Struktur- und Funktionsanalyse virulenzassoziierter listerieller Oberflächenproteine

The cell wall of the Gram-positive human pathogen Listeria monocytogenes is decorated with surface-associated proteins, a subset of which are virulence factors crucial for host-pathogen interactions. This thesis comprises two sections, each of which concentrates on a distinct class of proteins involved in listerial pathogenesis. The first section covers the biochemical and structural characterization of the autolysin Auto, a recently identified virulence factor. Its lytic activity and substrate specificity identify Auto as a broad-spectrum N-acetylglucosaminidase. It is structurally related to g-type lysozyme, but possesses a unique N-terminal extension, which forms an alpha-helical plug to obstruct the substrate-binding site, revealing a novel mode of autoinhibition. Auto is activated by proteolytic cleavage and elicits highest activity at pH 4.8. The combined features of Auto allow for its spatiotemporal activation during bacterial infection, providing the basis for the dynamic adaptation of the virulence-promoting surface proteome, presumably during the phagosomal step of infection. In the second section, the crystal structures of the functional domains of InlJ and InlG, two members of the internalin family of cell-surface proteins, are described. Whereas InlG is the smallest cell-wall bound internalin with 5 leucine-rich repeats (LRRs), InlJ bears 15 LRRs, the same number as InlA, the prototypical internalin family member. The repeat consensus sequence of InlJ differs from that of other internalins in that each repeat consists of 21 instead of 22 residues, and by replacing one hydrophobic residue by a conserved cysteine. Cysteines in consecutive repeats stack to form a regular intramolecular ladder. Quantitative analysis of the curvature, twist and lateral bending angles of InlJ and InlG, and comparison of these to other LRR proteins, provides a means for a systematic, geometric analysis of LRR-structures.Die Zellwand des Gram-positiven Humanpathogens Listeria monocytogenes ist mit oberflächenassoziierten Proteinen bedeckt, von denen einige als Virulenzfaktoren zentrale Wirt-Pathogen-Interaktionen vermitteln. Diese Arbeit umfasst zwei Teile, wobei der Fokus auf zwei verschiedenen, an listerieller Virulenz beteiligten Proteinklassen liegt. Der erste Teil umfasst die biochemische und strukturelle Charakterisierung des Autolysins Auto, eines kürzlich identifizierten Virulenzfaktors. Seine lytische Aktivität und Substratspezifität kennzeichnen Auto als Breitspektrum-N-Acetylglucosaminidase. Strukturell ähnelt Auto dem G-Typ Lysozym, besitzt jedoch eine N-terminale Extension, die in Form eines alpha-helikalen Stopfens die Substratbindetasche blockiert und eine neue Art der Autoinhibition darstellt. Die Aktivierung erfolgt duch Proteolyse, das pH-Optimum liegt bei pH 4.8. Diese Eigenschaften von Auto erlauben eine orts- und zeitabhängige Aktivierung während der bakteriellen Infektion und bilden so die Basis für eine dynamische Anpassung des virulenzfördernden Oberflächenproteoms, vermutlich im phagosomalen Infektionsschritt. Der zweite Teil der Arbeit beschreibt die Kristallstrukturen der funktionellen Domänen von InlJ und InlG, zweier Mitglieder der Oberflächenprotein-Familie der Internaline. Während InlG das kleinste zellwandgebundene Internalin mit 5 leucinreichen Repeats (LRRs) darstellt, weist InlJ 15 LRRs auf, ebensoviele wie der Prototyp der Familie, InlA. Die Konsensussequenz der InlJ-Repeats unterscheidet sich von der anderer Internaline durch die Länge von 21 statt 22 Resten sowie den Austausch eines hydrophoben Restes durch ein konserviertes Cystein. Die Cysteine in aufeinanderfolgenden Repeats bilden eine intramolekulare Leiter aus. Die quantitative Analyse der Krümmungs- Schraubungs- und seitlichen Biegungswinkel von InlJ und InlG sowie deren Vergleich mit anderen LRR-Proteinen bietet die Möglichkeit einer systematischen geometrischen Analyse von LRR-Proteinen

Membrane binding of antimicrobial peptides is modulated by lipid charge modification

Peptide interactions with lipid bilayers play a key role in a range of biological processes and depend on electrostatic interactions between charged amino acids and lipid headgroups. Antimicrobial peptides (AMPs) initiate the killing of bacteria by binding to and destabilizing their membranes. The multiple peptide resistance factor (MprF) provides a defense mechanism for bacteria against a broad range of AMPs. MprF reduces the negative charge of bacterial membranes through enzymatic conversion of the anionic lipid phosphatidyl glycerol (PG) to either zwitterionic alanyl-phosphatidyl glycerol (Ala-PG) or cationic lysyl-phosphatidyl glycerol (Lys-PG). The resulting change in the membrane charge is suggested to reduce the binding of AMPs to membranes, thus impeding downstream AMP activity. Using coarse-grained molecular dynamics to investigate the effects of these modified lipids on AMP binding to model membranes, we show that AMPs have substantially reduced affinity for model membranes containing Ala-PG or Lys-PG. More than 5000 simulations in total are used to define the relationship between lipid bilayer composition, peptide sequence (using five different membrane-active peptides), and peptide binding to membranes. The degree of interaction of a peptide with a membrane correlates with the membrane surface charge density. Free energy profile (potential of mean force) calculations reveal that the lipid modifications due to MprF alter the energy barrier to peptide helix penetration of the bilayer. These results will offer a guide to the design of novel peptides, which addresses the issue of resistance via MprF-mediated membrane modification

Hydrogen bond rotations as a uniform structural tool for analyzing protein architecture

Proteins fold into three-dimensional structures, which determine their diverse functions. The conformation of the backbone of each structure is locally at each Cα effectively described by conformational angles resulting in Ramachandran plots. These, however, do not describe the conformations around hydrogen bonds, which can be non-local along the backbone and are of major importance for protein structure. Here, we introduce the spatial rotation between hydrogen bonded peptide planes as a new descriptor for protein structure locally around a hydrogen bond. Strikingly, this rotational descriptor sampled over high-quality structures from the protein data base (PDB) concentrates into 30 localized clusters, some of which correlate to the common secondary structures and others to more special motifs, yet generally providing a unifying systematic classification of local structure around protein hydrogen bonds. It further provides a uniform vocabulary for comparison of protein structure near hydrogen bonds even between bonds in different proteins without alignment

Crystal Structure of the Vanadate-Inhibited Ca2+\mathrm{Ca^{2+}}-ATPase

Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca$^{2+}$-ATPase with bound vanadate in the absence of Ca$^{2+}$. Vanadate is bound at the catalytic site as a planar VO$_3$− in complex with water and Mg$^{2+}$ in a dephosphorylation transition-state-like conformation. Validating bound VO$_3$− by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl− site near the dephosphorylation site. Crystallization was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca$^{2+}$-ATPase. A comparison of the configurations of bound nucleotide analogs in the E2·VO$_3$− structure with that in E2·BeF$_3$− (E2P ground state analog) reveals multiple binding modes to the Ca$^{2+}$-ATPase

Membrane binding of antimicrobial peptides is modulated by lipid charge modification

Peptide interactions with lipid bilayers play a key role in a range of biological processes and depend on electrostatic interactions between charged amino acids and lipid headgroups. Antimicrobial peptides (AMPs) initiate the killing of bacteria by binding to and destabilizing their membranes. The multiple peptide resistance factor (MprF) provides a defense mechanism for bacteria against a broad range of AMPs. MprF reduces the negative charge of bacterial membranes through enzymatic conversion of the anionic lipid phosphatidyl glycerol (PG) to either zwitterionic alanyl-phosphatidyl glycerol (Ala-PG) or cationic lysyl-phosphatidyl glycerol (Lys-PG). The resulting change in the membrane charge is suggested to reduce the binding of AMPs to membranes, thus impeding downstream AMP activity. Using coarse-grained molecular dynamics to investigate the effects of these modified lipids on AMP binding to model membranes, we show that AMPs have substantially reduced affinity for model membranes containing Ala-PG or Lys-PG. More than 5000 simulations in total are used to define the relationship between lipid bilayer composition, peptide sequence (using five different membrane-active peptides), and peptide binding to membranes. The degree of interaction of a peptide with a membrane correlates with the membrane surface charge density. Free energy profile (potential of mean force) calculations reveal that the lipid modifications due to MprF alter the energy barrier to peptide helix penetration of the bilayer. These results will offer a guide to the design of novel peptides, which addresses the issue of resistance via MprF-mediated membrane modification

S-palmitoylation and s-oleoylation of rabbit and pig sarcolipin.

International audienceSarcolipin (SLN) is a regulatory peptide present in sarcoplasmic reticulum (SR) from skeletal muscle of animals. We find that native rabbit SLN is modified by a fatty acid anchor on Cys-9 with a palmitic acid in about 60% and, surprisingly, an oleic acid in the remaining 40%. SLN used for co-crystallization with SERCA1a (Winther, A. M., Bublitz, M., Karlsen, J. L., Moller, J. V., Hansen, J. B., Nissen, P., and Buch-Pedersen, M. J. (2013) Nature 495, 265-2691; Ref. 1) is also palmitoylated/oleoylated, but is not visible in crystal structures, probably due to disorder. Treatment with 1 m hydroxylamine for 1 h removes the fatty acids from a majority of the SLN pool. This treatment did not modify the SERCA1a affinity for Ca(2+) but increased the Ca(2+)-dependent ATPase activity of SR membranes indicating that the S-acylation of SLN or of other proteins is required for this effect on SERCA1a. Pig SLN is also fully palmitoylated/oleoylated on its Cys-9 residue, but in a reverse ratio of about 40/60. An alignment of 67 SLN sequences from the protein databases shows that 19 of them contain a cysteine and the rest a phenylalanine at position 9. Based on a cladogram, we postulate that the mutation from phenylalanine to cysteine in some species is the result of an evolutionary convergence. We suggest that, besides phosphorylation, S-acylation/deacylation also regulates SLN activity

A systematic approach to membrane protein crystallization in bilayers

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The SERCA residue Glu340 mediates interdomain communication that guides Ca2+ transport

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+-binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA's 10 transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+ and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca2+-binding transmembrane region