PD Research Report for the 2015 year
Name（Research group）Le Gao (Fujiwara research group, Graduate School of Science and Technology)
Research Theme

Archaeal chaperonin

Research PeriodApril 1, 2015 ~ March 31, 2016

Research Results（about 2,500 characters in Japanese, about 65 lines times 90 characters in English）
There were two major parts of our works in the last year. One is the study of cold-inducible archaeal
chaperonin CpkA. Thermococcus kodakarensis grows optimally at 85°C and possesses two chaperonins,cold-inducible CpkA and heat-inducible CpkB, which are involved in adaptation to low and hightemperatures, respectively. The two chaperonins share high sequence homology (77%), except in their
C-terminal regions. CpkA, which contains tandem repeats of a “GGM” motif, shows highest ATPase
activity at 60°C ~ 70°C, whereas in CpkB shows highest activity at temperatures higher than 90°C. To
clarify the effects of changes in ATPase activity on chaperonin function at lower temperatures, various
CpkA variants were constructed by introducing single point mutations into the C-terminal region. TheCpkA mutant, in which Glu530 was replaced with Gly (CpkA-E530G), showed increased ATPase
activity, with greatest activity at 50°C. The efficacy of the CpkA mutants against denatured
indole-3-glycerol-phosphate synthase (TrpC Tk ), which is a CpkA target, was then examined in vitro.
CpkA-E530G was more effective at facilitating the refolding of chemically unfolded TrpCTk at 50°C
than wild-type CpkA. The effect of cpkA-E530G on cell growth was then examined by introducing
cpkA-E530G into the genome of T. kodakarensis KU216 (pyrF). The mutant strain, DA4 (pyrF,cpkA-E530G), grew as well at 60°C as the parental KU216 strain. By contrast, DA4 grew morevigorously than KU216 at 50°C. These results suggested that the CpkA-E530G mutation prevented cold
denaturation of proteins under cold-stress conditions, thereby enabling cells to grow in coolerenvironments. Thus, a single base pair substitution in a chaperonin gene allows cells to grow vigorously
in a new environment.
The duplication and divergence of α and β chaperonin subunit genes such as cpkA and cpkB in T.
kodakarensis were thought to have occurred in a common ancestor; the α subunit was subsequently lostfrom the Pyrococcus lineage. However, the results of the present study suggest another possibility. The
natural habitat of Thermococcus is a solfatara, which fluctuate between middle (around solfatara, ~60°C)
and high temperatures (at the center of solfatara, ~100°C); therefore, these thermophiles have evolved anextra chaperonin that allows them to adapt to a colder environment. Living in an environment that moves
between temperature extremes facilitates natural selection; therefore, such thermophile can evolve
quickly. Thus, Thermococci adapted to lower temperatures by duplicating the chaperonin subunit α(CpkA) which acts as an “adaptive allele” and enables T. kodakarensis to adapt to its changing habitat.
Cold tolerance and cold adaptation are related, but different, behaviors. Cold tolerance usually occurs inresponse to a temporary drop in temperature, whereupon cold shock proteins (CSPs) enable their hosts tosurvive. Cold adaptation refers to a longer period of low temperature, triggering a response by not onlyCSPs but also by many non-stress proteins. However, these non-stress proteins are unstable andincorrectly folded under cold-stress conditions. Chaperonin plays an important role in protein foldingand refolding; however, CpkB is not suited to this task under cold-stress conditions because it has low
ATPase activity below 60°C. Thus, CpkA evolved as a unique cold-inducible chaperonin. CpkA andCpkB differ at the C-terminus, which forms part of equatorial domain. According to its C-terminalregion, CpkA, a cold-inducible thermosome, was classified as a CpkA-type chaperonin belonging togroup I chaperonins (which include GroEL of E. coli. By contrast, MM_1798, which encodes amesophilic chaperonin, was classified as a CpkB-type chaperonin along with those thermosomes from
Pyrococcus. Moreover, the growth temperature-dependent distribution of chaperonins and the growth
profile of the T. kodakarensis disruptants of cpkA and cpkB suggests that CpkA-type chaperonins play a
key role in cold adaptation. Without cpkA, T. kodakarensis disruptant DA1 was hard to grow at 60°C. An
extra C-type chaperonin in microorganisms might respond to other situations; an example is the TCP1(CCT)-containing chaperonin in eukaryotic cells, which has up to eight different subunits that interact
with different substrates. The wide variation in the C-terminal regions of chaperonins enables
microorganisms to adapt to changing environments.
In summary, we identified two growth temperature-dependent trends (correlation between theCpkA-type chaperonin gene copy number and growth temperature) in group II chaperonins, both ofwhich are driven by the initial duplication and subsequent mutation of a chaperonin gene. The tandem
repeats within the C-terminal region of CpkA are a mutational hotspot. Here, we showed that a single
base pair substitution (E530G) in the C-terminal region of T. kodakarensis CpkA allowed the organism
to adapt to a lower growth temperature (50°C). Because chaperonins regulate protein-protein interactions
within cells, especially under stress conditions, changes in the activity of chaperonins will lead tochanges in cell properties, such as cold adaptation. GroEL has a marked effect on the refolding ofproteins called mutators, which are involved in DNA repair; thus, GroEL might indirectly increase themutation rate of the host cell. Because GroEL and CpkA have a similar substrate bias, we would expect
the role of CpkA in T. kodakarensis to resemble that of GroEL. Further in vivo studies should aim toderive a mesophilic lineage from hyperthermophiles to better understand cold adaptive mechanisms inarchaea.
This work was published at Journal of Bacteriology, vol 197 (16), Aug 2015.
The other work is about development of novel apparatus for protein trapping using thermostablechaperonin CpkB. Our purpose is to develop a new molecular apparatus to specifically recognize andstabilize proteins, especially heterologous proteins in vivo and in vitro. To achieve this goal, weconstructed a chaperonin - labeled protein pair that chaperonin could specifically recognize and stabilize
the target proteins through the signal-tail attached to the end of the proteins. Our plan is carried out asfollowing route:(1)

Design and screen out the most efficient signal-tails which are recognized by chaperonins. It

would take about 6 months.
(2)

Optimize the chaperonin - signal-tail pair and their working condition. This would take about 3months.
(3)

Immobilize chaperonin. This would take about 3 months.

Through the procedure above, we would obtain a set of signal-tails which could be fussed into target
protein and the labeled target proteins would be specifically recognized and stabilized by modified

Group II chaperonins — CpkA and CpkB. Using ATP analogs — BeF x , the target proteins would beblocked selectively in the cavities of chaperonin from background proteins to avoid any cruel condition.
By adding in excessive ATP, the target proteins would be released from chaperonin at last. We then could
obtain the purified and functional target proteins in solution. We already obtained several functionalsignal-tails to enhance the specificity of CpkB.