ADP-ribose and analogues bound to the deMARylating macrodomain from the bat coronavirus HKU4.

Macrodomains are proteins that recognize and hydrolyze ADP ribose (ADPR) modifications of intracellular proteins. Macrodomains are implicated in viral genome replication and interference with host cell immune responses. They are important to the infectious cycle of Coronaviridae and Togaviridae viruses. We describe crystal structures of the conserved macrodomain from the bat coronavirus (CoV) HKU4 in complex with ligands. The structures reveal a binding cavity that accommodates ADPR and analogs via local structural changes within the pocket. Using a radioactive assay, we present evidence of mono-ADPR (MAR) hydrolase activity. In silico analysis presents further evidence on recognition of the ADPR modification for hydrolysis. Mutational analysis of residues within the binding pocket resulted in diminished enzymatic activity and binding affinity. We conclude that the common structural features observed in the macrodomain in a bat CoV contribute to a conserved function that can be extended to other known macrodomains.

Sidedness of interfacial arginine residues and anti-atherogenicity of apolipoprotein A-I mimetic peptides.

To test the hypothesis that sidedness of interfacial arginine (Arg) in apoA-I mimetic peptides, similar to that observed in apoA-I (Bashtovyy, D. et al. 2011. Sequence conservation of apolipoprotein A-I affords novel insights into HDL structure-function. J. Lipid Res. 52: 435-450.), may be important for biological activity, we compared properties of 4F and analogs, [K4,¹5>R]4F and [K9,¹³>R]4F, with Lys>Arg substitutions on the right and left side, respectively, of the 4F amphipathic helix. Intraperitoneal administration of these peptides into female apoE null mice (n = 13 in each group) reduced en face lesions significantly compared with controls; 4F and [K4,¹5>R]4F were equally effective whereas [K9,¹³>R]4F was less effective. Turnover experiments indicated that [K4,¹5>R]4F reached the highest, whereas [K9,¹³>R]4F had the lowest, plasma peak levels with a similar half life as the [K4,¹5>R]4F analog. The half life of 4F was two times longer than the other two peptides. The order in their abilities to associate with HDL in human plasma, generation of apoA-I particles with pre-ß mobility from isolated HDL, lipid associating ability, and sensitivity of lipid complexes to trypsin digestion was: 4F>[K4,¹5,>R]4F>[K9,¹³>R]4F. These studies support our hypothesis that the sidedness of interfacial Arg residues in the polar face of apoA-I mimetics results in differential biological properties.

Structural and biochemical characterization of Staphylococcus aureus clumping factor B/ligand interactions.

Clumping factor B (ClfB) from Staphylococcus aureus is a bifunctional protein that binds to human cytokeratin 10 (K10) and fibrinogen (Fg). ClfB has been implicated in S. aureus colonization of nasal epithelium and is therefore a key virulence factor. People colonized with S. aureus are at an increased risk for invasive staphylococcal disease. In this study, we have determined the crystal structures of the ligand-binding region of ClfB in an apo-form and in complex with human K10 and Fg α-chain-derived peptides, respectively. We have determined the structures of MSCRAMM binding to two ligands with different sequences in the same site showing the versatile nature of the ligand recognition mode of microbial surface components recognizing adhesive matrix molecules. Both ligands bind ClfB by parallel ß-sheet complementation as observed for the clumping factor A·Î³-chain peptide complex. The ß-sheet complementation is shorter in the ClfB·Fg α-chain peptide complex. The structures show that several residues in ClfB are important for binding to both ligands, whereas others only make contact with one of the ligands. A common motif GSSGXG found in both ligands is part of the ClfB-binding site. This motif is found in many human proteins thus raising the possibility that ClfB recognizes additional ligands.

TLR4-mediated activation of dendritic cells by the heat shock protein DnaK from Francisella tularensis.

Francisella tularensis is the causative agent of tularemia, a severe, debilitating disease of humans and other mammals. As this microorganism is also classified as a "category-A pathogen" and a potential biowarfare agent, there is a need for an effective vaccine. Several antigens of F. tularensis, including the heat shock protein DnaK, have been proposed for use in a potential subunit vaccine. In this study, we characterized the innate immune response of murine bone marrow-derived dendritic cells (DC) to F. tularensis DnaK. Recombinant DnaK was produced using a bacterial expression system and purified using affinity, ion-exchange, and size-exclusion chromatography. DnaK induced the activation of MAPKs and NF-kappaB in DC and the production of the proinflammatory cytokines IL-6, TNF-alpha, and IL-12 p40, as well as low levels of IL-10. DnaK induced phenotypic maturation of DC, as demonstrated by an up-regulation of costimulatory molecules CD40, CD80, and CD86. DnaK stimulated DC through TLR4 and the adapters MyD88 and Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) that mediated differential responses. DnaK induced activation of MAPKs and NF-kappaB in a MyD88- or TRIF-dependent manner. However, the presence of MyD88- and TRIF-dependent signaling pathways was essential for an optimal, DnaK-induced cytokine response in DC. In contrast, DnaK induced DC maturation in a TRIF-dependent, MyD88-independent manner. These results provide insight about the molecular interactions between an immunodominant antigen of F. tularensis and host immune cells, which is crucial for the rational design and development of a safe and efficacious vaccine against tularemia.

A structural model for the inhibition of calpain by calpastatin: crystal structures of the native domain VI of calpain and its complexes with calpastatin peptide and a small molecule inhibitor.

The Ca(2+)-dependent cysteine protease calpain along with its endogenous inhibitor calpastatin is widely distributed. The interactions between calpain and calpastatin have been studied to better understand the nature of calpain inhibition by calpastatin, which can aid the design of small molecule inhibitors to calpain. Here we present the crystal structure of a complex between a calpastatin peptide and the calcium-binding domain VI of calpain. DIC19 is a 19 residue peptide, which corresponds to one of the three interacting domains of calpastatin, which is known to interact with domain VI of calpain. We present two crystal structures of DIC19 bound to domain VI of calpain, determined by molecular replacement methods to 2.5A and 2.2A resolution. In the process of crystallizing the inhibitor complex, a new native crystal form was identified which had the homodimer 2-fold axis along a crystallographic axis as opposed to the previously observed dimer in the asymmetric unit. The crystal structures of the native domain VI and its inhibitor PD150606 (3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid) complex were determined with the help of molecular replacement methods to 2.0A and 2.3A resolution, respectively. In addition, we built a homology model for the complex between domain IV and DIA19 peptide of calpastatin. Finally, we present a model for the calpastatin-inhibited calpain.

A novel variant of the immunoglobulin fold in surface adhesins of Staphylococcus aureus: crystal structure of the fibrinogen-binding MSCRAMM, clumping factor A.

We report here the crystal structure of the minimal ligand-binding segment of the Staphylococcus aureus MSCRAMM, clumping factor A. This fibrinogen-binding segment contains two similarly folded domains. The fold observed is a new variant of the immunoglobulin motif that we have called DE-variant or the DEv-IgG fold. This subgroup includes the ligand-binding domain of the collagen-binding S.aureus MSCRAMM CNA, and many other structures previously classified as jelly rolls. Structure predictions suggest that the four fibrinogen-binding S.aureus MSCRAMMs identified so far would also contain the same DEv-IgG fold. A systematic docking search using the C-terminal region of the fibrinogen gamma-chain as a probe suggested that a hydrophobic pocket formed between the two DEv-IgG domains of the clumping factor as the ligand-binding site. Mutagenic substitution of residues Tyr256, Pro336, Tyr338 and Lys389 in the clumping factor, which are proposed to contact the terminal residues (408)AGDV(411) of the gamma-chain, resulted in proteins with no or markedly reduced affinity for fibrinogen.

Crystallization and preliminary X-ray crystallographic analysis of Ace: a collagen-binding MSCRAMM from Enterococcus faecalis.

Ace is a collagen-binding bacterial cell surface adhesin from Enterococcus faecalis. The collagen-binding domain of Ace (termed Ace40) and its truncated form Ace19 have been crystallized by the vapor-diffusion hanging-drop method. Ace19 was crystallized in two different crystal forms. A complete 1.65 A data set has been collected on the orthorhombic crystal form with unit cell parameters a=38.43 b=48.91 and c=83.73 A. Ace40 was crystallized in the trigonal space group P3(1)21 or P3(2)21 with unit cell parameters a=b=80.24, c=105.91 A; alpha=beta=90 and gamma=120 degrees. A full set of X-ray diffraction data was collected to 2.5 A. Three heavy atom derivative data sets have been successfully obtained for Ace19 crystals and structural analysis is in progress.