Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif.

Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors.

Crystallization and preliminary crystallographic characterization of the iron-regulated outer membrane lipoprotein FrpD from Neisseria meningitidis.

Fe-regulated protein D (FrpD) is a Neisseria meningitidis outer membrane lipoprotein that may be involved in the anchoring of the secreted repeat in toxins (RTX) protein FrpC to the outer bacterial membrane. However, the function and biological roles of the FrpD and FrpC proteins remain unknown. Native and selenomethionine-substituted variants of recombinant FrpD43-271 protein were crystallized using the sitting-drop vapour-diffusion method. Diffraction data were collected to a resolution of 2.25 A for native FrpD43-271 protein and to a resolution of 2.00 A for selenomethionine-substituted FrpD43-271 (SeMet FrpD43-271) protein. The crystals of native FrpD43-271 protein belonged to the hexagonal space group P6(2) or P6(4), while the crystals of SeMet FrpD43-271 protein belonged to the primitive orthorhombic space group P2(1)2(1)2(1).

Structural and molecular mechanism for autoprocessing of MARTX toxin of Vibrio cholerae at multiple sites.

The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP(6)). In this study, we demonstrated that InsP(6) is not simply an allosteric cofactor, but rather binding of InsP(6) stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-A crystal structure of this InsP(6)-bound unprocessed form of CPD was determined and revealed the scissile bond Leu(3428)-Ala(3429) captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP(6), but was reactivated for high affinity binding of InsP(6) by cooperative binding of both a new substrate and InsP(6). Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.

Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin.

The Gram-negative bacterium Vibrio cholerae is the causative agent of a severe diarrheal disease that afflicts three to five million persons annually, causing up to 200,000 deaths. Nearly all V. cholerae strains produce a large multifunctional-autoprocessing RTX toxin (MARTX(Vc)), which contributes significantly to the pathogenesis of cholera in model systems. The actin cross-linking domain (ACD) of MARTX(Vc) directly catalyzes a covalent cross-linking of monomeric G-actin into oligomeric chains and causes cell rounding, but the nature of the cross-linked bond and the mechanism of the actin cytoskeleton disruption remained elusive. To elucidate the mechanism of ACD action and effect on actin, we identified the covalent cross-link bond between actin protomers using limited proteolysis, X-ray crystallography, and mass spectrometry. We report here that ACD catalyzes the formation of an intermolecular iso-peptide bond between residues E270 and K50 located in the hydrophobic and the DNaseI-binding loops of actin, respectively. Mutagenesis studies confirm that no other residues on actin can be cross-linked by ACD both in vitro and in vivo. This cross-linking locks actin protomers into an orientation different from that of F-actin, resulting in strong inhibition of actin polymerization. This report describes a microbial toxin mechanism acting via iso-peptide bond cross-linking between host proteins and is, to the best of our knowledge, the only known example of a peptide linkage between nonterminal glutamate and lysine side chains.

Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin.

Vibrio cholerae secretes a large virulence-associated multifunctional autoprocessing RTX toxin (MARTX(Vc)). Autoprocessing of this toxin by an embedded cysteine protease domain (CPD) is essential for this toxin to induce actin depolymerization in a broad range of cell types. A homologous CPD is also present in the large clostridial toxin TcdB and recent studies showed that inositol hexakisphosphate (Ins(1,2,3,4,5,6)P(6) or InsP(6)) stimulated the autoprocessing of TcdB dependent upon the CPD (Egerer, M., Giesemann, T., Jank, T., Satchell, K. J., and Aktories, K. (2007) J. Biol. Chem. 282, 25314-25321). In this work, the autoprocessing activity of the CPD within MARTX(Vc) is similarly found to be inducible by InsP(6). The CPD is shown to bind InsP(6) (K(d), 0.6 microm), and InsP(6) is shown to stimulate intramolecular autoprocessing at both physiological concentrations and as low as 0.01 microm. Processed CPD did not bind InsP(6) indicating that, subsequent to cleavage, the activated CPD may shift to an inactive conformation. To further pursue the mechanism of autoprocessing, conserved residues among 24 identified CPDs were mutagenized. In addition to cysteine and histidine residues that form the catalytic site, 2 lysine residues essential for InsP(6) binding and 5 lysine and arginine residues resulting in loss of activity at low InsP(6) concentrations were identified. Overall, our data support a model in which basic residues located across the CPD structure form an InsP(6) binding pocket and that the binding of InsP(6) stimulates processing by altering the CPD to an activated conformation. After processing, InsP(6) is shown to be recycled, while the cleaved CPD becomes incapable of further binding of InsP(6).

A novel "clip-and-link" activity of repeat in toxin (RTX) proteins from gram-negative pathogens. Covalent protein cross-linking by an Asp-Lys isopeptide bond upon calcium-dependent processing at an Asp-Pro bond.

Clinical isolates of Neisseria meningitidis produce a repeat in toxin (RTX) protein, FrpC, of unknown biological activity. Here we show that physiological concentrations of calcium ions induce a novel type of autocatalytic cleavage of the peptide bond between residues Asp(414) and Pro(415) of FrpC that is insensitive to inhibitors of serine, cysteine, aspartate, and metalloproteases. Moreover, as a result of processing, the newly generated amino-terminal fragment of FrpC can be covalently linked to another protein molecule by a novel type of Asp-Lys isopeptide bond that forms between the carboxyl group of its carboxyl-terminal Asp(414) residue and the epsilon-amino group of an internal lysine of another FrpC molecule. Point substitutions of negatively charged residues possibly involved in calcium binding (D499K, D510A, D521K, and E532A) dramatically reduced the self-processing activity of FrpC. The segment necessary and sufficient for FrpC processing was localized by deletion mutagenesis within residues 400-657, and sequences homologous to this segment were identified in several other RTX proteins. The same type of calcium-dependent processing and cross-linking activity was observed also for the purified ApxIVA protein of Actinobacillus pleuropneumoniae. These results define a protein cleavage and cross-linking module of a new class of RTX proteins of Gram-negative pathogens of man, animals, and plants. In the calcium-rich environments colonized by these bacteria this novel activity is likely to be of biological importance.