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.

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.

Insertion of a chaperone domain converts FKBP12 into a powerful catalyst of protein folding.

The catalytic activity of human FKBP12 as a prolyl isomerase is high towards short peptides, but very low in proline-limited protein folding reactions. In contrast, the SlyD proteins, which are members of the FKBP family, are highly active as folding enzymes. They contain an extra "insert-in-flap" or IF domain near the prolyl isomerase active site. The excision of this domain did not affect the prolyl isomerase activity of SlyD from Escherichia coli towards short peptide substrates but abolished its catalytic activity in proline-limited protein folding reactions. The reciprocal insertion of the IF domain of SlyD into human FKBP12 increased its folding activity 200-fold and generated a folding catalyst that is more active than SlyD itself. The IF domain binds to refolding protein chains and thus functions as a chaperone module. A prolyl isomerase catalytic site and a separate chaperone site with an adapted affinity for refolding protein chains are the key elements for a productive coupling between the catalysis of prolyl isomerization and conformational folding in the enzymatic mechanisms of SlyD and other prolyl isomerases, such as trigger factor and FkpA.

Human cytomegalovirus-induced reduction of extracellular matrix proteins in vascular smooth muscle cell cultures: a pathomechanism in vasculopathies?

Human cytomegalovirus (HCMV) infection appears to be linked to the pathogenesis of atherosclerosis. An association between HCMV infection and an enhanced restenosis rate as well as the induction of vasculopathies after solid organ transplantation has been documented. Knowledge of the cellular and molecular basis of these findings is limited, however. By Northern blot and RT-PCR analysis of human foreskin fibroblasts (HFF) and human coronary artery smooth muscle cells (SMC), we identified extracellular matrix (ECM) genes that were downregulated after HCMV infection, including collagen type I and fibronectin. Quantitative immunoassays showed a significant reduction of soluble collagen type I and fibronectin proteins in supernatants of both cell types. This was shown to be a direct effect of HCMV infection and not due to a response to interferons released from infected cells, since neutralization of alpha and beta interferon activity could not block virus-induced downregulation of matrix proteins. As the amount of ECM depends on both synthesis and degradation, we also assessed the influence of HCMV on the activity of matrix metalloproteinases (MMP). Interestingly, a significant difference in virus-induced matrix degradation could be shown between the two cell types. HCMV upregulated MMP-2 protein and activity in SMC but not in HFF. Thus, HCMV infection of SMC reduces ECM dramatically by inducing two independent mechanisms that influence synthesis as well as degradation of ECM. These may represent molecular mechanisms for HCMV-induced pathogenesis of inflammatory vasculopathies and may facilitate dissemination of HCMV by promoting the detachment of infected cells in vivo.

SlyD proteins from different species exhibit high prolyl isomerase and chaperone activities.

SlyD is a putative folding helper protein from the Escherichia coli cytosol, which consists of an N-terminal prolyl isomerase domain of the FKBP type and a presumably unstructured C-terminal tail. We produced truncated versions without this tail (SlyD) for SlyD from E. coli, as well as for the SlyD orthologues from Yersinia pestis, Treponema pallidum, Pasteurella multocida, and Vibrio cholerae. They are monomeric in solution and unfold reversibly. All SlyD variants catalyze the proline-limited refolding of ribonuclease T1 with very high efficiencies, and the specificity constants (kcat/KM) are equal to approximately 10(6) M(-1) s(-1). These large values originate from the high affinities of the SlyD orthologues for unfolded RCM-T1, which are reflected in low KM values of approximately 1 microM. SlyD also exhibits pronounced chaperone properties. Permanently unfolded proteins bind with high affinity to SlyD and thus inhibit its prolyl isomerase activity. The unfolded protein chains do not need to contain proline residues to be recognized and bound by SlyD. The conservation of prolyl isomerase activity and chaperone properties within the SlyD family suggests that these proteins might act as true folding helpers in the bacterial cytosol. The SlyD proteins are also well suited for biotechnological applications. As fusion partners they facilitate the refolding and increase the solubility of aggregation-prone proteins such as the gp41 ectodomain fragment of HIV-1.

Functional solubilization of aggregation-prone HIV envelope proteins by covalent fusion with chaperone modules.

The envelope proteins of human immunodeficiency virus (HIV) and human T-cell lymphotrophic virus (HTLV) mediate cell attachment and membrane fusion. For HIV-1, the precursor protein gp160 is cleaved proteolytically into two fragments, the surface-associated receptor binding subunit gp120 and the membrane spanning subunit gp41, which is involved in membrane fusion during virus entry. Soluble and immunoreactive variants of gp41 are essential for the reliable diagnosis of HIV-1 infections. Hitherto, gp41 was solubilized by adding detergents, or in acidic or alkaline solvents. We find that covalent fusions with SlyD or FkpA, two homodimeric Escherichia coli chaperones with peptidyl-prolyl isomerase activity, solubilize retroviral envelope proteins without compromising their immunological reactivity. gp41 from HIV-1, gp36 from HIV-2 and gp21 from HTLV could be expressed in large amounts in the Escherichia coli cytosol when fused with one or two subunits of SlyD or FkpA. The fusion proteins could be easily isolated and refolded, and showed high solubility and immunoreactivity, thus providing sensitive and reliable tools for diagnostic applications. Covalent fusions with SlyD or FkpA might be valuable generic tools for the solubilization and activation of aggregation-prone proteins.

Upregulation of functionally active vascular endothelial growth factor by human cytomegalovirus.

Human cytomegalovirus (HCMV) infection is known to modulate host gene expression and has been linked to the pathogenesis of vasculopathies; however, relevant pathomechanisms are still unclear. It was shown that HCMV infection leads to upregulation of vascular endothelial growth factor (VEGF) expression in human foreskin fibroblasts and coronary artery smooth muscle cells (SMC). Activation of VEGF transcription by HCMV infection was confirmed by transient-expression experiments, which revealed that a short promoter fragment, pLuc135 (-85 to +50), is sufficient for activation. Site-directed mutagenesis of Sp1-recognition sites within this fragment abolished the upregulation of transcription. Functional VEGF protein is released into the culture supernatant of infected SMC. Incubation of endothelial cells with supernatants from HCMV-infected SMC cultures induced upregulation of VEGF receptor-2 expression on endothelial cells, as well as a significant upregulation of DNA synthesis, implicating cell proliferation. The mean incline of DNA synthesis at 48 and 72 h post-infection was 148 and 197 %, respectively. Addition of neutralizing antibodies against VEGF completely abolished this effect. Supernatants from SMC cultures incubated with UV-inactivated virus induced a comparable effect. This virus-induced paracrine effect may represent a molecular mechanism for HCMV-induced pathogenesis, such as inflammatory vasculopathies, by inducing a proatherogenic phenotype in SMC.

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.