Structure-based modification of a Clostridium difficile-targeting endolysin affects activity and host range.

Endolysin CD27L causes cell lysis of the pathogen Clostridium difficile, a major cause of nosocomial infection. We report a structural and functional analysis of the catalytic activity of CD27L against C. difficile and other bacterial strains.

Expression, purification, and functional analysis of murine ectodomain fragments of CD8alphaalpha and CD8alphabeta dimers.

Soluble mouse CD8alphaalpha and CD8alphabeta dimers corresponding to the paired ectodomains (CD8(f)) or their respective component Ig-like domains (CD8) were expressed in Chinese hamster ovary cells or the glycosylation variant Lec3.2.8.

The CD8beta ectodomain contributes to the augmented coreceptor function of CD8alphabeta heterodimers relative to CD8alphaalpha homodimers.

Within the lymphoid compartment, CD8 is expressed either as an alphaalpha homodimer or as an alphabeta heterodimer. Prior functional characterization of CD8alpha transfectants has demonstrated that CD8alphaalpha homodimers can reconstitute T cell responses in the absence of the CD8beta subunit. In order to now examine the role of CD8beta in TCR recognition, the CD8alpha cDNA alone or in combination with CD8beta cDNA was transfected into the mouse T cell hybridoma, N15wt, specific for VSV8/Kb.

Structural basis of CD8 coreceptor function revealed by crystallographic analysis of a murine CD8alphaalpha ectodomain fragment in complex with H-2Kb.

The crystal structure of the two immunoglobulin variable-like domains of the murine CD8alphaalpha homodimer complexed to the class I MHC H-2Kb molecule at 2.8 A resolution shows that CD8alphaalpha binds to the protruding MHC alpha3 domain loop in an antibody-like manner. Comparison of mouse CD8alphaalpha/H-2Kb and human CD8alphaalpha/HLA-A2 complexes reveals shared as well as species-specific recognition features.

Topology of T cell receptor-peptide/class I MHC interaction defined by charge reversal complementation and functional analysis.

The molecular interactions between the CD8 co-receptor dependent N15 and N26 T cell receptors (TCRs) and their common ligand, the vesicular stomatitis virus octapeptide (VSV8) bound to H-2Kb, were studied to define the docking orientation(s) of MHC class I restricted TCRs during immune recognition. Guided by the molecular surfaces of the crystallographically defined peptide/MHC and modeled TCRs, a series of mutations in exposed residues likely contacting the TCR ligand were analyzed for their ability to alter peptide-triggered IL-2 production in T cell transfectants. Critical residues which diminished antigen recognition by 1000 to 10,000-fold in molar terms were identified in both N15 Valpha (alphaE94A or alphaE94R, Y98A and K99) and Vbeta (betaR96A, betaW97A and betaD99A) CDR3 loops.