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IspH – RPS1 “Rosetta Stone” Protein
Presented By Armaan Haleem, Oldfield Group
Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois at Urbana-Champaign
Introduction
.
Cytoscape Visualization and Comparison of
IspH Protein Sizes
IspH-RPS1 is a fusion protein that contains the IspH
domain which functions as the last essential reducing
enzyme in the methylerithritol phosphate pathway. This
pathway is part of the isoprenoid biosynthesis pathway
and is an alternative to the mevalonate pathway found in
humans. The IspH-RPS1 protein is found in organsims
that cause severe diseases such as Clostridium tetani
and Clostridium botulinism
MALDI-TOF Spectrum, UV-Vis Spectrum, Michaelis
Mentin Kinetics, Activity Comparison, EPR
Spectrum
Proposed Function of IspH-RPS1
Possible IspH-RPS1 Bacterial Ribosome Interaction
Comparison of the Mevalonate and Methylerithritol
Pathways
Fig. 1 Sequence similarity network of the IspH family
proteins with 15660 members constructed at an
expectation-value (e-value) of 10−120. A single node
represents sequences with at least 90% similarity.
Major clusters of interest include: (a)
gammaproteobacteria (dark cyan, upper left) and
betaproteobacteria (blue, lower right); (b)
alphaproteobacteria (cyan); (c) firmicutes (purple);
(d) firmicutes (with very long sequences, red); (e)
bacteroidetes (yellow); (f) actinobacteria (orange);
(g) cyanobacteria and plants (green); (h)
apicomplexa (pink, genus Plasmodium, the malaria
parasites) and (i) desulfobacterales (brown, an
order of deltaproteobacteria) (j) the architectures of
IspH–RPS1 with 4 (top) and 6 (bottom) S1 repeats.
Immethun, C. M., Hoynes-O’Connor, A. G., Balassy, A., & Moon, T. S. (2013). Microbial Production of
Isoprenoids Enabled by Synthetic Biology. Front. Microbiol. Frontiers in Microbiology, 4.
doi:10.3389/fmicb.2013.00075
The methylerithritol pathway and mevalonate pathway
are parallel pathways in isoprenoid biosynhesis. The
methylerithritol pathway does not exist in humans and
thus serves as a potential drug target.
Analysis
Proposed Structure of IspH-RPS1
The IspH-RPS1 fusion protein is decidedly a “Rosetta”
stone protein because of the unaffected function of the
IspH domain. This is further shown through the
retained Kcat of the IspH domain on the fusion protein
from Clostridium thermocellum [56min-1] which is very
similar to the Kcat of IspH expressed from aquifex
aeolicus. Additionally, no other genes were found on
the genomes to code for these proteins indicating that
the fusion is likely due to complementary functions of
the domain.
The IspH Catalzyed Reaction of HMBPP  DMAPP
and IPP
Scheme 1 IspH catalysis. The reaction involves the
2H+/2e− reductive dehydroxylation of (E)-1-hydroxy-2methylbut-2-enyl-4-diphosphate (3) to dimethylallyl
diphosphate (1) and isopentenyl diphosphate (2).
Fig.3 Characterization of CthIspH–RPS1 and its
truncation mutants. (a) MALDI-TOF spectrum of purified
CthIspH. M1: CthIspH–RPS1; M2: EcRho, see text for
more details. (b) UV-Vis spectrum of CthIspH–RPS1 after
iron–sulfur cluster reconstitution (black) and after
dithionite reduction (red). (c) Michaelis–Menten kinetics of
CthIspH–RPS1. (d) Cartoon of the truncation proteins. D0:
no S1 repeat, D1: one S1 repeat, D2: two S1 repeats, D3:
three S1 repeats. WT has the four S1 repeats. (e)
Relative activities of truncation mutants compared to wildtype protein. Error bars are from n = 3 replicates. (f) EPR
of reduced CthIspH–RPS1, its truncation mutants, and E.
coli IspH. (g) EPR of CthIspH–RPS1_1-171, reduced
protein, with substrate HMBPP (1) and several ligands.
The small peak at g = 2.0 in the oxidized protein sample
(blue) is due to the small amount of [Fe3S4]+.
Fig. 2 IspH structures and IspH–fusion protein
structure predictions. (a) X-ray structure of E. coli
IspH (PDB ID code: 3KE8) (b) X-ray structure of P.
falciparum IspH (PDB code: 4N7B) (c) Phyre2
structure prediction of CthIspH–RPS1. The IspH
domains are in orange while the 4 S1 repeats are
colored green, purple, blue, or cyan.
The identification of the IspH domain function as
unaltered in the IspH-RPS1 fusion is essential to the
classification of this protein as a “Rosetta Stone”
protein.
Fig. 4 Possible IspH–RPS1/RelA/Rho ribosome
interactions. Upon binding to the ribosome through the
RPS1 domain, IspH could interact with RelA and affect its
activity. The ppGpp released modulates a series of cell
activities, for example inhibiting RNA polymerase
(RNAP). The transcription termination factor Rho could
also be affected upon interacting with IspH–RPS1 (as
suggested from the MALDI-TOF experiments).
Conclusions
The IspH-RPS1 was first given attention due to the
identification of an IspH domain in a protein 300-550
amino acid residues longer than the IspH domain. When
analyzed using various methods, the domain was
identified to have the same function in the fusion protein
as an independent IspH. This leads to the conclusion that
IspH-RPS1 is a “Rosetta Stone” protein because it is
likely that these two proteins have complementary
functions to each other.
Acknowledgments
Guodong
Rao
Bing O’
Dowd
Dr. Eric Oldfield Jikun Li
Ke Wang
IspH Poster Presentation
Introduction
IspH is (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP) reductase (essential reducing enzyme) that contains a 4Fe-4S cluster.
In this cluster, the fourth iron will carry out the reducing activity with the substrate. This enzyme is the last reducing enzyme in the
methylerithritol phosphate pathway, which is an alternative to the mevalonate isoprenoid biosynthesis pathway that exists in humans. The
pathway that exists in humans is called the mevalonate pathway. The substrate for IspH is HMBPP which will produce both DMAPP
(dimethylallyl pyrophosphate) + IPP (Isopentenyl diphosphate). The subject of this presentation is the analysis of the fusion of IspH to
another protein known as Ribosomal Protein S1. Many anaerobic bacteria found in the human stomach utilize IspH-RPS1 and some of
these bacteria are pathogenic.
IspH domains were found on proteins that were twice the size of usual IspH proteins. IspH has been studied before and found to typically
be around 300 residues and the IspH’s of concern were found in proteins with a size of 600-850 residues. Analysis was done using the
Enzyme Similarity Tool to find sequence similarity. Additionally, the location of the clusters was used to visualize the (similarities? – in
reference to Enzyme Similarity Tool) through the software “Cytoscape”.
Growing/Purifying/Re-folding IspH proteins
CthispH-RPS1 and mutants were amplified from genomic DNA of Clostridium thermocellum. This was done because thermophilic bacteria
were intended to produce enzymes that are more stable due to the nature of their environments. The DNA was then digested with SacIHF
and SalIHF and cloned into the pET28a(+) vector. The vector was transformed into E. coli – RIPL (used for “rare” proteins, and is different
due to large amounts of tRNA allow for the rare codons to be translated). Then the bacteria was grown to the necessary OD and induced
with IPTG (which inhibits the lac operon to allow transcription). Afterwards, the pellets were collected through centrifugation and lysed by
sonication. Then the lysate was run through a Ni 2+ column to bind with the histidine tag. These Ni-NTA binding sites are washed with
imidazole in varying concentrations to wash the column and elute the protein.
The iron sulfur clusters need to be reconstituted after this process. Azobacter vinelandii IscS protein is utilized. This enzymes hsCBA
(domain?) acts as a chaperone to mature Fe-S clusters. IspH-RPS1 was concentrated down to 2 mL and is degassed by bubbling nitrogen
and transferred to a glovebox. The buffer used to reconstitute is .5mM Fe(NH4)2(SO4)2, 2.5 mM L-cysteine, 5mM DTT and .1mM IscS (this
is not dialysed out because it is not considered a significant portion of the total protein).
Analysis of IspH-RPS1
As stated before, the first noticeable difference between the fusion IspH’s was that the protein contained many more residues but still
contained an IspH domain. These additional residues were found to comprise 4-6 RPS1 domains. These were found in anaerobic
bacteria. [majority of these IspH-RPS1 containing bacteria are firmicutes – Gram +, low G/C, and also members of Clostridium] 37% of
IspH-RPS1s are present in human gut bacteria. Clostridium is the 7th most prevalent gut bacteria and Eubacterium is the 9th most
prevalent. The function of RPS1 in the RPS1-IspH fusion is not proven but the high level of hybrids indicates a related functioning.
Of the RPS1-IspH containing bacteria, it was found that the Gram + bacteria had four repeats of RPS1 and the Gram – bacteria had six
repeats. The first two repeats bound to the ribosome and the last four were for mRNA binding. The fusion protein was simulated to have a
trefoil or cloverleaf structure (E. coli and P. falciparum). Alpha and beta domains composed the trefoil/cloverleaf with the catalytic 4Fe-4S
cluster located in the center. Linked to this protein were three Cys thiols that are essential for function. The other coordinating sites were
bound by substrate/inhibitor/water molecules.
The bacteria that contained the IspH-RPS1 fusion proteins did not show any other RPS1 or IspH genes. This would indicate that IspH does
perform it’s previously assumed role as these bacteria do not utilize the mevalonate pathway (they lack the genes to code for necessary
proteins). The Kcat [56min-1] of the fusion protein from Clostridium thermocellum proved to be very similar to the Kcat of IspH (not fused)
that was expressed from aquifex aeolicus, and the Km [15 uM] was 2 fold larger. These measurements were taken using the methylviolagen assay, where the substrate HMBPP is used by the enzyme to create the products at a 1:5 ratio. (determine mechanism of
violagen assay –to create the violagen radical 606 nm). The results were further confirmed by liquid chromatography-mass spectroscopy
and NMR. When an EPR spectrum was taken of the IspH-RPS1 fusion protein, the longer sequence was again identified to contain IspH
as they demonstrated a similar signal as “normal” IspHs. To further prove that RPS1 does not affect the function of the IspH domain, four
truncations were performed of the RPS1 domains and none of these affected the IspH activity. This collective data suggests that the
proteins are true Rosetta-Stone proteins in that their complement the other’s activity.
Role of IspH-RPS1
These complementary roles are only hypothesized and not proven. It has been shown that IspH-RPS1 binds close to the RelA site on
bacterial ribosomes. RelA is responsible for the biosynthesis of alarmone, which is a regulator for the bacterial stringent response. The
stringent response is activated when stress is detected by the bacteria and will cause the bacteria to stop spreading and instead focus on
the survival of it’s current cells. It is possible that IspH-RPS1 would detect stressors such as oxygen or iron and contribute to the response.
This would be logical due to the location of binding on the bacterial ribosome.
Conclusion
IspH is an essential protein in the methylerythritol isoprenoid biosynthesis pathway. This experiment establishes IspH-RPS1 as a RosettaStone protein in which the functions of the two proteins are complementary. In analyzing the hybrid IspH-RPS1 further studies and
experiments can be done to determine the exact function of this fusion protein.
References
1. Oldfield, E., Dr., O'Dowd, B., Rao, G., Li, J., & Wang, K. (2015). IspH–RPS1 and IspH–UbiA: “Rosetta stone” proteins. Chemical
Science, 6813-6822. doi:10.1039/c5sc02600h