Salt-inducible Protein Splicing in cis and trans by Inteins from Extremely Halophilic Archaea as a

16 Pages·2016·1.62 MB·English

Communication
Salt-inducible Protein Splicing in cis and
trans by Inteins from Extremely Halophilic
Archaea as a Novel Protein-Engineering Tool
Annika Ciragan, A. Sesilja Aranko, Igor Tascon and Hideo Iwaï
ResearchPrograminStructuralBiologyandBiophysics,InstituteofBiotechnology,UniversityofHelsinki,P.O.Box65,Helsinki,
FI-00014,Finland
Correspondence toHideo Iwaï:[email protected]
http://dx.doi.org/10.1016/j.jmb.2016.10.006
Editedby S. Koide
Abstract
Intervening protein sequences (inteins) from extremely halophilic haloarchaea can be inactive under low
salinity but could be activated by increasing the salt content to a specific concentration for each intein. The
halo-obligatoryinteinsconferhighsolubilityunderbothlowandhighsalinityconditions.Weshowedthebroad
utilityofsalt-dependentproteinsplicingincisandtransbydemonstratingbackbonecyclization,self-cleavage
for purification, and scarless protein ligation for segmental isotopic labeling. Artificially split MCM2 intein
derived from Halorhabdus utahensis remained highlysoluble and was capableof protein trans-splicing with
excellentligationkineticsbyreassemblyunderhighsalinityconditions.Importantly,theMCM2inteinhasthe
activesiteresidueofSeratthe+1position,whichremainsintheligatedproduct,insteadofCysasfoundin
manyotherefficientsplitinteins.SinceSerismoreabundantthanCysinproteins,thenovelsplitinteincould
widen the applications of segmental labeling in protein NMR spectroscopy and traceless protein ligation by
exploiting a Ser residue in the native sequences as the +1 position of the MCM2 intein. The split
halo-obligatoryinteinwassuccessfullyusedtodemonstratetheutilityinNMRinvestigationofintactproteins
by producingsegmentally isotope-labeled intactTonB protein from Helicobacterpylori.
©2016ElsevierLtd.Allrightsreserved.
Introduction molecules, temperature, lights, redox potential, and
domain swapping to activate the host protein
Halophiles thrive under high salinity conditions by functionsfordevelopingnewbiotechnologicalappli-
deploying various approaches to counter the in- cations (Fig. 1b) [9–13]. Conditional protein splicing
creased osmotic pressure caused by high salinity (CPS) allows us, for example, to have spatial and
[1]. Halo-adaptation of halophilic proteins at the temporal control of the host proteins of inteins [14].
molecular level is a fascinating research topic [2,3]. Protein-fragment complementation by split inteins
Inteins catalyzing protein splicing are prevalent in has been a powerful tool for both in vivo protein
halophilicorganisms[4,5].Proteinsplicingcatalyzed engineering and in vitro protein ligation, because
by inteins is a post-translational auto-processing splitinteinscanligatepolypeptidechainsviaprotein
modification in which an intervening sequence trans-splicing (PTS) (Fig. 1c) [15–17]. Therefore,
(intein)excisesitselffromthehostprecursorprotein, therehavebeenmanyattemptstoidentifynovelsplit
simultaneously ligating the two flanking sequences inteins with different lengths and robust splicing
(exteins) with a peptide bond (Fig. 1a) [6,7]. This activity for various in vivo and in vitro applications
process is self-catalytic, requiring neither additional [18–23]. However, artificially splitting inteins to
co-factorsnorenergy,andhasbecomeanimportant create robust split inteins has not been very
protein-engineering tool. A number of inteins have successful, because splitting a folded protein into
been identified in extremely halophilic archaea, and two pieces makes them less soluble due to the
some of them have been reported to be inactive in increase in exposed hydrophobic area caused by
Escherichia coli [5,8]. There has been growing disrupting the three-dimensional structure [24,25].
interest in controlling protein splicing using small Introducing a split site too close to the termini could
0022-2836/©2016ElsevierLtd.Allrightsreserved. JMolBiol(2016)428,4573–4588
4574 Salt-inducible Protein Splicing incis andtrans
substrate specificity (various junction sequences),
suchasthenaturallyoccurringsplitDnaEinteinfound
in Nostoc punctiforme (NpuDnaE intein) [18,20–22].
Inteins are unique enzymes having only a single
turnoverbecausesubstrates(exteins)are covalently
boundtotheenzyme(intein).Wepreviouslyreported
the comparison of protein-splicing activity among
relatively small inteins from various organisms to
identifypromiscuousinteinsbearingdifferentjunction
sequences with high splicing efficiencies [28]. We
noticedthatsomeinteinsdidnotspontaneouslysplice
when they were overexpressed in E. coli using a
modelsystemoftheB1domainofIgGbindingprotein
G(GB1)astheflankingexteins(Fig.2a)[28].Among
them, two inteins from Halobacterium salinarum
NRC-1 (ATCC 700922), HsaPolII and HsaCDC21
inteins,caughtourattention.H.salinarumNRC-1has
anoptimalgrowthat4.3 MNaClandanintercellular
potassium concentration of 4.6 M and produces
intein-less mature host proteins where inteins are
inserted [8,28]. We therefore tested protein-splicing
activitiesinthepresenceof4 MNaCl(Fig.2b).Both
Hsa inteins could produce the expected spliced
Fig. 1. Protein splicing in cis and trans. (a) Protein product in vitro only at a high salt concentration,
splicing ligates the flanking sequences between the −1 suggesting that the solution condition is responsible
and +1 positions at the splicing junctions, concomitantly for protein splicing rather than the host protein
excisingouttheinteinafterproteintranslationandfolding. contexts, namely the substrate (junction sequences)
(b) CPS in cis. Intein is unfolded or inactive before the specificityoftheinteins(Fig.2bandc).Cis-splicingof
induction of protein splicing by, for example, light, pH, these inteins under high salinity condition has also
small molecule ligand, salts, redox potential, or tempera-
been recently reported [8,29]. The required high
ture. (c) Protein ligation by protein trans-splicing (PTS)
salt concentration indicates that these inteins from
uses split intein fragments (Int and Int ) fused with the
N C extreme halophilic archaea are strictly halophilic
targetfragments(exteins).
(or halo-obligatory) instead of halo-tolerant (Fig. 2b
andc).Thisobservationpromptedustoscreenother
also induce partial splicing activity, resulting in inteinsfromdifferenthalophilicorganismsinorderto
premature cleavages [19,26]. The most widely identifyinteinsthatwouldbeusefulforproteinligation,
used split inteins for biotechnological applications with robust splicing activity and less side-products
have been naturally occurring split inteins found in by cleavages, because both Hsa inteins produced
cyanobacteria [18,23,27]. higheramountsofcleavedproductsthanthespliced
Here,wereportsalt-inducibleproteinsplicingincis product(Fig.2bandc).WediscoveredthattheMCM2
and trans using inteins from extreme halophilic inteinfromH.utahensis(HutMCM2intein)possesses
archaea that are inactive under low salinity but can better protein expression and splicing activity in the
be activated under high salinity as a novel CPS presenceofhighsaltconcentrationsthanHsainteins
systemforbackbonecyclization,proteinpurification, (Fig. 2b). Notably, the HutMCM2 intein could be
and scarless protein ligation for segmental isotopic activated at a lower concentration of NaCl without
labeling of an intact protein. We designed a novel, itproducingnotablequantitiesofside-productsusing
highly efficient split MCM2 intein from Halorhabdus a model system (Fig. 2c and Supplementary Figs. 1
utahensis for PTS and demonstrated its practical and2).
utility for segmental isotopic labeling of intact TonB
protein from Helicobacter pylori by highlighting the Protein purification by salt-induced cleavage
importance of segmental isotopic labeling with the
native sequence for NMR investigation. Canonical protein-splicing reaction involves the
four concerted reaction steps of (1) N-S(O) acyl
shift, (2) transesterification, (3) Asn cyclization, and
Results (4)S(O)-Nacylshift[30].AmutationofAsntoAlaat
thelastresidueoftheinteinshasbeenintroducedto
To expand the practical applications of protein createself-cleavableaffinitytagsforefficientprotein
ligationusingPTS,ithasbeencriticaltoidentifyrobust purification by halting the protein-splicing reaction
split inteins with better ligation efficiency and wider after the first step of N-S acyl shift, thereby making
Salt-inducible Protein Splicing incis andtrans 4575
Fig.2. Salt-dependentCPS.(a)Saltinductionofproteincis-splicingusingGB1asexteins.(b)Proteincis-splicingof
threeinteinsfromextremehalophilicarchaea.Thesamplesweretakenimmediatelyafterinductionwith4MNaCl(0h)
andafterovernightincubation(O.N.)andwereanalyzedbySDS-PAGE.Greyandblackarrowsindicatetheprecursorand
splicedproduct,respectively.(c)Cis-splicingefficiencyversusNaClconcentrationsfortheHutMCM2intein(solidline)and
theHsaCDC21intein(dottedline).Themeanvalues(filledcirclesandsquares)anderrorswereobtainedfromtriplicated
measurements.
the intermediate thioester susceptible to cleavage migration under low salinity conditions, thereby
by reducing agents such as DTT or by hydrolysis preventingprematurecleavagesfrequentlyobserved
(Fig. 3a) [31].Thisself-cleavableaffinity tagsystem for other inteins. To verify this hypothesis, we
has been commercially available as IMPACT™ introduced two Ala mutations at the lastresidue and
system (New England Biolabs) [31]. However, pre- atthe+1positionofHutMCM2inteinandaC-terminal
maturecleavageswithoutanycleavingagentsdueto purificationtag(octa-histidinetag)inthefusionprotein
hydrolysis of the thioester intermediate have been (Fig. 3a). The fusion protein could be easily purified
observed, depending on the chemical stability of with the C-terminal His-tag without any premature
thioesterintermediate[32,33].Thissuggeststhatthe cleavages. The self-cleavage of the fusion protein
three-dimensionalstructureoftheinteinissufficientto was then induced by the addition of a high concen-
promote the first step of N-S acyl shift. The tration of NaCl (Fig. 3b). The intein tag bearing the
halo-obligatoryHutMCM2inteinisinactiveunderlow C-terminalHis-tagcouldbesubsequentlyremovedby
salinity conditions due to unfolded conformation as anadditionalpurificationstepusingimmobilizedmetal
observed by NMR spectroscopy (data not shown). chelating (IMAC) chromatography (Fig. 3b). This
Therefore, it is implausible that the unstructured result confirms that the variant of a halo-obligatory
halophilic intein could induce the first step of N-S inteinbearingthemutationsattheC-terminaljunction
4576 Salt-inducible Protein Splicing incis andtrans
can be usedasa novel intein-mediated proteinpuri- efficiency, increasing salt concentration without any
fication system exploiting the salt-induced cleavage. reducing agents was sufficient to cleave the fusion
Importantly, the salt-controlled structure formation protein (Fig. 3c). This cleavage system might be
ofhalo-obligatoryinteinscancircumventanyprema- beneficialtoproteinscontainingdisulfidebonds.
ture cleavages without the optimization of protein
expression conditions, for example, the expression Salt-induced protein cyclization
temperature [31–33]. Despite the lower cleavage
Thenextquestionwaswhetherwecoulddevelop
asplitintein system fromahalo-obligatory intein for
salt-inducible protein ligation by PTS. First, we
tested in vitro protein cyclization by using a split
halo-obligatory intein because it is a simpler intra-
molecular splicing reaction. As reported previously,
wecreatedafusionproteinbyintroducingacircular
permutation of an intein-containing precursor bear-
ing green fluorescent protein (GFP) as the target
protein (Fig. 4)[34]. Backbone cyclization has been
shown to increase the stabilities of peptides and
proteinswithoutanychangesintheprimarystructure
when both the N- and C-termini are structurally in
closeproximity[35,36,38].However,intein-mediated
proteincyclizationusingasplitinteinspontaneously
producesacircularproteinalreadywithintheE.coli
cells [34,36]. Therefore, a purification tag needs to
be incorporated in the target protein/peptide for the
convenientpurification.Suchapurificationtagmight
not be always desirable or possible to incorporate
e.g., for smaller proteins and peptides. Inducible
protein/peptidecyclizationwouldbeabletofacilitate
the purification step of circular proteins/peptides by
implementingapurificationtagonlyintheprecursor
but not in the target (Fig. 4). Backbone cyclization
can be induced in vitro by salt induction after the
purification step of the precursor fusion protein
(Fig. 4a). For salt-inducible backbone cyclization,
Fig.3. Proteinpurificationbysalt-inducedself-cleavage.
(a)Cleavagereactionbysalt-inducibleself-cleavageusing
HutMCM2inteinwiththedoublemutationsofAlaatthelast
residue and at the +1 position of the intein; Step 1,
Salt-induced protein folding; Step 2, N-S acyl shift by the
foldedinteinfragment;Step3,Hydrolysisbyhydroxylorthiol
reagents; Step 4, Elution of the cleaved product. (b)
SDS-PAGE analysis of the expression and purification
steps. Lane M, molecular marker; lane 0, total cell lysate
beforeproteininduction;lane2,2hafterinduction;laneS,
supernatantaftercentrifugationofcelllysate;laneE,elution
fromNi-NTAcolumn;laneC,afterinducedcleavagebyNaCl
incubation. Right panel: SDS-PAGE analysis of second
purification step using the Ni-NTA column to remove the
inteintagandunreactedfusionprotein.LaneM,molecular
marker;laneSup,reactionmixturebeforeloading;laneFT,
flow-through fraction from the Ni-NTA column. (c)
SDS-PAGEanalysisoftheproteinexpression,purification,
andcleavage.LaneM,molecularmarker;lane0,totalcell
lysate before protein induction; lane 4, 4h afterinduction;
laneE,elutionfromtheNi-NTAcolumn;lane-,cleavagein
4MNaClwithoutanyreducingagent;laneTCEP,cleavage
in4MNaClwith0.5mMTCEP;laneDTT,cleavagein4M
NaClwith1mMDTT.
Salt-inducible Protein Splicing incis andtrans 4577
we tested HsaCDC21 intein that was split after site of the NpuDnaE intein as predicted from the
residue 120 and fused with GFP because the sequence alignment [25]. The full-length precursor
intramolecular reaction can be less prone to side protein was purified without any in vivo cyclization
reactions (Fig. 4a). The split site was selected reaction from E. coli using a His-tag at the
because the site corresponds to the naturally split N-terminus of the precursor protein. Backbone
cyclization using the precursor protein was then
inducedinvitrobyadditionofafinalconcentrationof
3.5 MNaCl.Figure4bshowsthesalt-inducedinvitro
backbone cyclization of GFP bearing a thrombin
cleavage site. Interestingly, only circular GFP was
produced by the salt induction, judging from the
SDS-PAGEanalysis.Thecircularformwasrelinear-
ized by the digestion of the GFP using thrombin to
confirm the cyclization, which migrated slower than
thecircularformaspreviouslyreported(Fig.4band
c) [34]. This result demonstrated the feasibility of
backbone cyclization of proteins/peptides by salt
induction using halo-obligatory split inteins.
Salt-induced protein ligation by PTS
Encouraged by the result from backbone cycliza-
tion, we next tested bimolecular protein ligation by
salt-inducible PTS using the HutMCM2 intein. This
intein was split into two halves after residue 117,
similar to HsaCDC21 intein [25]. Each of the split
halveswasfusedwithaGB1domainasanexteinfor
testing PTS (Fig. 5a). Trans-splicing kinetics of the
artificiallysplitHutMCM2inteinwas4.5 ± 0.6 × 10−4
(s−1) for the model system of GB1s, with one
N-terminal and three C-terminal native junction
sequences (“R” and “SED”, respectively) including
Seratthe+1position(Fig.5a).Thisligationkineticsof
the split HutMCM2 intein is as good as that of the
well-characterized robust split NpuDnaE intein with
the same model system of GB1, even though
NpuDnaEhasmorenucleophilicCysthanSeratthe
+1position[18,19].Becausethe+1positionofinteins
willremainintheligatedproduct,the+1residueoften
resultsasascarintheligatedproteinbyPTS.Since
Ser is more abundant than Cys, HutMCM2 intein
havingSeratthe+1positioncanbewidelyusedfor
various applications by exploiting Ser residues in
intact proteins. Moreover, the split HutMCM2 intein
wasmoresolublethanthewidelyusedNpuDnaEand
Fig. 4. Protein cyclization by salt-inducible protein
splicingusingHsaCDC21intein.(a)Schematicdrawingof
proteincyclizationbyinducibleproteinsplicing.Theinverse
triangleindicatesaspecificcleavagesiteofthrombinused
forthebackbonelinearization.(b)Timecourseofcyclization
ofGFPinthepresenceof3.5MNaCl.Lanes0,1,2,and3
indicate 0h, 1h, 3h, and 19h after the salt-induction,
respectively.Greyandblackarrowsindicatetheprecursor
and cyclized product, respectively. (c) Linearization of
circularGFPbythrombindigestion.Thr-andThr+indicate
withoutorwiththrombindigestion,respectively.Blackand
greyarrowsindicatecircularandlinearGFP,respectively.
4578 Salt-inducible Protein Splicing incis andtrans
Fig. 5. PTS using the halo-
obligatory split HutMCM2intein.(a)
In vitro protein ligation by inducible
PTS using a model system of GB1.
Lane0,beforesalt-induction;lanes1,
2, 3, 4, 5, and 6 indicate samples
taken 10min, 30min, 1h, 2h, 3h,
and6hafterthe4MNaClinduction,
respectively. (b) Comparison of the
solubleandinsolublefractionsofthe
N-terminalprecursor(Int fusedwith
N
the identical N-extein of GB1). Int
N
wasfromHutMCM2(HutMCM2ΔC62),
NpuDnaE,orSspDnaEinteins.M,P,
and S stand for marker, pellet, and
supernatant,respectively.
SspDnaE inteins when they were fused with an proteinshaveawell-foldedglobulardomain.However,
identicalextein(Fig.5bandSupplementaryFig.3a). significant structural differences in the
This is presumably because of the high solubility of three-dimensional structures of E. coli CTD have
strictly halophilic inteins as observed for other been reported for differently dissected CTDs with
halophilic proteins [37,39]. Salt-inducible split inteins various lengths [41–43]. Therefore, the
derived from halo-obligatory inteins could thus over- three-dimensional structure of the CTD of the TonB
come the solubility issues of many artificially split protein in the full-length context could shed light on
inteins, which have been used for applications howintactTonBproteinfunctions.ThePro-richregion
includingsegmentalisotopiclabeling[40]. ishighlyflexibleasevidenced bysevere NMRsignal
overlaps,whichcouldhinderthestructuralcharacter-
Scarless segmental isotopic labeling of TonB izationofthefull-lengthHpTonBindetailbybothNMR
from H. pylori spectroscopy and crystallography (see below).
HpTonB was split between the Pro-rich region and
To demonstrate the practicality of salt-inducible the CTD (after residue 154) in order to use Ser155
inteins, we used salt-induced in vitro protein ligation residueasthe+1positionofHutMCM2inteinandwas
for segmental isotopic labeling. We chose the TonB fused with two halves of a split HutMCM2 intein to
protein from H. pylori (HpTonB) for protein ligation, create N- and C-terminal precursors (Fig. 6b and c).
which was conducted using a split HutMCM2 intein FortheN-terminalprecursor,theN-terminalfragment
system.TonBproteinisamembrane-anchoredprotein containing the Pro-rich region (residues 36–154) of
essentialforTonB-dependentoutermembranetrans- HpTonB was fused with the N-terminal fragment
porters, consisting of a transmembrane (TM) region, (residues 1–117) of HutMCM2 intein (HutMCMΔC62)
a Pro-rich region, and a C-terminal domain (CTD) togetherwiththeC-terminalocta-histidinetag(Fig.6b
(Fig. 6a) [41–43]. The dissected CTDs of TonB and c). For the C-terminal precursor, CTD (residues
Fig. 6. (a) Primary structure and domains of HpTonB. TM region is highlighted in grey. Pro-rich region and CTD are
indicatedbyclosedrectanglesandbrokenrectanglesingrey,respectively.(b)Sequencealignmentofthejunctionregionsof
HpTonB, twoprecursors,andtheexpectedligationproduct.Thesplit siteusedfor segmental labelingisindicatedby“/”.
(c)ProcedureforproteinligationofHpTonBusinginduciblePTSforsegmentalisotopiclabeling.ResiduenumbersofHpTonB
areshownonthetop.(d)ProteinligationofHpTonBusingsplitHutMCM2inteinwithArgatthe−1position.LaneN,purified
N-terminalprecursorbeforemixing;laneC,purifiedC-terminalprecursorbeforemixing;lane0h,immediatelyaftermixingthe
N-andC-terminalprecursorsin3.5MNaCl;lane16h-,after16-hdialysisin0MNaCl;lane16h+,after16-hdialysisin3.5M
NaCl;lane22h-,after22-hdialysisin0MNaCl;lane22h+,after22-hdialysisin3.5MNaCl;lane39h-,after39-hdialysisin
0MNaCl;lane39h+,after39-hin3.5MNaCl;laneLP,theligationproductaftertheIMACpurification.
Salt-inducible Protein Splicing incis andtrans 4579
155–285) of HpTonB was fused with the C-terminal dinetagfor 15N-labeling(Fig.6bandc).BothN-and
fragment (residues 145–186) of HutMCM2 intein C-terminal precursors were cloned in the expression
(HutMCM2 )togetherwiththeN-terminalhexahisti- vectors under the control of a T7 promoter and were
C42
Fig.6(legend onprevious page)
4580 Salt-inducible Protein Splicing incis andtrans
expressedinE.coli.Theprecursorswereindividually
purifiedbyIMACwithoutanysolubilizationsteps(Fig.
6c and Supplementary Fig. 3b). The two purified
precursors in phosphate-buffered saline (PBS) were
mixed at a final concentration of ~0.1 mM in a total
volumeof4.5 mL.The solutionmixture wasdialyzed
against 1 L of 3.5 M NaCl, 0.5 M NaPi, and 0.5 mM
tris(2-carboxyethyl)phosphine (TCEP) at pH 7.0 for 1
to 2 days at room temperature, followed by a buffer
exchangetoPBSbufferbeforefurtherpurification.The
ligatedproductwasfurtherpurifiedbyanotherroundof
IMAC because all of the unreacted precursors and
excised split intein fragments remained tagged with
poly-histidine tags and could be efficiently removed.
The flow-through fractions from IMAC were collected
and analyzed by SDS-PAGE (Fig. 6d). Typically, the
ligatedHpTonB(36–285)withN95%purity(~3 mgof
theligatedproductfrom0.5 Lofeachcellculture)was
obtainedafter1to2 daysofdialysis.Thesegmentally
labeled sample was also confirmed by mass spec-
trometry(SupplementaryFig.4).Theligationefficiency
was estimated to be ~30% after a 1-day dialysis for
HpTonBwiththenativesequence(Lysatresidue154).
Theligationefficiencywasbetter(N~80%aftera2-day
dialysis)whenthenative−1residue(Arg)ofHutMCM2
intein was used instead of Lys at the −1 position
(residue 154 in HpTonB). The lower efficiency and
slowerkineticscomparedtothemodelsystemsuggest
that both the junction sequences (the −1 and +2
positions) and the target protein still significantly
influence the final yield, as has been previously
observed with other inteins [18,22]. However, the
precursors and split intein fragments were efficiently
removedbypurificationtagsinthesplitinteins,despite
the lower efficiency for the ligation of intact HpTonB
(Fig. 6c and d). Importantly, the native sequence of
HpTonBwasproducedwithoutanyscarbecausethe
“KS”sequenceinHpTonBwasutilizedasthe−1and
+1 positions of HutMCM2 intein for protein ligation,
therebyresultinginthenativesequenceofHpTonBat
thesplicingjunction.
Figure 7 shows the comparison of the uniformly
15N-labeled HpTonB(36–285) and the segmentally
[15N, (155–285)]-labeled intact HpTonB(36–285) pro- Fig. 7. [1H, 15N]-HSQC spectra from (a) uniformly
duced by salt-induced PTS. The uniformly labeled 15N-labeled HpTonB(36–285) and (b) segmentally [15N,
sample shows the heavily overlapped strong NMR (155–285)]-labeled HpTonB(36–285). (c) Comparison of
signalswithinanarrowrangearound8–8.5 ppminthe 1D 1H–NMR spectrum (thin line) and 1D 1H projection
[1H, 15N]-heteronuclear single quantum coherence from[1H, 15N]-HSQCspectrumoftheidenticalsampleof
(HSQC) spectrum due to the highly flexible Pro-rich segmentally[15N,(155–285)]-labeledHpTonB(36–285).
region(Fig.7a).Ontheotherhand,the[1H,15N]-HSQC
spectrum of the segmentally isotope-labeled sample sequence could thus facilitate the detailed NMR
presents the well-dispersed peaks with homogenous analysisofCTDinthepresenceoftheflexiblePro-rich
intensitiesoriginatingonlyfromtheCTD(residues155– region.
285) of HpTonB (Fig. 7b). Moreover, the 1H 1D
spectrumofthesegmentallylabeledsamplestillexhibits The comparison between CTD and the
strong 1H signals from the Pro-rich region, validating full-length HpTonB without TM
thattheflexiblePro-richregionisnotlabeledwith 15N
due to the segmental isotopic labeling (Fig. 7c). The ToevaluatethestructuralinfluencesofthePro-rich
segmentally isotope-labeled HpTonB with the native region on the CTD of HpTonB, we determined the
Salt-inducible Protein Splicing incis andtrans 4581
NMR structure of HpTonB(194–285), which was becausetheC-terminalstrandisfoundtointeractwith
constructed based on the sequence homology with the TonB-dependent outer membrane transporters
other TonB proteins (Fig. 9a). The NMR structure of [43–46].Thisexampleofsegmentalisotopiclabelingof
HpTonB(194–285)(PDB entry: 5LW8) revealed the intact HpTonB with the native sequence clearly
presenceof an additionalN-terminalhelix compared emphasizestheimportanceofstructuralinvestigation
with the CTD of E. coli TonB(152–239)(PDB entry: inthefull-lengthcontextandthattruncateddomainsof
1XX3[43]),whichisdistinctlydifferentfromanyother proteins,whichareoftenusedforstructuralinvestiga-
previouslyreportedTonBproteinsbutmoresimilarto tion, might not represent actual biological
therelatedHasBprotein[42–44].Thecomparisonof conformation.
the[1H, 15N]-HSQCspectraofthetruncatedCTDof
HpTonB(194–285) and the segmentally labeled
HpTonB(36–285) identified clear differences in the Discussion
[1H, 15N]-HSQCspectra(Fig.8).Thechemicalshifts
differencesbetweenthetwoconstructsofHpTonBare This study reports that inteins from extremely
visualizedontheNMRstructureofHpTonB(194–285) halophilic archaea can be halo-obligatory and are
(Fig. 9b and c). The observed large chemical shift thusinactiveunderlowsalinityconditionsbutcanbe
changes are located at the N terminus as expected activated by increasing the salt concentration. This
andarealsoobservedfortheβ-strands,particularlyfor salt-dependent activation of halo-obligatory inteins
the C-terminal strand. This data suggests that the opens new opportunities to control protein splicing.
prolonged N-terminal region of HpTonB(36–285) has Forexample,asalt-inducibleintein-mediatedprotein
structuralinfluencesontheremainingCTD.Wethink purification system could both prevent premature
thattheN-terminalPro-richregionaffectsthedynam- cleavages and be used for purification without any
ics of the first helix, which causes chemical shift reducingagents(Fig.3c).Theuseofhalo-obligatory
changes and might influence the biological function inteins for backbone cyclization facilitates the
Fig.8. Anoverlayoftwo[1H,15N]-HSQCspectraofHpTonB(194–285)(green)andsegmentally[15N,(155–285)]-labeled
HpTonB(36–285)(darkgrey).TheresonanceassignmentsofthebackboneamidesofHpTonB(194–285)aremarkedbythe
residuenumberandone-letteraminoacidcodes.Side-chainamideresonancesofGlnandAsnareindicatedby“sc”.
4582 Salt-inducible Protein Splicing incis andtrans
Fig.9. (a)Astereoviewofanensem-
ble of 20 NMR solution structures of
CTD, HpTonB(194–285). α-helices and
β-sheets are colored in red and blue,
respectively.(b)Chemicalshiftperturba-
tionofHpTonB(194–285)bythePro-rich
region. The average chemical differ-
ences between HpTonB(194–285) and
HpTonB(36–285) were calculated with
the formula Δδ = [(Δδ )2+
av H
0.154*(Δδ )2]1/2andplottedagainstthe
N
residue number. (c) The locations with
larger chemical shift changes
(Δδ ≥ 0.2) are highlighted on the
av
schematic drawing of NMR structure
of HpTonB(194–285). Residues
with Δδ ≥ 0.4 ppm and 0.4 ppm N
av
Δδ ≥0.2ppm are colored in red and
av
yellow,respectively.TheNandCtermini
areindicatedbyNandC,respectively.
Residues withambiguous assignments
in the segmentally [15N, (155–285)]-la-
beled HpTonB(36–285) are shown in
dark gray in the structure and are
indicated by red asterisk in the plot.
Drawingsofthestructurewereproduced
byPyMOL[62].
production of cyclic peptides and proteins in vitro smaller bioactive peptides as any tag could poten-
because spontaneous cyclization in vivo can be tiallyreduce their biological activities.
prevented,therebymakingitpossibletoincorporate These halo-obligatory inteins were found to be
purification tags in the precursor instead of the highlysolublebothwithandwithoutahighsaltcon-
cyclizedpeptide(Fig.4)[34].Thiswillbeparticularly centration because the two strictly halophilic inteins
advantageous for the backbone cyclization of couldbeeasilyconcentratedtoN4 mM(N80 mg/ml).