Peter Schaarschmidt

NMR Solution Structure of SlyD from Escherichia coli: Spatial Separation of Prolyl Isomerase and Chaperone Function

SlyD (sensitive to lysis D) is a putative folding helper from the bacterial cytosol and harbors prolyl isomerase and chaperone activities. We determined the solution NMR structure of a truncated version of SlyD (1-165) from Escherichia coli (SlyD*) that lacks the presumably unstructured C-terminal tail. SlyD* consists of two well-separated domains: the FKBP domain, which harbors the prolyl isomerase activity, and the insert-in-flap (IF) domain, which harbors the chaperone activity. The IF domain is inserted into a loop of the FKBP domain near the prolyl isomerase active site. The NMR structure of SlyD* showed no distinct orientation of the two domains relative to each other. In the FKBP domain, Tyr68 points into the active site, which might explain the lowered intrinsic prolyl isomerase activity and the much lower FK506 binding affinity of the protein compared with archetype human FKBP12 (human FK506 binding protein with 12 kDa). The thermodynamics and kinetics of substrate binding by SlyD* were quantified by fluorescence resonance energy transfer. NMR titration experiments revealed that the IF domain recognizes and binds unfolded or partially folded proteins and peptides. Insulin aggregation is markedly slowed by SlyD* as evidenced by two-dimensional NMR spectroscopy in real time, probably due to SlyD* binding to denatured insulin. The capacity of the IF domain to establish an initial encounter-collision complex, together with the flexible orientation of the two interacting domains, makes SlyD* a very powerful catalyst of protein folding.

Mutations in the UL97 ORF of ganciclovir-resistant clinical cytomegalovirus isolates differentially affect GCV phosphorylation as determined in a recombinant vaccinia virus system.

Mutations in the human cytomegalovirus (HCMV) UL97 phosphotransferase have been associated with ganciclovir (GCV) resistance due to an impairment of GCV monophosphorylation. Vaccinia virus recombinants (rVV) were generated that encoded different HCMV UL97 proteins (pUL97) with mutations previously detected in resistant HCMV clinical isolates at codons 460, 520, 592, 594, 595, 598 and 607. These rVVs allowed quantification of GCV phosphorylation catalyzed by the different mutated pUL97s. When compared to rVV-UL97 wild type, mean levels of residual intracellular GCV phosphorylation differed by a factor of 10 for the mutated UL97 proteins ranging from 5.2 to 51.8%. Mutations M460V (located in a UL97 region homologous to domain VIb of protein kinases) and H520Q (located in a cytomegalovirus-specific, functionally critical domain) were responsible for the lowest levels of residual GCV phosphorylation (9.3 and 5.2%). Mutations in a region homologous to the domain IX had a lower impact on GCV phosphorylation (15.8-51.8%). The relevance of pUL97 mutation G598S in inducing GCV resistance was demonstrated for the first time.

Comparison between Human Cytomegalovirus pUL97 and Murine Cytomegalovirus (MCMV) pM97 Expressed by MCMV and Vaccinia Virus: pM97 Does Not Confer Ganciclovir Sensitivity

ABSTRACT The UL97 protein (pUL97) of human cytomegalovirus (HCMV) is a protein kinase that also phosphorylates ganciclovir (GCV), but its biological function is not yet clear. The M97 protein (pM97) of mouse cytomegalovirus (MCMV) is the homolog of pUL97. First, we studied the consequences of genetic replacement of M97 by UL97. Using the infectious bacterial plasmid clone of the full-length MCMV genome (M. Wagner, S. Jonjic, U. H. Koszinowski, and M. Messerle, J. Virol. 73:7056–7060, 1999), we replaced the M97 gene with the UL97 gene and constructed an MCMV M97 deletion mutant and a revertant virus. In addition, pUL97 and pM97 were expressed by recombinant vaccinia virus to compare both for known functions. Remarkably, pM97 proved not to be the reason for the GCV sensitivity of MCMV. When expressed by the recombinant MCMV, however, pUL97 was phosphorylated and endowed MCMV with the capacity to phosphorylate GCV, thereby rendering MCMV more susceptible to GCV. We found that deletion of pM97, although it is not essential for MCMV replication, severely affected virus growth. This growth deficit was only partially amended by pUL97 expression. When expressed by recombinant vaccinia viruses, both proteins were phosphorylated and supported phosphorylation of GCV, but pUL97 was about 10 times more effective than pM97. One hint of the functional differences between the proteins was provided by the finding that pUL97 accumulates in the nucleus, whereas pM97 is predominantly located in the cytoplasm of infected cells. In vivo testing revealed that the UL97-MCMV recombinant should allow evaluation of novel antiviral drugs targeted to the UL97 protein of HCMV in mice.

Altered Expression of Extracellular Matrix in Human-Cytomegalovirus-Infected Cells and a Human Artery Organ Culture Model to Study Its Biological Relevance

The influence of human cytomegalovirus (HCMV) on the transcription of 11 selected, representative extracellular matrix genes was investigated in cell culture. Northern blot hybridization indicated the downregulation of all mRNAs investigated. Based on our results and the known repression of other extracellular matrix transcripts and the β-actin transcription during HCMV infection, we suggest that one molecular mechanism contributing to the cytopathic effect may be the transcriptional downregulation of genes encoding proteins involved in cell structure and intercellular connection. To further study the biological relevance of this and other pathogenetic mechanisms, we established a human renal artery organ culture system and characterized this new infection model for HCMV. Our model is a new suitable system for the investigation of molecular as well as functional consequences of HCMV infection in a more physiological microenvironment.

Chaperone-Aided in Vitro Renaturation of an Engineered E1 Envelope Protein for Detection of Anti-Rubella Virus IgG Antibodies

The envelope glycoproteins of Rubella virus, E1 and E2, mediate cell tropism, and E1 in particular plays a pivotal role in the fusion of the virus with the endosomal membrane. Both are the prime targets of the humoral immune response. Recombinant variants of the E1 ectodomain as well as E1 antigen preparations from virus lysates are commonly used to detect anti-Rubella immunoglobulins in human sera. Hitherto, recombinant E1 for diagnostic applications has been produced chiefly in eukaryotic expression systems. Here, we report the high-yield overproduction of an engineered E1 ectodomain in the Escherichia coli cytosol and its simple and convenient renaturation into a highly soluble and immunoreactive conformation. C-Terminal fusion to one or two units of the E. coli chaperone SlyD enhances expression, facilitates in vitro refolding, and improves the overall solubility of Rubella E1. As part of this fusion protein, the E1 ectodomain fragment of residues 201-432 adopts an immunoreactive fold, providing a promising tool for the sensitive and specific detection of anti-E1 IgG in Rubella serology. Two disulfide bonds in the membrane-adjacent part of the E1 ectodomain are sufficient to generate conformations with a high and specific antigenicity. The covalently attached chaperone modules do not impair antibody recognition and binding of Rubella E1 when assessed in a heterogeneous immunoassay. SlyD and related folding helpers are apparently generic tools for the expression and refolding of otherwise unavailable proteins of diagnostic or medical importance.