title: Calcium binding to leptospira outer membrane antigen LipL32 is not necessary for its interaction with plasma fibronectin, collagen type IV, and plasminogen.
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.
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CEP 05513-970, São Paulo.
authors.forename: Pricila
authors.initials: P
authors.lastname: Hauk
authors.forename: Angela Silva
authors.initials: AS
authors.lastname: Barbosa
authors.forename: Paulo Lee
authors.initials: PL
authors.lastname: Ho
authors.forename: Chuck Shaker
authors.initials: CS
authors.lastname: Farah
mesh_terms: D019943:Amino Acid Substitution; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D024141:Collagen Type IV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005353:Fibronectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007919:Leptospira / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010958:Plasminogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D002413:Cations, Divalent; D024141:Collagen Type IV; D005353:Fibronectins; C406589:LipL32 protein, Leptospira; D008074:Lipoproteins; D010958:Plasminogen; D002118:Calcium