RESUMO
The ability of enzymes to distinguish between fatty acyl groups can involve molecular measuring devices termed hydrocarbon rulers, but the molecular basis for acyl-chain recognition in any membrane-bound enzyme remains to be defined. PagP is an outer membrane acyltransferase that helps pathogenic bacteria to evade the host immune response by transferring a palmitate chain from a phospholipid to lipid A (endotoxin). PagP can distinguish lipid acyl chains that differ by a single methylene unit, indicating that the enzyme possesses a remarkably precise hydrocarbon ruler. We present the 1.9 A crystal structure of PagP, an eight-stranded beta-barrel with an unexpected interior hydrophobic pocket that is occupied by a single detergent molecule. The buried detergent is oriented normal to the presumed plane of the membrane, whereas the PagP beta-barrel axis is tilted by approximately 25 degrees. Acyl group specificity is modulated by mutation of Gly88 lining the bottom of the hydrophobic pocket, thus confirming the hydrocarbon ruler mechanism for palmitate recognition. A striking structural similarity between PagP and the lipocalins suggests an evolutionary link between these proteins.
Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Endotoxinas/química , Endotoxinas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Hidrocarbonetos/metabolismo , Palmitatos/metabolismo , Acilação , Aciltransferases/genética , Aciltransferases/isolamento & purificação , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Transporte Biológico , Cristalografia por Raios X , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Expressão Gênica , Hidrocarbonetos/química , Ligantes , Metabolismo dos Lipídeos , Lipídeos/química , Modelos Moleculares , Dados de Sequência Molecular , Palmitatos/química , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
The enzymology of palmitate addition to lipid A can be traced to the early discovery of monosaccharide lipid A precursors, but the functional importance of lipid A palmitoylation in bacterial resistance to the host immune response has emerged only recently. Lipid A palmitoylation in enterobacteria is determined by a PhoP/PhoQ-activated gene pagP, which encodes an unusual outer membrane enzyme of lipid A biosynthesis. PagP structure and dynamics have now been elucidated by both NMR spectroscopy and X-ray crystallography. PagP is an 8-stranded antiparallel beta-barrel preceded by an N-terminal amphipathic alpha-helix. The PagP barrel axis is uniquely tilted by 30 degrees with respect to the membrane normal. An interior hydrophobic pocket in the upper half of the molecule functions as a hydrocarbon ruler, which allows the enzyme to distinguish palmitate from other acyl chains found in phospholipids. Internalization of a phospholipid palmitoyl group within the barrel appears to occur by lateral diffusion from the outer leaflet through non-hydrogen bonded regions between beta-strands. The MsbA-dependent trafficking of lipids from the inner membrane to the outer membrane outer leaflet is necessary for lipid A palmitoylation in vivo. Efforts to determine the PagP catalytic mechanism may lead to the development of inhibitors for the treatment of infections.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Lipídeo A/metabolismo , Ácido Palmítico/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Configuração de Carboidratos , Membrana Celular/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
The bacterial outer membrane enzyme PagP transfers a palmitate chain from a phospholipid to lipid A. In a number of pathogenic Gram-negative bacteria, PagP confers resistance to certain cationic antimicrobial peptides produced during the host innate immune response. The global fold of Escherichia coli PagP was determined in both dodecylphosphocholine and n-octyl-beta-d-glucoside detergent micelles using solution NMR spectroscopy. PagP consists of an eight-stranded anti-parallel beta-barrel preceded by an amphipathic alpha helix. The beta-barrel is well defined, whereas NMR relaxation measurements reveal considerable mobility in the loops connecting individual beta-strands. Three amino acid residues critical for enzymatic activity localize to extracellular loops near the membrane interface, positioning them optimally to interact with the polar headgroups of lipid A. Hence, the active site of PagP is situated on the outer surface of the outer membrane. Because the phospholipids that donate palmitate in the enzymatic reaction are normally found only in the inner leaflet of the outer membrane, PagP activity may depend on the aberrant migration of phospholipids into the outer leaflet. This finding is consistent with an emerging paradigm for outer membrane enzymes in providing an adaptive response toward disturbances in the outer membrane.
