Structural plasticity of ll-37 indicates elaborate functional adaptation mechanisms to bacterial target structures

The human cathelicidin LL-37 is a multifunctional peptide of the human innate immune system. Among the various functions of LL-37, its antimicrobial activity is important in controlling the microorganisms of the human body. The target molecules of LL-37 in bacteria include membrane lipids, lipopolysaccharides (LPS), lipoteichoic acid (LTA), proteins, DNA and RNA. In this mini-review, we summarize the entity of LL-37 structural data determined over the last 15 years and specifically discuss features implicated in the interactions with lipid-like molecules. For this purpose, we discuss partial and full-length structures of LL-37 determined in the presence of membrane-mimicking detergents. This constantly growing structural database is now composed of monomers, dimers, tetramers, and fiber-like structures. The diversity of these structures underlines an unexpected plasticity and highlights the conformational and oligomeric adaptability of LL-37 necessary to target different molecular scaffolds. The recent co-crystal structures of LL-37 in complex with detergents are particularly useful to understand how these molecules mimic lipids and LPS to induce oligomerization and fibrillation. Defined detergent binding sites provide deep insights into a new class of peptide scaffolds, widening our view on the fascinating world of the LL-37 structural factotum. Together, the new structures in their evolutionary context allow for the assignment of functionally conserved residues in oligomerization and target interactions. Conserved phenylalanine and arginine residues primarily mediate those interactions with lipids and LPS. The interactions with macromolecules such as proteins or DNA remain largely unexplored and open a field for future studies aimed at structures of LL-37 complexes. These complexes will then allow for the structure-based rational design of LL-37-derived peptides with improved antibiotic properties

N-Acetilglutamato sintasa : correlaciones estructura-función

La N–acetilglutamato sintasa (NAGS) cataliza la formación del N-acetilglutamato (NAG) usando como sustratos glutamato y acetil-CoA. En microorganismos y plantas, esta reacción es el primer paso de la ruta de biosíntesis de la arginina, mientras que, en animales, está implicada en el control del ciclo de la urea, ya que el NAG es un activador esencial de la carbamilfosfato sintetasa I encargada de la formación del carbamilfosfato, intermedio en el ciclo de la urea. En humanos, la deficiencia de NAGS produce hiperamonemia y, desde 2002, año en el que el gen de la NAGS humana fue identificado, varias mutaciones han sido publicadas. Esta tesis trata, principalmente, de la NAGS bacteriana clásica de Pseudomonas aeruginosa (PaNAGS), la cual se organiza en dos dominios: uno N-terminal llamado aminoácido quinasa (AAK) que presenta el plegamiento característico de esta familia (α3β8α4) unido por un corto conector a un dominio acetiltransferasa (GNAT). Al inicio de esta tesis, pocos estudios se habían hecho con esta enzima y no se conocía nada de su estructura, pero sí se tenían datos estructurales de la N-acetilglutamato quinasa (NAGK) enzima que cataliza el segundo paso en la ruta de síntesis de la arginina. Esta enzima, cuyo monómero presentaba el plegamiento AAK, era un hexámero, concretamente, un trímero de dímeros conectados por sus hélices N-terminales, siendo éstas responsables, a su vez, de la inhibición por la arginina. En esta tesis, se delinea, mediante mutagénesis dirigida, el sitio para la arginina en la PaNAGS, mostrando la misma localización que en la NAGK. Además, basándose en la conservación de los residuos clave de unión de arginina en la NAGS bacteriana y humana, se propone que el sitio para la arginina sea el mismo en la forma humana. También por medio de mutagénesis dirigida, se demuestra que el dominio GNAT es el que presenta el sitio de unión de los sustratos y el centro catalítico. Una mutación localizada en el dominio AAK, muestra el carácter modulador de este dominio sobre el dominio GNAT, al que le confiere mayor afinidad por el glutamato. Esta hipótesis se ve reforzada con la obtención de los dominios aislados, mostrando que el dominio AAK es hexamérico y no presenta actividad sintasa ni quinasa y que el dominio GNAT cataliza la reacción por sí solo pero con un Km para el glutamato muy alto y sin ser inhibido por la arginina. Para determinar qué dominio AAK confiere afinidad por el glutamato al dominio GNAT, se obtiene una forma truncada de la hélice N-terminal de la PaNAGS, la cual es responsable de la hexamerización de la enzima, obteniendo entonces dímeros. Esta forma, con baja afinidad por el glutamato e insensible a la inhibición por arginina, demuestra que el dominio AAK que modula la actividad del GNAT no es el de la propia subunidad, sino el de la subunidad vecina. Por homología con el mecanismo de inhibición de la NAGK, se propone un mecanismo de inhibición para la NAGS, en el que, la unión de la arginina, produce una expansión del anillo hexamérico de dominios AAK de la NAGS, lo cual provoca que el conector interdominios ejerza una tracción sobre el dominio catalítico GNAT, cambiando su posición y dificultando la catálisis. El mecanismo propuesto, se somete a prueba mediante mutantes de elongación (mostrando abolición de la inhibición) y acortamiento del conector (con inhibición constitutiva, la cual se caracteriza en la forma silvestre por mostrar un aumento en el Km del glutamato y un descenso acusado de la Vmax). Además, modificando la secuencia del conector interdominios, conseguimos que la arginina pase de ser un inhibidor a un activador de la NAGS, como ocurre en la Naturaleza ya que la arginina inhibe las NAGSs bacterianas mientras que activa las NAGSs de mamíferos. Mediante la introducción de dos mutaciones en el dominio AAK de la NAGS, conseguimos recuperar la actividad quinasa, demostrando que el dominio AAK de la NAGS es una NAGK ancestral

The role of bacterial transport systems in the removal of host antimicrobial peptides in Gram-negative bacteria.

Antibiotic resistance is a global issue that threatens our progress in healthcare and life expectancy. In recent years, antimicrobial peptides (AMPs) have been considered as promising alternatives to the classic antibiotics. AMPs are potentially superior due to their lower rate of resistance development, since they primarily target the bacterial membrane ("Achilles´ heel" of the bacteria). However, bacteria have developed mechanisms of AMP resistance, including the removal of AMPs to the extracellular space by efflux pumps such as the MtrCDE or AcrAB-TolC systems, and the internalisation of AMPs to the cytoplasm by the Sap transporter, followed by proteolytic digestion. In this review, we focus on AMP transport as a resistance mechanism compiling all the experimental evidence for the involvement of efflux in AMP resistance in Gram-negative bacteria and combine this information with the analysis of the structures of the efflux systems involved. Finally, we expose some open questions with the aim of arousing the interest of the scientific community towards the AMPs - efflux pumps interactions. All the collected information broadens our understanding of AMP removal by efflux pumps and gives some clues to assist the rational design of AMP-derivatives as inhibitors of the efflux pumps

The structure of the antimicrobial human cathelicidin LL-37 shows oligomerization and channel formation in the presence of membrane mimics

The human cathelicidin LL-37 serves a critical role in the innate immune system defending bacterial infections. LL-37 can interact with molecules of the cell wall and perforate cytoplasmic membranes resulting in bacterial cell death. To test the interactions of LL-37 and bacterial cell wall components we crystallized LL-37 in the presence of detergents and obtained the structure of a narrow tetrameric channel with a strongly charged core. The formation of a tetramer was further studied by cross-linking in the presence of detergents and lipids. Using planar lipid membranes a small but defined conductivity of this channel could be demonstrated. Molecular dynamic simulations underline the stability of this channel in membranes and demonstrate pathways for the passage of water molecules. Time lapse studies of E. coli cells treated with LL-37 show membrane discontinuities in the outer membrane followed by cell wall damage and cell death. Collectively, our results open a venue to the understanding of a novel AMP killing mechanism and allows the rational design of LL-37 derivatives with enhanced bactericidal activity

Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity

Chitin deacetylases (CDAs) act on chitin polymers and low molecular weight oligomers producing chitosans and chitosan oligosaccharides. Structurally-defined, partially deacetylated chitooligosaccharides produced by enzymatic methods are of current interest as bioactive molecules for a variety of applications. Among Pochonia chlamydosporia (Pc) annotated CDAs, gene pc_2566 was predicted to encode for an extracellular CE4 deacetylase with two CBM18 chitin binding modules. Chitosan formation during nematode egg infection by this nematophagous fungus suggests a role for their CDAs in pathogenicity. The P. chlamydosporia CDA catalytic domain (PcCDA) was expressed in E. coli BL21, recovered from inclusion bodies, and purified by affinity chromatography. It displays deacetylase activity on chitooligosaccharides with a degree of polymerization (DP) larger than 3, generating mono- and di-deacetylated products with a pattern different from those of closely related fungal CDAs. This is the first report of a CDA from a nematophagous fungus. On a DP5 substrate, PcCDA gave a single mono-deacetylated product in the penultimate position from the non-reducing end (ADAAA) which was then transformed into a di-deacetylated product (ADDAA). This novel deacetylation pattern expands our toolbox of specific CDAs for biotechnological applications, and will provide further insights into the determinants of substrate specificity in this family of enzymes.This work was supported by the European Commission NANO3BIO project, grant agreement n° 613931 (to A.P.), and grants BFU2016–77427-C2-1-R (to A.P.) and AGL2015-66833-R (to L.L.) from MINECO, Spain. Pre-doctoral contracts are acknowledged to Generalitat Valenciana (to A.A.), Generalitat de Catalunya (to H.A.), and European Commission NANO3BIO project (to L.G.)

Structural basis for selective recognition of acyl chains by the membrane-associated acyltransferase PatA

The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We report here the crystal structures of PatA from Mycobacterium smegmatis in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl-CoA analog. The structures reveal an alpha/beta architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design.This work was supported by the European Commission Contract HEALTH-F3-2011-260872, the Spanish Ministry of Economy and Competitiveness Contract BIO2013-49022-C2-2-R, and the Basque Government (to M.E.G.); Slovak Research and Development Agency Contract No. DO7RP-0015-11 (to K.M.) and the NIH/NIAID grant AI064798 (to M.J.). D.A.-J. acknowledges the support from Fundacion Biofisica Bizkaia. We gratefully acknowledge Sonia Lopez-Fernandez (Unit of Biophysics, CSIC, UPV/EHU, Spain), Drs E. Ogando and T. Mercero (Scientific Computing Service UPV/EHU, Spain) for technical assistance. We thank the Swiss Light Source (SLS), and the Diamond Light Source (DLS) for granting access to synchrotron radiation facilities and their staff for the onsite assistance. We specially thank the BioStruct-X project to support access to structural biology facilities. We also acknowledge all members of the Structural Glycobiology Group (Spain) for valuable scientific discussions. The following reagent was obtained through BEI Resources, NIAID, NIH: Mycobacterium tuberculosis, Strain H37Rv, Purified Phosphatidylinositol Mannosides 1 and 2 (PIM1,2), NR-14846

The antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of Mycobacterium tuberculosis

open20openRosado, Leonardo Astolfi; Wahni, Khadija; Degiacomi, Giulia; Pedre, Brandã¡n; Young, David; De la Rubia, Alfonso G.; Boldrin, Francesca; Martens, Edo; Marcos-Pascual, Laura; Sancho-Vaello, Enea; Albesa-Jové, David; Provvedi, Roberta; Martin, Charlotte; Makarov, Vadim; Versã©es, Wim; Verniest, Guido; Guerin, Marcelo; Mateos, Luis M.; Manganelli, Riccardo; Messens, JorisRosado, Leonardo Astolfi; Wahni, Khadija; Degiacomi, Giulia; Pedre, Brandã¡n; Young, David; De la Rubia, Alfonso G.; Boldrin, Francesca; Martens, Edo; Marcos-Pascual, Laura; Sancho-Vaello, Enea; Albesa-Jové, David; Provvedi, Roberta; Martin, Charlotte; Makarov, Vadim; Versã©es, Wim; Verniest, Guido; Guerin, Marcelo; Mateos, Luis M.; Manganelli, Riccardo; Messens, Jori

N-acetilglutamato sintetasa: correlaciones estructura–función

142 pág.Para la realización de esta Tesis, Enea Sancho Vaello ha disfrutado de una Beca lanzadera del CIBER de enfermedades raras (CIBERer) y de una Ayuda predoctoral de formación en investigación en salud del Instituto de Salud Carlos III. El trabajo se ha enmarcado dentro de los proyectos: “Complejos macromoleculares, proteínas multifuncionales, pluriempleo y enfermedades raras en la familia aminoácido quinasa” (BFU2008-05021) financiado por el Ministerio de Ciencia e Innovación, “Bases estructurales de enfermedades raras metabólicas, endovasculares y de la hemostasia", financiado por el CIBERER-Instituto de Salud Carlos III y el Ministerio de Sanidad y Consumo y “Caracterización molecular de la patogénesis de los errores del ciclo de la urea: déficit de la acetilglutamato sintasa” (AP-082/10) financiado por la Conselleria de Sanitat- Generalitat Valenciana. El interés del grupo en la N-acetilglutamato sintasa se relaciona directamente con las patologías del ciclo de la urea que constituyen una de sus líneas de actuación dentro del CIBERER-ISCIII.Peer reviewe

Functional dissection of N-acetylglutamate synthase (ArgA) of Pseudomonas aeruginosa and restoration of its ancestral N-acetylglutamate kinase activity

11 páginas, 7 figuras, 1 tabla.In many microorganisms, the first step of arginine biosynthesis is catalyzed by the classical N-acetylglutamate synthase (NAGS), an enzyme composed of N-terminal amino acid kinase (AAK) and C-terminal histone acetyltransferase (GNAT) domains that bind the feedback inhibitor arginine and the substrates, respectively. In NAGS, three AAK domain dimers are interlinked by their N-terminal helices, conforming a hexameric ring, whereas each GNAT domain sits on the AAK domain of an adjacent dimer. The arginine inhibition of Pseudomonas aeruginosa NAGS was strongly hampered, abolished, or even reverted to modest activation by changes in the length/sequence of the short linker connecting both domains, supporting a crucial role of this linker in arginine regulation. Linker cleavage or recombinant domain production allowed the isolation of each NAGS domain. The AAK domain was hexameric and inactive, whereas the GNAT domain was monomeric/dimeric and catalytically active although with ∼50-fold-increased and ∼3-fold-decreased K(m)(glutamate) and k(cat) values, respectively, with arginine not influencing its activity. The deletion of N-terminal residues 1 to 12 dissociated NAGS into active dimers, catalyzing the reaction with substrate kinetics and arginine insensitivity identical to those for the GNAT domain. Therefore, the interaction between the AAK and GNAT domains from different dimers modulates GNAT domain activity, whereas the hexameric architecture appears to be essential for arginine inhibition. We proved the closeness of the AAK domains of NAGS and N-acetylglutamate kinase (NAGK), the enzyme that catalyzes the next arginine biosynthesis step, shedding light on the origin of classical NAGS, by showing that a double mutation (M26K L240K) in the isolated NAGS AAK domain elicited NAGK activityThis work was supported by grants BFU2008-05021 of the Spanish Ministry of Science (MEC and MICINN) and Prometeo/2009/051 of the Valencian Government. ES-V and . MLF-M were supported by contracts from the Instituto de Salud Carlos III and the JAE-DOC Programme of the Consejo Superior de Investigaciones Científicas.Peer reviewe