X-ray crystallographic and SAXS studies on Penicillium marneffei MP1 protein revealing its strong arachidonic acid binding property as a putative novel virulence factor

Abstract

Penicillium marneffei (P. marneffei) infection is the second most deadly opportunistic systematic mycosis called penicilliosis, in immuno-compromised patients, especially in Southeast Asia. Different virulence mechanisms in P. marneffei had been discovered and investigated. In the sera of patients infected by P. marneffei, MP1 protein (MP1p), a highly secretory galactomannoprotein antigen with two consecutive ligand-binding domains, MP1p-LBD1 and MP1p-LBD2, was found to be very abundant. A published crystal structure had shown that the MP1p-LBD2 formed a closed hydrophobic cavity to bind a co-purified palmitic acid (PLM) tightly (PDB: 3L1N). It is then hypothesized that capturing of lipids from human hosts may be a possible virulence mechanism of P. marneffei to remain infectious inside the hosts, by expressing large amount of MP1p. In our previous study by X-ray crystallography, it was shown that MP1p-LBD2 can accommodate up to two arachidonic acid (AA) molecules, the sole precursor of many paracrine signalling molecules for regulations of inflammatory responses against different types of infections, in its closed central cavity replacing PLM.

In this study, crystal structures of MP1p-LBD1 complexed with a co-purified palmitic acid molecule and two arachidonic acid molecules were solved at resolution 1.80 Å and 2.60 Å, respectively. The structures of MP1p-LBD1 complexed with either PLM or AAs resembled the structure of MP1p-LBD2 complexed with AAs, but not PLM. This finding revealed that MP1p-LBD1 has the equal fatty acid binding function very similar to MP1p-LBD2. A 4.20 Å resolution crystal structure of dimeric double-domains construct from MP1p-LBD1 to MP1p-LBD2 complexed with endogenous fatty acids revealed the relative spatial orientation between the two domains. Surprisingly, SAXS experiments suggested that the monomeric form of the double domain construct was the dominant form in solution, regardless of ligands bound. The presence of this dominant monomeric form was also confirmed by static light scattering (SLS) experiments.

Taken together, this structural study on MP1p-LBD1 and the double-domain construct strongly support that the highly expressed MP1p is a potent fatty acid-capturing protein having two equally strong fatty acid binding domains in one single protein. Supported by in vivo functional data from collaborators, P. marneffei and potentially other pathogens secreting similar proteins can severely disturb hosts’ signalling cascades during pro-inflammatory responses for their better survival in hosts, by removing important hydrophobic paracrine signalling molecules.