Abstract
LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca(2+). Recent crystal structures have been obtained for the protein in the apo- and Ca(2+)-bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca(2+) and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca(2+) binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca(2+) affinity as the wild-type protein. We then evaluated if Ca(2+) binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca(2+) ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.
Highlights
Leptospirosis is a zoonosis caused by spirochetes belonging to the genus Leptospira
Because the apo-structure did not unambiguously reveal the Ca2ϩ-binding site, we produced three LipL32 mutants designed to disrupt two originally proposed candidate binding sites on opposite sides of the protein: (i) LipL32Q67A and LipL32S247A have mutations in a cavity that coincides with the Ca2ϩ-binding site from the ColG collagenase from Clostridium histolyticum and (ii) LipL32D163–168A has five aspartates in a conserved acidic loop all changed to alanines
Our previous observation that many of the proteins with the closest structural homology to LipL32 bind both calcium ions and extracellular matrix proteins led us to demonstrate for the first time that LipL32 binds Ca2ϩ, but not other divalent metals such as Mg2ϩ, Zn2ϩ, and Cu2ϩ (14)
Summary
Wild-type LipL32His tag LipL32Q67A or LipL32Q67A_His tag LipL32S247A or LipL32S247A_His tag LipL32D163–168A or LipL32D163–168A_His tag LipL32185–272_His tag. 14 12 This study This study This study 12 results indicate that LipL32 binds to target proteins in a manner that does not depend on metal binding to the Ca2ϩ-binding site
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