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NMR structure of capistruin
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 Ribosomal peptides represent an emerging
class of bioactive natural products. As in nonribosomally synthesized
peptides macrocyclization is a strategy to improve the stability
against elevated temperatures, chemical denaturants and degradation by
proteases, as well as to reduce the entropy loss upon binding of the
biological target molecule. Within the class of macrocyclized ribosomal
peptides lasso peptides share a unique three-dimensional structure.
They are composed of an N-terminal 8/9-residue macrolactam ring, which
is formed upon a condensation reaction between the
a-amino group of Gly/Cys at position 1 and the side
chain carboxyl group of Asp/Glu at position 9, and a linear C-terminal
tail. This tail is threaded through the N-terminal macrocycle and
trapped by steric hindrance of bulky side chains stabilizing the
entropically disfavored lasso structure. In addition to this rigid
structure lasso peptides display a large variety of biological
activities ranging from inhibition of HIV replication to antibacterial
activity by obstructing the NTP uptake channel of bacterial RNA
polymerase. Their biosynthetic machinery is encoded by a gene cluster
consisting of four genes, which code for: (I) the lasso peptide
precursor protein, (II) two processing enzymes, which transform the
precursor into the bioactive lasso structured peptide, and (III) the
export protein, which transports the mature peptide outside the cell
and further acts as an immunity protein. Up to now six peptides are
proven by NMR spectroscopy to be lasso structured peptides, and the
latest one was capistruin, a lasso peptide from Burkholderia
thailandensis E264, which was found by a genome mining approach
within our group.
To contribute to the lasso
peptide research field we are investigating the biosynthesis,
biological activities and protein engineering potential of lasso
peptides, especially of capistruin from Burkholderia
thailandensis E264 and MccJ25 from Escherichia coli.
Furthermore we are developing the genome mining based lasso peptide
isolation approach further to present new members of this fascinating
class of peptides, which are solely composed of proteinogenic amino
acids, but due to their unique topology display outstanding
stability.
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Nonribosomal peptide synthesis
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Biosynthesis of the cyclic decapeptide
Tyrocidine
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A large number of therapeutically useful cyclic and linear peptides of
bacterial or fungal origin are synthesized via a template-directed,
nucleic acid-independend nonribosomal mechanism. This assembly line
mechanism is carried out on megaenzymes called nonribosomal peptide
synthetases (NRPSs). They are organised in iterative modules, one for
each amino acid to be built into the peptide product. Usually the order
of such modules in NRPSs is colinear to the sequence of the synthesized
peptide, exploring an assembly line way comparable to a linear
workflow. A typical module usually comprises about 1000 residues and is
responsible for one reaction cycle of selective substrate recognition
and activation as adenylate, covalent intermediate fixation as
enzyme-bound thioester, and peptide bond formation (figure 1). This is
accomplished by division of the working steps to specialised
semi-autonomous domains. A minimal elongation module consists of a 55
kDa adenylation (A-) domain, responsible for substrate selection and
activation through ATP hydrolysis, a 10 kDa downstream
peptidyl-carrier-protein (PCP) domain for the covalent fixation as a
thioester, and a 50 kDa condensation (C-) domain, located upstream of
the A-domain. The C-domain catalyses the peptide bond formation between
an activated aminoacyl- and a peptidyl-bound intermediate of two
adjacent modules. The result is a peptide elongated by one residue
fixed to the PCP and regeneration of the preceding module's PCP domain.
The basic set of domains within a module can be extended by optional
modifying domains, including domains for substrate epimerisation,
N-methylation, and heterocyclic ring formation, which are inserted at
specific locations into the module. This enlarges the broad spectrum of
possible products that results from the incorporation of
non-proteinogenic substrates (far over 100 carboxylic acids are known
as substrates). Further variability is also achieved through product
cyclisation and post-assembling modifications. In their modular
organisation the NRPSs give a parallel to fatty acid (FAS) and
polyketide (PKS) synthesis which are both carried out on similarly
organised multienzyme complexes. Also the cofactor used for
intermediate fixation and downstream transport is in all three cases a
4'-phosphopantetheine (4'PP-) moiety that is linked to an invariant
serine residue of the PCP or its counterparts, the acyl carrier
proteins (ACPs) of PKS and FAS. The cofactor is deriven from Coenzyme A
and posttranslationally attached to the apo-enzymes of all three
families by dedicated 4'PP-transferases. The progress that has been
made in the past decades towards understanding of the molecular
principles of nonribosomal peptide synthesis has been extended in the
last years to the structural level. With structural information now
available for a prototype of a specificity conferring A-domain of an
NRPS and for a PCP domain, we have in hand structural data on a minimal
initiation module. In addition, the crystal structure of the first
enzyme that converts a NRPS from its inactive apo-form to the active
holo-form, the 4'PP-transerase Sfp, has been solved recently. This
information will help understanding the struture/function relationship
of modules, the elementary building blocks of a NRPS.
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Bacterial metal homeostasis
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FeuA in complex with
Fe(III)-bacillibactin
Metal ions play a major role in
biological systems by acting as cofactors for redox processes and
electron transfer chains and by stabilizing protein structures in many
functional aspects. Low concentrations of metals such as iron and
copper causes growth limitations in many microbes, while their excess
can lead to severe problems of toxicity. Thus, metal homeostasis has to
be tightly regulated to ensure optimal balances of the intracellular
metal ion pools. We are currently investigating proteins which are
associated with bacterial iron and copper acquisition and channelling
including extra- and intracellular metal sensing, cellular uptake, and
intracellular targeting. Although iron is one of the most abundant
metals in the earth’s crust, its insolubility at physiological pH and
in presence of oxygen causes serious problems virtually to all
microorganisms. Bacteria have evolved several mechanisms to overcome
this problem. The secretion of low-molecular weight compounds with
enormous iron binding affinities, called siderophores, is one of the
most prominent iron acquisition strategies. Siderophores represent a
large and diverse group of microbial compounds and often play an
essential role in virulence development of important human, animal, and
plant pathogens. As a model system, we are investigating
siderophore-mediated iron acquisition and its physiological regulation
in the Gram-positive soil bacterium Bacillus subtilis, which
produces the triscatecholate-trilactone siderophore bacillibactin that
has an iron-binding constant of 1048
m−1. Bacteria are not only able to take up
their own siderophores, but can also use exogenous, so called
xenosiderophores. Identification and characterization of new
siderophore uptake systems by semi-chemical approaches and genome
mining is a further aspect of our research. Protein evolution of the
bacterial siderophore binding proteins shall lead to molecular and
structural insights of siderophore binding mechanisms and may provide
new protein tools for competitive high affinity sequestration of
virulence-associated compounds.
To enlarge our current view of cellular metal pathways,
quantitative metal proteomics and two-hybrid interaction studies are
initiated to identify the intracellular metal channeling routes
including the major direct and indirect target systems such as the
iron-sulfur cluster biogenesis machinery, metal chaperons,
metal-dependent regulators or metal storage proteins. |
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Mechanisms of Cold Shock Adaptation
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 Over 80% of
earth's biosphere is governed by temperatures below 5ƒC.Therefore, it
is not surprising that many organisms evolved sophisticated mechanisms
to resist the life-endangering influence of low temperatures. As a
model organism for Gram-positive bacteria, we study the cold adaptation
mechanisms of the soil bacterium Bacillus subtilis. After a
sudden temperature drop, a number of physico-chemical problems arise
that severely affect cellular functions and require appropriate
adaptation. The sum of all cellular reactions required for efficient
adaptation to a sudden decrease in environmental temperature is defined
as cold shock response (CSR). With a growth optimum temperature of 39ƒC
and the capacity to grow within an overall temperature interval of 11ƒC
to 53ƒC, B. subtilis is well suited for analysis of CSR. By
powerful phenotypic analysis methods like 2-D gel electrophoresis, we
identified over 35 proteins involved in cold shock adaptation of B.
subtilis. Among these are proteins required for protein
biosynthesis (S6, L7/L12), protein folding (PPiB), chemotaxis (CheY),
carbohydrate uptake (Hpr), amino acid biosynthesis (CysK, IlvC),
iron-uptake (DhbA) and general metabolism like glycolysis (GapDH, TIM)
to mention only a few. Most highly cold-induced proteins, however, are
the so-called cold shock proteins (CSPs) CspB, CspC and CspD that
belong to a family of small acidic proteins that were since found in a
wide range of bacteria (over 50 species). They also have significant
homology to eucaryotic Y-box and glycine-rich proteins. The crystal and
NMR structures of CspB, a prototype of this superfamily, have been
solved, defining a new fold. CspB has been subsequently
characterization in vitro as a single-stranded nucleic acid
binding molecule. We have proven for the first time that the presence
of at least one of these three CSPs found in B. subtilis is
essential for survival even at optimal growth temperature. Although a
large number of valuable biochemical and structural data on CSP is
available, the precise mode of action of this protein family is far
from being understood. Functional involvement of CSPs in such important
processes like transcriptional gene regulation, chromosome
condensation, protein synthesis and RNA chaperone activity are
discussed controversially. However, through the analysis of CSP
deletion mutants in B. subtilis, it has demonstrated that
removal of CSP genes results in pleiotrophic alteration of protein
synthesis, cell lysis during entry of stationary growth phase and the
inability to differentiate to endospores. In addition to this, recently
we initiated a new series of investigations dealing with the processes
coupled to the cytoplasmic membrane during cold shock. In the future,
our work will continue to focus on the examination of CSP
structure-function relationship, regulation, cellular location and
interaction with the translation and transcription machinery and as
well as on processes coupled to the cytoplasmic membrane. |
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 Microorganisms use a number of small basic
proteins for organization and compaction of their DNA. By their
interaction with the genome, these proteins do have a profound effect
on gene expression, growth behavior, and viability. More than 30
members of the family of histone-like proteins, all with a size of
about 90 amino acids and an overall basic net charge, have been
identified so far in organisms of virtually every branch of the
eubacterial kingdom, archaea, cyanobacteria, plant chloroplasts, and
bacteriophages. In contrast, the B. subtilis genome encodes only
for one histone-like protein by the hbs gene and there is no
homolog to the E. coli H-NS protein. This correlates with findings that
the hbs gene in B. subtilis is essential, and attempts to
construct a knock out mutation have failed so far . The construction of
a HBsu-GFP fusion protein allowed us to visualize for the first time
the cellular localization of this protein as in vitro analysis
has shown its ability to bind DNA. Using fluorescence microscopy,
HBsu-GFP fluorescence was observed inside the cell at exactly the same
location as the DAPI fluorescence which localized the nucleoid. The
results and investigations of using HBsu protein mutants with reduced
DNA binding activity emphasize its principal role in DNA packaging. As
HBsu from the mesophilic B. subtilis displays 87% identity and
94% similarity to its thermophilic couterpart from B.
sterothermophilus (Hbst, for which the 3D-structure is known), a
nearly identical structure for both can be assumed. The amino terminal
half of the protein is composed of two alpha-helices connected by a
turn, followed by three beta-sheets. Whereas the first sheet is
involved in forming the body of the protein structure, the latter two
form a beta-ribbon extension called the "arm" that was shown to be
involved in DNA binding. The interface of the dimer is largely
dependent upon interactions between several hydrophobic residues
scattered along the primary sequence, an attribute that is conserved in
all members of this family of proteins.
We are also studying the regulatory role of several other proteins
involved in specific and non-specific DNA binding, including some
proteins that affect gene expression at the turn from vegetative to
stationary growth in B. subtilis. |
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Zuletzt aktualisiert:
01.12.2009
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Yinghui Wang
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