Protein Engineering: New Approaches and Applications
Engineering chimaeric proteins from fold
fragments: ‘hopeful monsters’ in protein design
Birte Höcker*1
*Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany
Abstract
Modern highly complex proteins evolved from much simpler and less specialized subunits. The same concept
can be applied in protein engineering to construct new well-folded proteins. Hybrid proteins or chimaeras
can be built from contemporary protein fragments through illegitimate recombination. Even parts from
different globular folds can be fitted together using rational design methodologies. Furthermore, intrinsic
functional properties encoded in the fold fragments allow rapid adaptation of the new proteins and thus
provide interesting starting scaffolds for further redesign.
Introduction
The diversity of proteins that make up today’s cells
and organisms is striking. To gain an overview of this
diversity we try to organize and sort the different observed
protein structures into separate bins. Many structures
are very complex, built from long polypeptide chains.
However, distinct folding units can usually be discerned.
These polypeptide chain segments that are able to fold
autonomously are called protein domains. When comparing
all of the different known protein domains, it easily becomes
apparent that the same structural architectures often show
up in varying contexts [1]. Some of these domains have
adapted large interfaces and form strong interactions between
each other, whereas many are merely positioned as beads
on a string. Based on these observations, it is commonly
understood that proteins evolved through the recruitment
and recombination of domains (Figure 1A).
Similarly, the protein domains themselves, which consist
of approximately 100–200 amino acids, appear to have
evolved through comparable mechanisms. Upon closer
scrutiny, we can distinguish smaller fragments, often called
supersecondary structures, from which the domains are built
up [2,3]. The smaller units are easily distinguishable in
solenoid proteins, e.g. the blades of the β-propeller or the
strand–turn–helix repeat of the LRR (leucine-rich repeat)
proteins, but were also identified in globular domains such as
the (βα)8 - [or TIM (triose phosphate isomerase)-] barrel [4]
and the β-trefoil proteins [5] (Figure 1B).
The hypothesis of the evolution of protein domains
from smaller intrinsically stable subunits via combinatorial
assembly has been explored by protein engineering [6].
Homologous recombination has been developed to (semi-)
randomly create hybrids from which successful candidates
can be selected [7–9]. However, protein chimaeras can
Key words: (βα)8 -barrel, computational design, evolution, flavodoxin-like fold, fold fragments,
protein chimaera.
Abbreviation used: PRAI, phosphoribosyl anthranilate isomerase.
1
email [email protected]
Biochem. Soc. Trans. (2013) 41, 1137–1140; doi:10.1042/BST20130099
also be generated through the recombination of very
divergent or even unrelated fragments. In the present minireview, I focus on the potential of illegitimate recombination
for the creation and diversification of protein folds, especially
the (βα)8 -barrel fold. The work is also discussed from an
evolutionary viewpoint.
Evolution and design of (βα)8 -barrel
enzymes from halves
The ubiquitous (βα)8 -barrel, which is a typical enzyme fold,
has been hypothesized to have evolved via gene duplication
and fusion from an ancestor half its size based on the striking
two-fold symmetry observed in the crystal structures of
two (βα)8 -barrel proteins from histidine biosynthesis, HisA
(phosphoribosylformimino-5-aminoimidazole carboxamide
ribotide isomerase) and HisF (imidazole glycerol phosphate
synthase) [10,11]. Experimental evidence for the hypothesis
was provided by the observation that separately produced
halves of HisF from Thermotoga maritima behaved as
independent folding units that together could form a
functional heterodimer [12]. The evolutionary scenario of
duplication and fusion of identical copies of a half-barrel were
further explored by protein engineering. A stable (βα)8 -barrel
was constructed by fusing two copies of the C-terminal half
of HisF in tandem and optimizing the half-barrels’ interface
by the reconstruction of a salt-bridge cluster [13]. Subsequent
improvement of the soluble expression of the construct [14]
later enabled elucidation of the proteins’ structures by Xray crystallography, which validated the highly symmetrical
nature of this artificial protein [15]. In a complementary
computational approach, a slightly different fragment of
HisF was identified whose duplication leads directly to a
stable symmetric (βα)8 -barrel [16]. Also in a less symmetrical
but related (βα)8 -barrel, namely PRAI (phosphoribosyl
anthranilate isomerase) (or TrpF) from Escherichia coli,
a stable half-barrel fragment was identified [17]. Tandem
duplication of the central (βα)3–6 fragment yielded a
predominantly monomeric and reasonably stable protein.
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Figure 1 Evolution of proteins
(A) Independently folding units, the protein domains (different colours),
are recruited to build up the complex multidomain proteins that we
observe today. Active sites are indicated. (B) The domains themselves
evolved through similar mechanism from smaller subdomain-sized
fragments: schematic representation of a globular protein in blue and a
solenoid protein in red.
Since the half-barrel can exist as a stable structure, an
alternative evolutionary route in (βα)8 -barrel evolution is
possible, namely the recombination of different half-barrels.
Cross-wise fusion of the halves of the related but sequentially
very divergent (βα)8 -barrel enzymes HisA and HisF from
Thermotoga maritima yielded the chimaeric proteins HisFA
and HisAF. Although the first protein turned out to be a
molten globule, the latter was expressed as a very stable
and compact monomeric protein that unfolded with high
co-operativity [13]. This HisAF protein was then submitted
to directed evolution towards the activity of PRAI from
tryptophan biosynthesis which is chemically similar to the
activity of the HisA enzyme. It turned out that a few
mutations in HisAF were sufficient to establish efficient TrpF
activity [18]. The experiments exemplify that recombination
of structurally complementary fragments can quickly bring
about new protein variants that are as stable and active as
naturally observed counterparts. Although this is a beautiful
proof of concept regarding the evolutionary hypothesis
of diversification through recombination, it furthermore
provides clear evidence that new and useful proteins can
be engineered from fragments of contemporary (βα)8 -barrel
proteins.
Chimaeras by recombination of fragments
from different folds
If the half-barrel is a progenitor of the (βα)8 -barrel fold,
we would expect to observe such fragments also in other
protein folds. A structure-based search of the PDB revealed
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Authors Journal compilation that the half-barrel topology strongly resembles the one
of the flavodoxin-like fold [19]. This is a doubly wound
(βα)5 -protein, which forms a central five-stranded β-sheet
sandwiched between helices on both sides. A comparison
of topologies shows an extra αβ element after β 1 in the
flavodoxin-like proteins, where in the symmetrical (βα)8 barrels there is a long loop of almost the same length. In
fact, the secondary-structural elements β 1 α 2 –β 5 of proteins
from the response regulator and the cobalamin-binding
domain superfamilies superimpose very well with β 1 –β 4
of the HisF half-barrels. The position of the last helix,
α 5 in the flavodoxin-like fold and α 4 in the half-barrels,
deviates significantly. The comparison shows that the similar
fragments are fixed in very different contexts. In the barrel,
the hydrophobic interior is shielded through the second half,
whereas in the flavodoxin-like fold shielding is achieved
through the first and last α-helices that fold on to the
other side of the β-sheet, thereby creating a three-layered
fold.
This similarity prompted us to test whether these
structurally similar parts could substitute for each other. We
created a chimaera in which we transplanted this part from
CheY (chemotaxis response regulator), a well-characterized
single-domain protein, into the (βα)8 -barrel enzyme HisF
creating the fold-chimaera CheYHisF [20]. The resulting
protein was a stable monomer that unfolds co-operatively and
that, based on CD and fluorescence spectroscopy, appeared
similar in structure to the wild-type HisF protein. It was
possible to crystallize the protein and to solve its crystal
structure to 3.1 Å (1 Å = 0.1 nm) resolution. The structure
confirmed that large parts of the protein retained the parent
structures; however, it also revealed unexpected details at the
interface of the combined fragments. An additional β-strand
was observed in the core of the barrel that invades between
β 1 and β 2 of the CheY part and is formed by residues of
the C-terminus of HisF and from the histidine tag included
for purification. A construct without these strand-forming
residues (CheYHisF-sfr) had a high tendency to aggregate,
thereby illustrating the importance of this structural element
for the integrity of the fold. This indicates that the two
recombined fragments do not optimally fit together and that
strain is released by widening of the barrel, which leaves space
for the insertion of the C-terminal residues [20].
Hence the next obvious question was which residues
were causing the ill-fitting of the fragments. In a followup approach, we used the computational design program
Rosetta to identify and fix problematic areas for the formation
of an eight-stranded CheYHisF chimaera [21]. An iterative
approach was applied of alternating rounds of backbone
perturbation with side-chain redesign and gradient-based
minimization on an eight-stranded CheYHisF model. In
addition, native residues were favoured in the calculations
to ensure that only mutations were selected that significantly
improve energy of the construct and to determine the fewest
mutations to rescue the structure. In the end, we decided
to introduce five mutations into CheYHisF-sfr targeted to
the interface of the combined CheY and HisF fragments.
Protein Engineering: New Approaches and Applications
The new variant, called CheYHisF-sfr_RM, performed really
well. Large amounts could be solubly expressed, the protein
was eluted as a sharp peak of an analytical size-exclusion
chromatography with an apparent molecular mass lower
than that of the nine-stranded CheYHisF, and it was
even more stable as judged by chemical unfolding studies.
Furthermore, the protein was amenable to structure determination by NMR spectroscopy, which confirmed the overall
arrangement of a classical (βα)8 -barrel as well as the computational predictions. Detailed characterization showed that
all five mutations contribute to the improved properties of
the chimaera and together enhance the packing and relieve
the steric hindrances at the interface [21].
The two chimaeras, the nine- and the eight-stranded, can
both bind phosphate or other phosphorylated compounds.
Already in the crystal structure of CheYHisF, a bound
sulfate ion proved that the phosphate-binding site, which
originates from the HisF fragment, was intact and functional
[20]. Binding tests with the improved CheYHisF-sfr_RM
construct further showed the protein’s affinity for a
phosphorylated product analogue of the enzyme PRAI
from tryptophan biosynthesis. This affinity could easily be
improved by two targeted mutations at the binding site [21].
To test the generalizability of our chimaerogenesis approach, another chimaera was successfully constructed from
analogous fragments of NarL (nitrite response regulator)
and HisF. This chimaeric NarLHisF protein performed far
better than CheYHisF in terms of solubility and stability.
The integrity of this protein was also dependent on the presence of the C-terminal residues of HisF including the
attached histidine tag, which leads to the conclusion that
this protein also forms a nine-stranded barrel just like the
CheYHisF chimaera [22].
Evolutionary considerations
Altogether, the results demonstrate how quickly new proteins
can develop from fragments and how recombination can
lead to the diversification of proteins without necessarily
developing a new protein fold. If the fragments that are
recombined carry some functionality, e.g. a binding site
that stays intact in the recombined chimaera, this can
instantly give the new protein an advantage for survival.
Accumulation of additional mutations can then lead to the
optimization and refinement of the function of this new
protein. Once evolutionary drift and adaptation occurred,
such proteins that arose through recombination would be
almost indistinguishable from proteins that diverged after the
duplication of a gene encoding a full protein domain.
However, just as easily, illegitimate recombination will not
yield the expected folded protein. Even if tiny fragments
are structurally as similar as those that we swapped from
CheY and HisF, there might be small hindrances that do not
allow the tight packing that renders the core of the globular
(βα)8 -barrel so stable. In many cases, the proteins will not
fold properly at all, but, in some cases, alternative solutions
can be found as observed in the nine-stranded CheYHisF
Figure 2 Design of chimaeras from parts of different folded
proteins: combination of fragments from HisF in blue and CheY in
green
The extra secondary-structure element in the CheYHisF chimaera is
shown in red. The chimaera was subsequently optimized at the interface
of the recombined fragments and in the ligand-binding site.
chimaera. The topology found in the CheYHisF protein had
not been observed before in any of the structures deposited
in the PDB. Nonetheless, it is a stable structure and might
be present in natural proteins whose structures have not yet
been determined. It is also conceivable that the protein fold
has been sampled in the course of evolution, but that it was
not fixed and instead returned to a proper eight-stranded
(βα)8 -barrel in a futile cycle just as we changed CheYHisF
through the addition of five targeted mutations (Figure 2).
On the other hand, in a rare event, the aberrant structure
could become established in a population and serve as a seed
of a new fold. Thus we call the unusual chimaeric structure
a ‘hopeful monster’, following a phrase that was used to
describe sudden jumps in speciation by geneticist Richard
Goldschmidt in 1940 [23], because it shows a route to new
and possibly unexplored protein topologies.
Conclusions and future challenges
According to Greek mythology, a chimaera is a monstrous
creature built up of parts of different animals. In the
protein world, hybrids or chimaeras are not as unusual.
Furthermore, dramatic reorganizations have been attributed
an important role in the natural evolution of proteins.
In this context, we view the protein chimaeras that we
engineered by recombination of fragments from different
folds as hopeful monsters that are worthwhile exploring.
The experiments so far have shown that chimaeras built from
fragments of different folds can open up unexpected routes
to new topologies and that fragments that carry functional
sites can allow easy adaptation to new functionalities. It
appears that the illegitimate recombination of interesting
protein fragments provides good starting scaffolds for
the development of new functions as well as a protocol
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to rapidly engineer proteins for new applications. We
developed a general strategy for the construction of
protein chimaeras, which combines rational observations
and computational design [24]. This methodology can be
used to build stable and functional proteins. Ultimately, we
would like to create chimaeras with various functionalities by
combining fragments bearing distinct functional features. The
engineering of new enzymes by illegitimate recombination
would be an innovative and valuable resource for applied
research.
Funding
Work in the Höcker laboratory is supported by funds of the Max
Planck Society and the German Research Foundation.
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Received 5 June 2013
doi:10.1042/BST20130099