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. 2019 Mar 25;87(4):e00679-18.
doi: 10.1128/IAI.00679-18. Print 2019 Apr.

Talaromyces marneffei Mp1 Protein, a Novel Virulence Factor, Carries Two Arachidonic Acid-Binding Domains To Suppress Inflammatory Responses in Hosts

Wai-Hei Lam #  1 , Kong-Hung Sze #  2   3   4   5 , Yihong Ke #  2   3   4   5 , Man-Kit Tse #  2   3   4   5 , Hongmin Zhang  1   6   7 , Patrick C Y Woo  2   3   4   5 , Susanna K P Lau  2   3   4   5 , Candy C Y Lau  2   3   4   5 , Simin Xu  2   3   4   5 , Pok-Man Lai  2   3   4   5 , Ting Zhou  2   3   4   5 , Svetlana V Antonyuk  8 , Richard Y T Kao  2   3   4   5 , Kwok-Yung Yuen  9   3   4   5 , Quan Hao  10   6
Affiliations

Talaromyces marneffei Mp1 Protein, a Novel Virulence Factor, Carries Two Arachidonic Acid-Binding Domains To Suppress Inflammatory Responses in Hosts

Wai-Hei Lam et al. Infect Immun. .

Abstract

Talaromyces marneffei infection causes talaromycosis (previously known as penicilliosis), a very important opportunistic systematic mycosis in immunocompromised patients. Different virulence mechanisms in T. marneffei have been proposed and investigated. In the sera of patients with talaromycosis, Mp1 protein (Mp1p), a secretory galactomannoprotein antigen with two tandem ligand-binding domains (Mp1p-LBD1 and Mp1p-LBD2), was found to be abundant. Mp1p-LBD2 was reported to possess a hydrophobic cavity to bind copurified palmitic acid (PLM). It was hypothesized that capturing of lipids from human hosts by expressing a large quantity of Mp1p is a virulence mechanism of T. marneffei It was shown that expression of Mp1p enhanced the intracellular survival of T. marneffei by suppressing proinflammatory responses. Mechanistic study of Mp1p-LBD2 suggested that arachidonic acid (AA), a precursor of paracrine signaling molecules for regulation of inflammatory responses, is the major physiological target of Mp1p-LBD2. In this study, we use crystallographic and biochemical techniques to further demonstrate that Mp1p-LBD1, the previously unsolved first lipid binding domain of Mp1p, is also a strong AA-binding domain in Mp1p. These studies on Mp1p-LBD1 support the idea that the highly expressed Mp1p is an effective AA-capturing protein. Each Mp1p can bind up to 4 AA molecules. The crystal structure of Mp1p-LBD1-LBD2 has also been solved, showing that both LBDs are likely to function independently with a flexible linker between them. T. marneffei and potentially other pathogens highly expressing and secreting proteins similar to Mp1p can severely disturb host signaling cascades during proinflammatory responses by reducing the availabilities of important paracrine signaling molecules.

Keywords: X-ray crystallography; arachidonic acid (AA); lipid-protein interaction; nuclear magnetic resonance; virulence factors.

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Figures

FIG 1
FIG 1
Crystallographic structure of Mp1p-LBD1 in complex with copurified PLM at 1.80-Å resolution. (A) Overall monomeric structure of Mp1p-LBD1 complexed with PLM (shown in cyan spheres). The length of this domain was about 47 Å. (B) Alignments of this monomeric Mp1p-LBD1 structure (green) with two domain-swapped, open Mp1p-LBD2 complexed with palmitic acid (PDB entry 3L1N, magenta and blue) (left, 762 main chain atoms with RMSD of 0.668 Å) and monomeric Mp1p-LBD2 complexed with arachidonic acid (PDB entry 5CSD, red) (right, 811 main chain atoms with RMSD of 0.538 Å). Helix 3 of Mp1p-LBD1 resembled the same helix in 5CSD but not 3L1N (regions highlighted in orange boxes), in which helix 3 turns and breaks the continuation at conserved glycine residues (indicated by arrows in corresponding colors). (C) 2mFo-DFc map (green; contour level, 1.0 σ) of PLM in refined model. (D) The amino acid residues involved in interaction between copurified PLM and Mp1p-LBD1.
FIG 2
FIG 2
Superposition of different Mp1p-LBD structures with different ligand positions revealed a possible reason for domain swapping in Mp1p-LBD2 in crystal structures. (A) Alignment of helix 3 from Mp1p-LBD1 complexed with PLM (orange) and Mp1p-LBD2 complexed with AA (5CSD, cyan) near the hinge regions. (B) Alignment of H3 helices from Mp1p-LBD1 complexed with PLM (orange) and LBD2 complexed with PLM (3L1N; green and purple for helix 3 from another Mp1p-LBD2 monomer) near the hinge regions. The PLM in Mp1p-LBD1 and AA in Mp1p-LBD2 locate near the hinge points of both Mp1p-LBDs, providing additional hydrophobic interactions around this region that may stabilize the closed configuration during crystallization. PLM in Mp1p-LBD2, however, locates on the other side of the binding cavity, leading to the absence of the hydrophobic interaction, and may lead to a domain-swapped open structure, as observed in 3L1N.
FIG 3
FIG 3
Mp1p-LBD1 complexed with 1 or 2 AAs. (A) Overall closed monomeric structures of Mp1p-LBD1 complexed with 1 AA molecule (left) and 2 AA molecules (right). Bound AAs are shown in cyan spheres at the middle of the five-helix bundles. (B) 2mFo-DFc maps (green; contour level, 1.0 σ) of AA in refined models. (Top) Singly bound AA. (Bottom) Two AAs in 2-AA bound form. (C) Detailed interaction between AA and Mp1p-LBD1. (Left) Singly bound AA in Mp1p-LBD1-AA form. (Middle) AAa in Mp1p-LBD1-2AA form. Ser165 also provides hydrogen bond. (Right) AAb in Mp1p-LBD1-2AA on the other side of the cavity.
FIG 4
FIG 4
Asn105 in Mp1p-LBD1 provides additional hydrogen bond to bind AA, leading to shifted AA positions compared to those of AAs in Mp1p-LBD2. (A) Superposition of Mp1p-LBD1-1AA (pink) and Mp1p-LBD1-2AAs (green). AAa in Mp1p-LBD1-2AA superimposes the singly bound AA. Asn105 is involved in forming a hydrogen bond with bound AA in both forms. (B) Superposition of Mp1p-LBD1-1AA (pink) and Mp1p-LBD2-1AAs (orange). It is clear that the interlude of Asn105 preferentially forms a hydrogen bond with singly bound AA in Mp1p-LBD1, while the singly bound AA in Mp1p-LBD2 penetrates deeper into the cavity and forms a hydrogen bond with the conserved glutamine residue (Gln298 in Mp1p-LBD2). (C) Superposition of Mp1p-LBD1-2AA (green) and Mp1p-LBD2-2AAs (blue). The conformation of the 2 bound AAs is largely conserved in both Mp1p-LBDs, but the positions of the head groups differ due to an additionally available hydrogen bond from Asn105 in Mp1p-LBD1.
FIG 5
FIG 5
Crystal structure of Mp1p-LBD1-LBD2 at 4.20-Å resolution. (A) Pseudodimeric structure of Mp1p-LBD1-LBD2. One monomer is green and the other is yellow. (B) Structure at Mp1p-LBD2 hinge point Gly259 of helix 3 (in full, shown in red) causing the open conformation of Mp1p-LBD2. Thus, this part is the same as the single-domain Mp1p-LBD2-PLM structure (PDB entry 3L1N). (C) The two modelled linkers (shown in red) built between two pairs of Mp1p-LBD1 and Mp1p-LBD2.
FIG 6
FIG 6
(A) MS/MS spectra of the precursor ions [M–H]− at m/z 303.2328 on arachidonic acid standard (upper trace) and that of organic extract from the in vitro pulldown experiment (lower trace) at the same collision energy (CE) of 30 to 50 V. (B) MS/MS precursor ion scan on lysophosphatidylcholine (LPC) head group (inset) at m/z 184.0715 at CE of 20 V (upper trace) and 30 to 50 V (lower trace).
FIG 7
FIG 7
NMR titration experiments of 15N-labeled Mp1p-LBD1 with AA. 1H-15N HSQC titration spectra of 15N-Mp1p-LBD1 in the absence (blue) and presence (red) of AA are shown. Mp1p-LBD1 was titrated against AA at pH 8.0. Most peaks displayed slow exchange (peak weakening), while some showed fast exchange (peak shifting).
FIG 8
FIG 8
ITC of AA binding to Mp1p-LBD1. (Top) Raw heat of binding obtained by ITC when AA was mixed with Mp1p-LBD1. (Bottom) Table of thermodynamic parameters obtained by fitting the ITC data to a two-state binding model (Kd, dissociation constant; ΔH, change in enthalpy; -TΔS, change in entropy; N, number of binding sites; subscripts 1 and 2 refer to the 1st and 2nd binding steps for data fit to a two-state model).
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