Comparative Genomics of Listeria Species Glaser, P.; Frangeul, L.; Buchrieser, C. ...
Science (American Association for the Advancement of Science),
10/2001, Letnik:
294, Številka:
5543
Journal Article
Recenzirano
Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. ...monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.
The peptide group between residues B24 and B25 of insulin was replaced by an ester bond. This modification only in the backbone was meant to eliminate a structurally important H‐bond between the ...amide proton of B25 and the carbonyl oxygen of A19. and consequently to enhance detachment of the C‐terminal B‐chain from the body of the molecule, exposing the underlying A‐chain. According to a model derived from the effects of side‐chain substitutions, main‐chain shortening, and crosslinking, this conformational change is prerequisite for receptor binding.
Contrary to the expectation that increased flexibility would increase receptor binding and activity, depsi‐insulin (B24‐B25 CO‐0insulin) has turned out to be only 3‐4% potent. In search of an explanation for this observation, the solution structure of depsi‐insulin was determined by two‐dimensional H‐NMR spectroscopy. It was found that the loss of the B25‐A19 H‐bond does not entail detachment of the C‐terminal B‐chain. On the contrary, it is overcompensated by a gain in hydrophobic interaction achieved by insertion of the Phe B25 side chain into the molecule's core. This is possible because of increased rotational freedom in the backbone owing to the ester bond. Distortion of the B20‐B23 turn and an altered direction of the distal B‐chain are consequences that also affect self‐association. The exceptional position of the B25 side chain is thus the key feature of the depsi‐insulin structure. Being buried in the interior, it is not available for guiding the interaction with the receptor, a crucial role attributed to it by the model. This seems to be the main reason why the structure of depsi‐insulin represents an inactive conformation.
The transmembrane glycoprotein gp130 is the common
signal transducing receptor subunit of the interleukin-6-type
cytokines. It is a member of the cytokine-receptor superfamily
predicted to consist of ...six domains in its extracellular
part. The second and third domain constitute the cytokine-binding
module defined by a set of four conserved cysteines and
a WSXWS motif, respectively. The three-dimensional structure
of the carboxy-terminal domain of this region was determined
by multidimensional NMR. The domain consists of seven β-strands
constituting a fibronectin type III-like topology. The
structure reveals that the WSDWS motif of gp130 is part
of an extended tryptophan/arginine zipper which modulates
the conformation of the CD loop.
This paper reports on an insulin analogue with 12.5-fold
receptor affinity, the highest increase observed for a
single replacement, and on its solution structure, determined
by NMR spectroscopy. The ...analogue is d-AlaB26des-(B27–B30)-tetrapeptide-insulin-B26-amide.
C-terminal truncation of the B-chain by four (or five)
residues is known not to affect the functional properties
of insulin, provided the new carboxylate charge is neutralized.
As opposed to the dramatic increase in receptor affinity
caused by the substitution of d-Ala for the wild-type
residue TyrB26 in the truncated molecule, this very substitution
reduces it to only 18% of that of the wild-type hormone
when the B-chain is present in full length. The insulin molecule in solution is visualized as an ensemble
of conformers interrelated by a dynamic equilibrium. The question
is whether the “active” conformation of the hormone,
sought after in innumerable structure/function studies, is or is
not included in the accessible conformational space, so that it
could be adopted also in the absence of the receptor. If there were
any chance for the active conformation, or at least a predisposed
state to be populated to a detectable extent, this chance should
be best in the case of a superpotent analogue. This was the motivation
for the determination of the three-dimensional structure of
d-AlaB26des-(B27–B30)-tetrapeptide-insulin-B26-amide.
However, neither the NMR data nor CD spectroscopic comparison
of a number of related analogues provided a clue concerning
structural features predisposing insulin to high receptor
affinity. After the present study it seems more likely
than before that insulin will adopt its active conformation
only when exposed to the force field of the receptor surface.
Ciliary neurotrophic factor (CNTF) promotes survival in vitro and in vivo of several neuronal cell types including sensory and motor neurons. The primary structure of CNTF suggests it to be a ...cytosolic protein with strong similarity to the alpha-helical cytokine family which is characterized by a bundle of four anti-parallel helices. CNTF exerts its activity via complexation with CNTF receptor (CNTF-R). This complex consists of a CNTF-binding protein (CNTF-R) and two proteins important for signal transduction gp130 and leukaemia inhibitory factor receptor (LIF-R). We have shortened the cDNA coding for CNTF at both the 5' and the 3' end and expressed the truncated proteins in bacteria. Biological activities of the protein preparations were determined by their ability to induce proliferation of BAF/3 cells that were stably transfected with CNTF-R, gp130 and LIF-R cDNAs. CNTF proteins with 14 amino acid residues removed from the N-terminus were biologically active whereas the removal of 23 amino acids resulted in an inactive protein. In addition, 18 amino acid residues could be removed from the C-terminus of the CNTF protein without apparent loss of bioactivity, but further truncation at the C-terminus yielded biologically inactive proteins. The introduction of two point mutations into the CNTF protein at a site that presumably interacts with one of the two signal-transducing proteins resulted in a CNTF mutant with no measurable bioactivity. In addition, a model of the three-dimensional structure of human CNTF was constructed using the recently established structural co-ordinates of the related cytokine, granulocyte colony-stimulating factor. CD spectra of CNTF together with our mutational analysis and our three-dimensional model fully support the view that CNTF belongs to the family of alpha-helical cytokines. It is expected that our results will facilitate the rational design of CNTF mutants with agonistic or antagonistic properties.
Distinct yet overlapping sets of STAT transcription factors are activated by different cytokines. One example is the differential activation of acute phase response factor (APRF, also called Stat3) ...and Stat1 by interleukin 6 and interferon-gamma. Interleukin 6 activates both factors while, at least in human cells, interferon-gamma recruits only Stat1. Stat1 activation by interferon-gamma is mediated through a cytosolic tyrosine motif, Y440, of the interferon-gamma receptor. In an accompanying paper (Gerhartz, C., Heesel, B., Sasse, J., Hemmann, U., Landgraf, C., Schneider-Mergener, J., Horn, F., Heinrich, P. C., and Graeve, L. (1996) J. Biol. Chem. 271, 12991-12998), we demonstrated that two tyrosine motifs within the cytoplasmic part of the interleukin 6 signal transducer gp130 specifically mediate APRF activation while two others can recruit both APRF and Stat1. By expressing a series of Stat1/APRF domain swap mutants in COS-7 cells, we now determined which domains of Stat1 and APRF are involved in the specific recognition of phosphotyrosine motifs. Our data demonstrate that the SH2 domain is the sole determinant of specific STAT factor recruitment. Furthermore, the SH2 domain of Stat1 is able to recognize two unrelated types of phosphotyrosine motifs, one represented by the interferon-gamma receptor Y440DKPH peptide, and the other by two gp130 YXPQ motifs. By molecular modeling, we propose three-dimensional model structures of the Stat1 and APRF SH2 domains which allow us to explain the different binding preferences of these factors and to predict amino acids crucial for specific peptide recognition.
The pleiotropic cytokine interleukin-6 (IL-6) interacts with the specific ligand binding subunit (IL-6Rα) of the IL-6 receptor, and this complex associates with the signal-transducing subunit gp130 ...(IL-6Rβ). Human IL-6 acts on human and murine cells, whereas murine IL-6 is only active on murine cells. The construction of a set of chimeric human/murine IL-6 proteins has recently allowed us to define a region (residues 43-55) within the human IL-6 protein, which is important for the interaction with gp130. Subdividing this region shows that mainly residues 50-55 of the human IL-6 are necessary for this interaction. Recently, another human IL-6 double mutant (Q159E and T162P) showed reduced affinity to gp130 but residual activity on the human myeloma cell line XG-1. Into this IL-6 mutant we introduced the murine residues 43-49 or 50-55 together with two point mutations, F170L and S176A, which had been reported to increase the affinity of IL-6 to the IL-6Rα. The resulting IL-6 molecule, which contained the murine residues 50-55, was inactive on human myeloma cells and in addition completely inhibited wild type IL-6 activity on these cells. Such an antagonist may be used as a specific inhibitor of IL-6 activity in vivo.
Listeria monocytogenes, an invasive opportunistic, food-borne pathogen, remains one of the leading causes of mortality from food-borne infections. The recently determined complete genome sequences of
...L. monocytogenes strain EGDe and of that of the closely related non-pathogenic species
Listeria innocua strain CLIP11262 enhance our knowledge of the genetic basis of the virulence of
L. monocytogenes and advance our understanding of the evolution of these
Listeria species. Both genomes encode a high number of surface, transport and regulatory proteins. Comparison of the genome organisation revealed a perfect synteny between the two
Listeria genomes. Comparison with other closely related bacteria also showed a high conservation in genome organisation among the
Listeria,
Staphylococcus and
Bacillus group of low G+C content bacteria. Distinct G+C content of a number of strain-specific genes suggests intensive lateral gene transfer. The identification of a 55-kb locus encoding proteins with high homology to
Salmonella enterica serovar Typhimurium vitamin B
12 synthesis proteins as well as those necessary for degradation of ethanolamine and propanediol further indicates acquisition of a complete metabolic pathway by horizontal gene transfer and a probable role of this locus in anaerobic growth in the host.
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