Elke Deuerling profile: a crouching dragon guarding new-born
proteins
By Geoffrey Montgomery
Elke Deuerling was sitting in her office at Heidelberg University in
the Spring of 2004 when the telephone rang. It was Nenad Ban,
her collaborator on her HFSP Young Investigator grant team.
“Elke,” said Ban, “I think you should come to Zurich.” At his ETH
Zurich laboratory, Ban had been trying to solve the crystal structure
of a bacterial protein called Trigger Factor which Deuerling had
been studying nearly non-stop since November, 1996. Ban told
Deuerling: “I think we have the structure”--a structure of Trigger
Factor bound to a domain just adjacent to a ribosome‟s exit tunnel,
the birth canal of all new proteins—“and it looks amazing.”
“And knowing Nenad,” says Deuerling, “I recognized immediately
that when he said, „It looks amazing,‟ it is really amazing.” The next day Deuerling, Bernd Bukau
and several of their graduate students boarded a train to Zurich to see a crystal structure whose
shape Ban likened to a crouching dragon.
When Deuerling first began studying Trigger Factor in late 1996 after joining Bukau‟s laboratory in
Freiburg as a post-doctoral fellow, “my goal was to unravel the function of this protein,” which was
then undetermined.
A decade before, Trigger Factor had been isolated as a hypothetical
“trigger” promoting the translocation of an E. coli secretory protein into vesicles in vitro; but this
hypothesis was refuted when cells depleted for Trigger Factor failed to show secretion defects.
Then in the mid-1990s, Trigger Factor was re-discovered by three groups, including Bukau‟s, who
were investigating different aspects of how newly-synthesized protein chains fold into their proper
three-dimensional form.
Trigger Factor was found to be associated with nascent chains on
ribosomes; it was hypothesized that Trigger Factor may be acting as a new kind of chaperone: a
special factor that might aid protein folding at the earliest stage of protein synthesis, on the
ribosome itself.
The founders of molecular genetics had not anticipated the need for chaperones. An essential
component of Francis Crick‟s “sequence hypothesis” was that the amino-acid sequence of a
nascent polypeptide held all the structural information needed to fold itself in three dimensions.
This hypothesis was later corroborated by Christian Anfinsen‟s demonstration that denatured
RNase enzyme can refold in vitro. In a 1998 interview with this writer in which Crick brought up
the topic of “these chaperones which help molecules fold up,” Crick reflected: “If you had asked
us way back [in the 1950s whether proteins needed external assistance to fold correctly], we
would have said, „No, no--the protein will fold itself up and maybe the ribosome will help a little.‟”
But in fact, a new-born protein faces grave challenges upon being translated from messenger
RNA. As the N-terminal end of the nascent protein emerges from the ribosomal tunnel in a
largely unfolded state, explains Deuerling, it “exposes hydrophobic patches that are very prone to
aggregation, especially in the crowded environment of the cytosol.” Because of these sticky,
exposed hydrophobic patches—which generally will become buried within the hydrophobic
interior of the mature, folded protein—newborn, unfolded proteins are highly vulnerable to
proteolysis or aggregation.
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“So nascent proteins need molecular chaperones to assist folding to the native state,” says
Deuerling. Chaperones possess their own hydrophobic patches to bind transiently to the
hydrophobic patches of nascent unfolded proteins, and thus prevent aggregation or proteolysis.
However, all chaperones studied previous to Trigger Factor were found to act in the cytosol or at
membranes, never at the ribosome, the actual seat of protein translation and synthesis.
Indeed, when Deuerling began working Trigger Factor in late 1996, “the chaperone theory of its
function was not well accepted.” The one part of the Trigger Factor protein with a recognizable
function was a domain in the middle of the protein that had homology to a class of enzymes
called PPIases. “And at that time many people considered Trigger Factor to be a PPIase that
may work on the ribosome, for example to maintain ribosomal architecture.” In her first set of
experiments, Deuerling decided to delete the Trigger Factor encoding-gene from the E. coli
genome. “But the outcome was pretty disappointing. There was no phenotype at all, the cells
grew fine, like wild-type cells. There was no obvious function I could deduce from this knock-out
strain.”
Deuerling continued to puzzle over this disappointing result when she went home that night. If
Trigger Factor had no essential function, why was the gene present across eubacterial species,
even in a species with an extremely minimal, stripped-down genome? What if, instead, Trigger
Factor was so important “that when you delete it the cell has a kind of back-up system that
complements this loss of Trigger Factor function?” Deuerling decided to create a series of double
mutants, combining the Trigger Factor knock-out with knock-outs for genes that might have
complementary functions. “And I was immediately lucky in doing that, because the first double
mutant I tried was a Trigger factor knock-out combined with a knock-out for DnaK”—the bacterial
homologue of the famous eukaryotic cytosolic chaperone Hsp[Heat shock protein]-70.
Deuerling found that double Trigger Factor-DnaK mutants are lethal at 30°C and above, and lead
to massive aggregation of over 340 different proteins, a result published in Nature in 1999
(Deuerling et al. Nature 400: 693-696). “That protein synthesis is directly linked to chaperoneassisted protein folding—that was, I think, quite new. And in addition, what was also new and
exciting is that this chaperone on the ribosome is part of a network and works together with
cytosolic chaperones to promote protein folding.... The current view is that you have a network
built by different types of chaperones that somehow cooperate or compensate for each other to
promote the folding of newly-synthesized proteins.”
By the time Deuerling boarded
the train to Zurich with her
Heidelberg colleagues in the
spring of 2004, pieces of the
puzzle
of
Trigger
Factor
protein‟s functional architecture
were demarcated in a diagram
they had published in Nature
two years before.
Deuerling
and
her
colleagues
had
compared
the
N-terminal
ribosome-binding domain of
Trigger Factor in several different bacterial species and identified a highly-conserved “TF
signature” motif.
By mass spectrometry, she found that Trigger Factor binds to two ribosomal proteins, including
one called L23, which lies immediately adjacent to the ribosome exit tunnel for nascent proteins.
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The exquisitely powerful tools of bacterial genetics enabled Deuerling to pinpoint the crucial
amino acid sites on the TF signature sequence that mediated binding to L23 (by making point
mutations to the Trigger Factor gene); and, in complementary fashion, the crucial amino acid
sites on the L23 ribosomal protein to which Trigger Factor binds (by expressing a mutant version
of L23 on a plasmid under the control of a chemically manipulable (IPTG) promoter). Mutations at
the site of this Trigger Factor-ribosomal L23 interaction acted like deletions of the entire TF gene;
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in a DnaK deficient genetic background, cells died at 37 C with an accompanying massive protein
aggregation (Kramer et al. Nature 419: 171-4 (2002)). However, deletion of the protein‟s middle,
PPIase domain did not affect its chaperone function. And most mysterious of all was the protein‟s
C-terminal “unknown function” domain, ranging between amino acids 248 to 432.
“At the point shortly before we had the Trigger Factor structure,” remembers Deuerling, “we really
had reached a point where we were almost at the end of what we could do with genetics and
biochemistry....” Seven years of experimentation with this protein had convinced them that
Trigger Factor uses its N-terminal tail to somehow bind to the ribosome, “that it is the first
chaperone to interact with the nascent chain…and that this interaction is very transient. Which
makes sense, because this is a highly dynamic process, and the interaction has to be balanced
and correlated with the dynamics of protein translation and protein folding.
But how all this
works mechanistically was completely unclear, and we had not really any idea what this
chaperone might look like. We could model the PPIase domain based on the structure of
homologous PPIases, and there was the structure of Trigger Factors N-terminal domain, but that
was all. We had no idea what the the major C-terminal portion would look like, nor did we know
its three-dimensional domain arrangement.”
On the train-trip to Nenad Ban‟s Zurich lab,
Deuerling was filled with anticipatory excitement to see these obscure protein domains visualized
and fitted into a crystal structure.
Deuerling had first met Ban some four years before at a Freiburg seminar Ban had given on his
high-resolution structure of the Haloarcula marismortui ribosome, a landmark in the history of Xray crystallography which he had solved as a post-doctoral fellow in Tom Steitz‟s lab [Ban et al.
Science 289:905-20 (2000)]. “He was working on the archaeon ribosome 50s [large subunit]
structure, and of course we were working on Trigger Factor—and archaeons do not have Trigger
Factor.” But over dinner, they began to talk and wonder whether a chimeric experiment might be
possible: “to try and see whether these archaeon ribosomes can bind E. coli Trigger Factor and
[by this means] look for a structure.” Shortly afterward, “Nenad called me up and said, you know,
we would need some funding for this really ambitious and challenging experiment, to crystallize
Trigger Factor in complex with the ribosome. Why don‟t we try to apply for an HFSP grant?
Deuerling thought that was a wonderful idea. “I really love the combination of genetics with
biochemistry and biophysical methodologies” that the grant enabled Deuerling and Ban to pursue,
“I think it‟s very powerful when you can have this kind of synergy.” The two young scientists were
awarded a Young Investigator‟s grant in 2002 to unravel how this chaperone sits on the ribosome
and performs its functions.
When Deuerling and her colleagues arrived in Zurich that spring of 2004, Ban and graduate
student Lars Ferbitz showed them a PowerPoint presentation of the Trigger Factor crystal
structure. With vivid wit—“Nenad Ban is not only a scientist, Nenad Ban is a kind of artist,” says
Deuerling—Ban compared the structure to a “crouching dragon” with head, tail, back and two
arms. “It was one of the best moments of my scientific career, to see this structure,” remembers
Deuerling.
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Crouching Dragon - Reprinted by
permission from Macmillan Publishers
Ltd: Nature Sep 30;431(7008):590-6.
Epub 2004 Aug 29, copyright (2004)
Her image of the linear domain structure of Trigger Factor protein was transformed, becoming
three-dimensional. Crucial experimental conclusions she had assiduously pursued over the
years—such as the precise site of Trigger Factor-ribosome binding—sprang into atomicresolution focus. And the structure was also replete with surprises, beginning with its remarkable
dragon-like extended fold, in which the protein domain order of the folded structure was quite
different from the protein‟s linear domain order. The mysterious C-terminal portion could now be
seen to twist back between the structure‟s PPIase “head” and N-terminal “tail” to form the
crouching dragon‟s “back” and “arms”, creating an arched inner surface laced with protective
hydrophobic patches.
Ribbon diagram of the trigger factor
fold - Reprinted by permission from
Macmillan Publishers Ltd: Nature Sep
30;431(7008):590-6. Epub 2004 Aug 29,
copyright (2004)
Indeed, like a fabled dragon guarding a treasure of gold in a cave, the Trigger Factor dragon-like
fold seemed ideally suited to guard precious new-born protein chains as they emerged from the
ribosome‟s exit tunnel. Ferbitz and Ban had solved the structure of the N-terminal tail domain of
this crouching dragon bound to the L23 component of the archaeon ribosome. Then, by
extrapolation, they used the position of this link between dragon-tail and ribosome to superpose
the full-length protein structure, in which the crouching dragon appears to hunch right over the
ribosome exit tunnel, exposing hydrophobic patches on its inner surface for protective interactions
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with the sticky hydrophobic patches of newly-translated nascent chains, thus forming what Ban
and his colleagues call “a cradle for new proteins.”
Fold for a nascent polypeptide chain Reprinted by permission from Macmillan
Publishers Ltd: Nature Sep 30;431(7008):5906. Epub 2004 Aug 29, copyright (2004)
Remarkably, in the superposed model, this cradle is large enough to allow a small, new-born
protein to fold inside it, in addition to providing protection from proteases and agglomeration. L23
seems to provide a universal docking site for ribosome associated proteins, such as the Signal
Recognition Particle involved in protein transport, and there is evidence that the mode of
ribosomal interaction revealed by the Trigger Factor model may apply to these other ribosomeassociated factors as well. This complex, dynamic interplay on the ribosomal surface is also
“something we want to investigate further in the future,” says Deuerling. “How does nature
coordinate these different processes, protein folding and transport, that are linked with protein
biogenesis, and which converge at the L23 protein through these ribosomal factors that bind at
L23? How can this interplay be organized to allow the correct fate of a nascent peptide to be
either folded in the cytosol or to be transported?”
Another aim in her ongoing research is to perform detailed
biophysical experiments on the kinetics of trigger factornascent chain interactions.
“Does Trigger Factor really
form a cavity or cradle, together with the ribosomal surface,
to accommodate large portions of nascent chains?” says
Deuerling. Or does this crouching dragon change its
structure upon binding, flattening its back like a stalking
tiger low to the ground, so that “it is bound more like a flat
lid on top of the ribosomal exit site with a small crevice for
nascent polypeptides to pass through”—a model favored by
other chaperone investigators . “I think this is still an open
question. And one has to perform further experiments to
unravel what is the truth.”
While Trigger Factor is not conserved in higher organisms, Deuerling‟s group at Heidelberg spent
nearly three years establishing a parallel system in yeast to study eukaryotic ribosome-associated
chaperones and factors. As in E. coli, “we now can use powerful yeast genetics, in combination
with biochemical and biophysical approaches, and hopefully be able to unravel what are the
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underlying principles of protein folding in eukaryotic cells.” Deuerling has discovered that the
NAC protein complex in yeast binds to the homologue of the L23 ribosomal protein, although the
function of NAC remains unknown.
“We think in eukaryotes there is also co-translationally
assisted protein folding, since there are factors that should help, but how they work, how they
function, why they are different from Trigger Factor—this is completely unclear.” Moreover, yeast
has become a powerful and elegant system to study protein-folding disorders related to aging and
prion disease, another of Deuerling‟s long-term interests.
Deuerling will be performing these multiple, interweaving studies in her new independent
laboratory at the University of Konstanz, where she has recently been appointed as a full
professor. “I have to say that this HFSP [Young Investigator] grant enormously contributed to
many different aspects of my career,” says Deuerling. “First, this funding allowed us to build up
and support the collaboration with Nenad, also including Bernd Bukau. And this collaboration
was very fruitful and allowed us to perform these demanding experiments. And certainly we will
continue this collaboration now that the grant period is completed. The second very important
aspect for me is that, receiving such funding by HFSP, which is a very prestigious organization,
was crucial to demonstrate in the scientific community that I can successfully initiate and perform
research independently. And this was a prerequisite to get additional funding, applying for new
grants. And I think it was also very helpful in getting three excellent job offers [to be a full
professor]. Certainly having this grant helped a lot.” Indeed, Deuerling concluded, “I‟m quite
pleased by how things have worked out so far,” and by all “the exciting new questions these
studies have raised.”
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