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Crystal Structure of the Mammalian Lipopolysaccharide Detoxifier

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Crystal Structure of the Mammalian Lipopolysaccharide Detoxifier Crystal structure of the mammalian PNAS PLUS lipopolysaccharide detoxifier Alexei Gorelika,b, Katalin Illesa,b, and Bhushan Nagara,b,1 aDepartment of Biochemistry, McGill University, Montreal, H3G0B1, Canada; and bGroupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, H3G0B1, Canada Edited by Vishva M. Dixit, Genentech, San Francisco, CA, and approved December 26, 2017 (received for review November 13, 2017) LPS is a potent bacterial endotoxin that triggers the innate immune and possibly on LPS solubilized by the proteins CD14 or LBP system. Proper recognition of LPS by pattern-recognition receptors (22). In mice, AOAH deficiency results in prolonged tolerance requires a full complement of typically six acyl chains in the lipid (over 2 months) (9), whereas its overexpression improves survival portion. Acyloxyacyl hydrolase (AOAH) is a host enzyme that and recovery from bacterial infection (23). It has been suggested removes secondary (acyloxyacyl-linked) fatty acids from LPS, that enhancing AOAH activity could improve outcomes in infec- rendering it immunologically inert. This activity is critical for tions and certain chronic conditions in humans (9, 23). recovery from immune tolerance that follows Gram-negative Although AOAH was discovered more than 30 years ago (13, infection. To understand the molecular mechanism of AOAH 14), an understanding of the molecular details of AOAH func- function, we determined its crystal structure and its complex with LPS. tion is still lacking. The determinants of LPS recognition by the The substrate’s lipid moiety is accommodated in a large hydrophobic protein are unknown, as is the basis of its selectivity toward pocket formed by the saposin and catalytic domains with a secondary secondary acyl chains. The specific role of its built-in saposin acyl chain inserted into a narrow lateral hydrophobic tunnel at the domain (19) is unclear, and the mechanism by which proteolytic active site. The enzyme establishes dispensable contacts with the processing of AOAH in acidic organelles increases its enzymatic phosphate groups of LPS but does not interact with its oligosaccha- activity on LPS (19) has not been determined. Here, we report ride portion. Proteolytic processing allows movement of an amphi- the crystal structures of AOAH and its complex with lipopoly- pathic helix possibly involved in substrate access at membranes. saccharide; these findings, together with biophysical and bio- chemical analyses, address these questions. BIOCHEMISTRY acyloxyacyl hydrolase | lipopolysaccharide | immune tolerance | saposin | calcium-binding protein Results Overall Structure. Endosomal AOAH undergoes differential ipopolysaccharide (LPS, endotoxin) is the major constituent posttranslational processing relative to its secreted counterpart. Lof the external face of the Gram-negative bacterial outer The N-terminal signal peptide is removed from both forms, but a membrane (1) and is the most potent agonist of innate immunity propeptide is further cleaved off only from the endosomal pro- in humans (1). This complex molecule is composed of a glycolipid tein (17, 19). Additionally, a proteolytic event in the acidic en- called “lipid A,” linked to a core oligosaccharide to which a long vironment of endosomes separates the enzyme into a small and a O-polysaccharide is attached (1, 2). Lipid A normally consists of a large subunit linked by a disulfide bridge (15, 17, 19). We de- diphosphorylated diglucosamine scaffold bearing several lipid termined the crystal structures of full-length human, murine, and tails. Stimulation of the MD-2–Toll-like receptor 4 (TLR4) im- rabbit AOAH lacking the signal peptide, including the structures mune receptor complex by LPS at the cell surface requires the of the intact and cleaved protein (Fig. 1 A and B). The large presence of six acyl chains on its lipid A moiety; penta- or hepta- subunit comprises the catalytic domain, a hitherto unrecognized acyl ligands are 100-fold less active, and tetra-acyl analogs are calcium-binding region, and an isolated amphipathic helix (α6). inactive (1–5). Hexa-acyl LPS is also necessary for the detection of – bacterial invasion by cytoplasmic caspases 4, 5, and 11 (6 8). Significance Following the acute response, the host can enter a tolerant state in which subsequent responses to LPS are dampened (5, 9, LPS is the major bacterial molecule recognized by the human 10), preventing excessive inflammation. Persistent exposure of innate immune system. It elicits a strong inflammatory re- macrophages to endotoxin down-regulates the expression of sponse followed by a state of immune tolerance. The human proinflammatory factors, including TNFα, and represses in- enzyme acyloxyacyl hydrolase (AOAH) then detoxifies LPS tracellular signal-transduction pathways initiated by TLR4 (11). to reestablish sensitivity for subsequent infections. We determined This involves regulators, including SOCS1, ST2, IRAKM, SHIP, the 3D structure of AOAH to better understand its function. The GILZ, and mir-146a, as well as transcriptional control (11). enzyme binds to LPS via a hydrophobic surface and contains a However, recovery from this state of immune tolerance is re- hydrophobic tunnel into which one fatty acyl chain of the LPS fits quired for proper defense against further Gram-negative infec- to be cleaved off. AOAH also interacts with the phosphate groups tions. Reestablishment of sensitivity to LPS is accomplished by of LPS but not its saccharide portion. Additionally, we report an its detoxification from the resolving infection (5, 9, 10, 12). This unexpected calcium-binding domain in this enzyme. process is carried out by a host enzyme, acyloxyacyl hydrolase (AOAH), which removes fatty acids from lipid A (13, 14), Author contributions: A.G. designed research; A.G. and K.I. performed research; A.G. and thereby rendering it inert to TLR4- or caspase-based detection. B.N. analyzed data; and A.G. and B.N. wrote the paper. Lipid A typically contains four primary acyl chains directly at- The authors declare no conflict of interest. tached to the diglucosamine backbone and a variable number of This article is a PNAS Direct Submission. C secondary fatty acids linked to the primary ones (Fig. 1 ); AOAH Published under the PNAS license. selectively hydrolyzes the secondary (acyloxyacyl-linked) chains Data deposition: The atomic coordinates and structure factors have been deposited in the (13–19). This protein is found in endosomes or possibly lysosomes Protein Data Bank (PDB), www.wwpdb.org (PDB ID codes 5W78 and 5W7A–5W7F). of phagocytic white blood cells (5, 13, 15, 18, 19) and liver-resident 1To whom correspondence should be addressed. Email: [email protected]. macrophages (20) as well as in the serum of certain animals (19, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 21) and acts on LPS aggregates, bacterial membrane remnants, 1073/pnas.1719834115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1719834115 PNAS Early Edition | 1of10 Downloaded by guest on September 30, 2021 A crystallized construct P SP P saposin ext. α6 Ca-binding esterase 5342 115 157 942971 362S 035H725D 575 O antigen B C core oligosaccharide 4’ 3’2’ 1 lipid A 3 2 N C Fig. 1. AOAH structure and substrate. (A) Domain organization of AOAH. The native proteolytic processing site is marked by a red triangle. Catalytic triad residues are represented by pink circles. α6, amphipathic helix; ext, saposin domain extension; PP, propeptide; SP, signal peptide. (B) Mature AOAH is composed of a small subunit containing a saposin domain (red) with an extension (orange) and a large subunit comprising an esterase domain (green), a calcium-binding region (dark blue), and an amphipathic helix α6 (cyan). The native proteolytic processing site is indicated by scissors, and a segment not visible in the structure is represented by a gray dashed line. The N and C termini are labeled (the propeptide is not visible in the structure). N-linked glycans (white sticks) are simplified for clarity. Calcium ions (yellow spheres) and disulfide bonds (yellow sticks) are displayed, including the interdomain bridge as thicker sticks. Catalytic triad residues are shown as pink sticks. (C) Chemical structure of E. coli LPS, with positions on the diglucosamine backbone of lipid A indicated in red. Secondary (acyloxyacyl) chains are in blue, with the bonds hydrolyzed by AOAH marked by blue arrows. The small subunit consists of a saposin domain (17, 19); saposins state and associate with the catalytic domain via a hydrophobic are lipid- and membrane-binding modules that assist the func- interface (27–29) or can adopt a closed conformation with fewer tion of lysosomal hydrolases, among other roles (24, 25). The contacts to the rest of the enzyme (27). In AOAH, the relative propeptide is not visible in the electron density of the structures. position of the saposin domain is invariant in all the crystal The protein bears four Asn-linked glycans, whereas the Asn336 structures reported here, including the intact and proteolytically sequon is buried and inaccessible for glycosylation. processed forms, and in presence of substrates and detergent. The small subunit is abutted against two long segments of the cata- Catalytic and Saposin Domains. The AOAH catalytic domain has a lytic domain (Fig. 1B), interacting with the latter via an extensive five-stranded parallel β-sheet at its core surrounded by 11 helices 1,165-Å2 hydrophobic interface involving 40 side chains,
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