subunits.
Understanding the activities and regulation principle of multi-isoforms
of ClpP in pathogenic bacteria may aid in intervening disease
outcomes particularly to the co-evolving antibiotic resistance strains.
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ClpP 2 subunitsco-evolving antibiotic resistance strainsClpP heptameric ringsSize exclusion chromatography fractionatesrClpP 1rClpP 2Isothermal titration calorimetry analysisATPase Chaperone ClpX Leptospira interrogans genomeATPase chaperone rClpXClpP 1rClpP isoforms bindrClpP isoform mixture fractionatesrClpP isoform mixturefluorogenic peptide substratesActive Leptospiral ClpP 1P Protease ComplexrClpP 2 isoformequivalent concentration exhibit
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