!
4.1$Two$CNB$domains$in$PKG$I$show$different$cyclic$nucleotide$selectivity$
!
4.1.1$CNB=A$has$no$cyclic$nucleotide$selectivity$
!
We!initially!hypothesized!that!the!high!affinity!CNB5A!domain!might!play!a!key!
role! in! cGMP! selectivity! of! PKG! I! because! the! CNB5A! domain! is! located! in! close!
proximity!to!the!auto5inhibitory!(AI)!sequence.!Since!dissociation!of!the!AI!sequence!
from!the!catalytic!core!is!one!of!the!key!features!of!allosteric!activation!of!all!cyclic!
nucleotide! (cNT)! dependent! protein! kinases,! we! assumed! that! the! CNB5A! domain!
should!modulate!the!kinase!activity!in!a!cGMP5specific!manner.!In!addition,!previous!
studies! also! supported! our! hypothesis! by! showing! that! cGMP! binding! at! CNB5A!
results!in!spontaneous!PKG!activation!(Hofmann!et!al.,!1985l!Smith!et!al.,!2000).!For!
these! reasons,! we! solved! crystal! structures! of! PKG! Iβ! CNB5A! (925227)! in! three!
different!states!with!cGMP,!cAMP,!and!no!ligand!to!demonstrate!the!cGMP5specific!
contacts! provide! cGMP! selectivity.! However,! all! three! structures! exhibited! an!
identical!conformation,!suggesting!that!the!domain!is!not!affected!by!different!ligand!
binding! (Figure! 4.1).! Unexpectedly,! the! structure! of! the! non5cNT! added! CNB5A!
domain!showed!a!clear!electron!density!of!cNT!in!two!of!the!four!CNB5A!molecules!in!
the!asymmetric!unit!(Figure!3.8).!These!results!raise!the!question!of!whether!or!not!
the!CNB5A!domain!is!really!selective!for!cGMP,!because!the!results!strongly!suggest!
that!the!CNB5A!domain!has!a!high!binding!affinity!for!cGMP!as!well!as!cAMP.!!!
Figure!4.1:! Structural!comparisons! of!four! different!ligand! bound!states! of! PKG!Iβ!
CNB0A.!Stereoview!of!all!four!structures!shows!identical!conformation.!!
Together! with! the! structural! observations,! our! ITC! measurements! of! cNT!
affinities!of!the!CNB5A!domain!revealed!that!CNB5A!is!not!selective!for!cGMP.!The!
ITC! data! showed! that! CNB5A! only! has! a! 25fold! higher! affinity! for! cGMP! than! for!
cAMP! (Table! 4.1).! The! binding! constant! (KD)! for! cGMP! is! similar! to! the! previously!
measured!KD!for! cGMP! in! CNB5A! (Hofmann! et! al.,! 1983,! 1985),! but! such! a! high!
binding! affinity! for! cAMP! had! never! been! observed! before! (Kim! et! al.,! 2011).! The!
results! also! showed! that! binding! of! both! cNTs! to! the! CNB5A! domain! is! strongly!
enthalpy5driven!(512.5!verse!512.4!kcal/mol),!suggesting!that!the!binding!mechanism!
is! mainly! caused! by! electrostatic! interactions,! most! likely! between! the! negatively5 charged! cyclic! phosphate! group! and! positively5charged! amino! acid! residues! in! the!
PBC!(Table!5.1).!In!contrast,!the!entropy!values!of!cGMP!and!cAMP!(54.7!and!56.1!
cal/mol/K,!respectively)!showed!a!slight!difference,!suggesting!that!the!hydrophobic!
interaction! between! the! different! configured! purine! base! rings! of! cGMP! and! cAMP!
and! the! residues! at! the! β5! strand! (Leu172! and! Cys173)! might! cause! this! slight!
difference!in!KD!between!cGMP!and!cAMP.!!
These!ITC!results!are!also!well!supported!by!our!observation!with!the!cAMP5!
bound! structures! of! the! CNB5A! domain.! The! cAMP! bound! structures! revealed! that,!
while! cGMP! only! binds! to! CNB5A! in!syn! conformation,! cAMP! binds! to! CNB5A! in!
either!anti!or!syn!conformation!(Figure!4.2).!Cyclic!GMP!and!cAMP!differ!only!in!their!
purine! base! groups.! Cyclic! AMP! has! an! amino! group! at! 6! position! of! the! adenine!
ring,!but!cGMP!contains!a!carbonyl!group!at!the!same!position!and!an!amino!group!
at!the!2!position!(Figure!4.3).!Because!of!these!differences,!their!equilibrium!states!in!
solution! are! different.! Both! cNTs! exist! in! both! conformations,! however! cGMP! stays!
favorable!in!the!syn,conformation!(syn!and!anti!ratio!=!95:5)!and!cAMP!is!favorable!in!
the, anti! (syn, and! anti, ratio=! 30:70)! (Fazakerley! et! al.,! 1977l! Yathindra! and!
Sundaralingam,! 1974).! While! cGMP! readily! stays! in! a! favorable! conformation! (syn)!
for! binding! to! CNB5A,! more! than! ⅔! of! cAMP! is! in! the!anti! conformation! in! solution,!
which!is!not!favorable!for!binding!to!the!cGMP5binding!pocket.!This!natural!difference!
in!conformation!of!cAMP!may!be!the!cause!of!the!two!states!of!the!bound!cAMP!in!
Table! 4.1:! Summary! of! ITC! measurements! of! human! PKG! Iβ! CNB0A! (920227)! with!
cGMP!and!cAMP.!
hPKG!Iβ!CNB5A!
(925227)!
KD! Enthalpy!(ΔH)! Entropy!(ΔS)!
cGMP! 12!±!2!nM! 512.5!kcal/mol! 54.7!cal/mol/K!
cAMP! 27!±!4!nM! 512.4!kcal/mol! 56.1!cal/mol/K!
the!cAMP!co5crystal!structures!(Figure!4.2).!The!anti!conformation!of!cAMP!bound!at!
the! cGMP5binding! pocket! causes! unfavorable! hydrophobic! interactions! with! the!
base5interacting!residues!(Leu172!and!Cys173)!in!the!β5!strands.!Moreover,!it!also!
causes!the!elimination!of!a!charged!interaction!between!the!unprotonated!N3!at!the!
adenosine! and! Thr193! (middle! and! right! panels! in! Figure! 4.2).! Thus,! the! cAMP!
bound!structures!of!CNB5A!also!provide!structural!explanation!of!the!slightly!lower!KD! for!cAMP!(Table!4.1).!Taken!all!together,!the!CNB5A!domain!itself!does!not!have!any!
selectivity! between! cGMP! and! cAMP,! but! the! nature! of! cNTs! may! cause! the! slight!
difference!of!the!cNT!binding!affinity!in!CNB5A.!
Figure! 4.2:! Cyclic! nucleotide! binding! site! of! CNB0A! with! different! cyclic! nucleotide!
binding! states.!Left:! cGMP! bound! state! (syn),! Middle:! cAMP! bound! state! (anti),! Right:!
cAMP!bound!state!(syn).!Modified!from!(Kim!et!al.,!2011).!
Figure!4.3:!cGMP!and!cAMP.!!cGMP!and!cAMP!only!differ!in!their!purine!base!rings.!(A)!! cGMP,!an!hydrogen!at!1!position,!an!amino!group!at!2!position,!and!a!carbonyl!group!at!6!
position! of! the! purine! are! distinctive! from! cAMP.! (B)! cAMP! has! an! amino! group! at! 6!
position!of!its!purine!base.!Generated!using!MarvinSketch.!
4.1.2$CNB=B$is$responsible$for$cGMP$selectivity$
Our!FP!competition!assay!results!revealed!that!the!CNB5B!domain!has!cGMP!
selectivity.!While!the!CNB5A!domain!showed!no!cGMP!selectivity,!the!CNB5B!domain!
showed! 2405fold! higher! binding! affinity! for! cGMP! than! for! cAMP! (Table! 4.2),!
suggesting! the! CNB5B! domain! is! responsible! for! cGMP! selectivity! in! PKG! I.! The!
CNB5A! and! the! CNB5B! domains! are! very! similar! with! the! high! sequence! similarity!
(35%,! the! sequence! similarity! between! the! residues! in! the! PBC! is! 57%),! so! the!
affinity!measurement!results!drew!a!question!!as!to!what!elements!of!CNB5B!causes!
the!cGMP!selectivity.!!
To! further! understand! details! of! the! structural! elements! of! CNB5B! that!
contribute! to! cGMP! selectivity,! we! solved! crystal! structures! of! the! truncated! CNB5B!
domain! (2195369)! with! and! without! cGMP! (Figures! 3.16! and! 3.17).! The! co5crystal!
structure!with!cGMP!showed!that!the!CNB5B!domain!shares!a!majority!of!the!cGMP!
interactions! that! were! seen! in! the! CNB5A! domain! (Figure! 4.4).! The! interactions!
between!the!ribose!and!cyclic!phosphate!are!conserved!between!two!CNB!domains!
in! PKG! I,! including! interactions! through! a! glutamate! (A:! Glu183,! B:! Glu307),! an!
arginine!(A:!Arg192,!B:!Arg316),!and!a!threonine!(A:!Thr193,!B:!Thr317).!In!addition,!
hydrophobic! interaction! with! a! leucine! (A:! Leu172,! B:! Leu296)! is! identical! between!
the!two!CNB!domains.!In!contrast,!the!interactions!through!Arg297!on!the!β5!strand!
and!Tyr351!at!the!C5terminus!are!unique!in!CNB5B!(Figure!4.4).!Interestingly,!Arg297!
interacts! with! the! 65position! carbonyl! oxygen! (O6),! which! is! an! unique! feature! of!
cGMP.! This! led! us! to! hypothesize! that! Arg297! may! be! a! key! residue! for! cGMP!
selectivity! in! PKG! I.! To! confirm! this! hypothesis,! we! generated! alanine! mutants! of!
cGMP5interacting!residues!in!CNB5B!and!tested!their!binding!affinities!for!cGMP!and!
cAMP!(Table!4.3).!Although!all!mutants!showed!various!ranges!of!reduction!in!both!
cGMP! and! cAMP! affinities,! the! Arg297Ala! mutant! showed! a! 235fold! higher! EC50!for!
Table! 4.2:! Affinity! measurements! of! PKG! Iβ! CNB0A! and! CNB0B!(Summarized! from!
(Huang! et! al.,! 2014l! Kim! et! al.,! 2011).! The! data! were! generated! by! Dr.! Nicholas! Gene!
Brown,!Dr.!Dar5Chone!Chow,!and!Dipl.5Biol.!Robin!Lorenz.!
Domain! Methods! CNB0A! CNB0B!
cGMP! ITC!(KD)! 12!±!2!nM!(3)! 5!
FP!(EC50)! 5! 215!±!13!nM!
SPR!(EC50)! 5! 181!±!8!nM!
cAMP! ITC!(KD)! 27!±!4!nM!(3)! 5!
FP!(EC50)! 5! 52!±!8!μM!
SPR!(EC50)! 5! 36!±!3!μM!
cGMP,!while!its!EC50!for!cAMP!decreased!45fold!(Table!4.3).!The!Arg297Ala!mutant!is!
the! only! mutant! to! show! a! significant! decrease! in! cGMP! affinity! while! showing!
increases! in! cAMP! affinity,! suggesting! that! the! bulky! positively! charged! Arg297! on!
the!β5!strand!is!a!key!residue!that!is!crucial!for!cGMP!selectivity.!
!
!
Figure!4.4:!Structural!comparison!between!the!CNB0A:cGMP!and!the!CNB0B:cGMP!
complexes.!(A)!The!cGMP5binding!pocket!in!CNB5A!(PDB!code:!3OD0).!(B)!The!cGMP5 binding! pocket! in! CNB5B! (PDB! code:! 4KU7).! The! key! cGMP! interacting! residues! are!
shown! as! black! sticks.! An! ordered! water! molecule! is! shown! as! a! blue! sphere.! (C)!
Sequence! alignment! between! CNB5A! and! CNB5B! of! PKG! Iβ.! Conserved! residues! are!
shaded!in!yellow,!and!cGMP!interacting!residues!through!their!side!chains!are!shaded!in!
red.!!!
4.1.3$Distinct$cyclic$nucleotide$interacting$residues$in$PKG$I$and$PKA$I$explain$their$
cNT$preference$$
!
The! structural! comparisons! between! the! CNB! domains! of! PKG! I! and! PKA! I!
RIα! provide! mechanical! insights! to! how! cGMP5binding! pockets! are! designed! to!
discriminate! cAMP! (Figure! 4.5).! While! PKG! I! has! a! free! cysteine! residue! (Cys173)!
and! bulky! residues! including! Leu172,! Leu296,! and! Arg297! at! the! β5! strand! of! the!
CNB! domains,! PKA! RΙα! has! residues! with! short! side! chains! at! the! corresponding!
positions:! Ala189,! Thr190,! Val313,! and! Gly314.! These! differences! affect! the! cNT!
accessibility.! As! discussed! earlier,! the! majority! of! cAMP! is! in! an!anti,conformation!
(Fazakerley!et!al.,!1977l!Yathindra!and!Sundaralingam,!1974)l!therefore,!in!PKA!RIα,!
the!anti5cAMP!can!easily!access!the!binding!pocket!due!to!the!short!side!chains!of!
the! residues! at! the! β5! strand.! In! contrast,! bulkier! residues! in! PKG! I! may! provide!
unfavorable!binding!conditions!for!the!anti5cAMP.!As!seen!in!Figure!4.6,!Arg297,!in!
the! apo! CNB5B! structure,! shows! an! elongated! conformation,! so! it! possibly! creates!
steric!hindrance!with!the!65position!amine!group!of!the!anti5cAMP.!If!this!is!the!case,!
Arg297!can!block!the!binding!of!the!anti5cAMP!into!the!cGMP5binding!pocket!(Figure!
4.6).! Furthermore,! the! positively5charged! guanidinium! group! of! Arg297! could! also!
disturb! the! binding! of! the! similarly! charged! adenine! group! of! cAMP,! supporting! our!
hypothesis!of!Arg297’s!role!as!a!cAMP!filter!in!PKG!I.!!
Table!4.3:!Cyclic!nucleotide!binding!affinities!of!PKG!Iβ!CNB0B!(2190329)!wild!type!and!
mutants!(Huang!et!al.,!2014).!(Measurements!were!done!by!Dipl.5Biol.!Robin!Lorenz).!
!
! KD!±!SEM*!(n)!measurements! EC50!±!SEM*!(n)!competition!
PKG!Iβ!(2195 369)!
85Fluo5cGMP! 85Fluo5cAMP! cGMP! cAMP!
Wild!type! 157!±!12!nM!(5)! 12!±!1!μM!(2)! 215!±!13!nM!(8)! 52!±!8!μM!(5)!
Leu296Ala! 907!±!50!nM!(2)! >!54!μM!(2)! 28!±!1!μM!(3)! 91!μM!(1)!
Arg297Ala! 9!±!1!μM!(2)! 19!±!1!μM!(2)! 5!±!0.5!μM!(2)! 12!±!2!μM!(2)!
Thr317Ala! >!9!μM!(2)! >!12!μM!(2)! 21!μM!(1)!†! 76!μM!(1)!†!
Try351Ala! 300!±!40!nM!(2)! >!8!μM!(2)! 6!±!0.2!μM!(2)! 130!±!25!μM!(3)!
*FP! measurements! were,! at! least,! in! duplicate.!KD! is! the! dissociation! constant,! EC50! is! the! half5maximal! effective!
concentration,!and!SEM!is!standard!error!of!measurement.!!
†!EC50!values!were!measured!with!SPR!competition!assay!as!FP!competition!was!not!possible.!
Figure! 4.5:! Cyclic! nucleotide! interacting! residues! in! CNB0domain! of! PKG! I! and! PKA!
RI.!(A)! cGMP! contact! residues! in! CNB5A! (left)! and! CNB5B! (right)! of! PKG! Iβ.! (B)! cAMP!
contact!residues!in!both!CNB5A!(left)!and!CNB5B!(right)!of!PKA!RIα.!
Figure!4.6:!Structural!comparisons!between!the!apo0!and!cGMP0bound!CNB0B!of!PKG!
Iβ.!The!apo!(gray)!and!the!cGMP!bound!(red)!structures!aligned!at!the!β5barrel!region.!An!
enhanced! view! of! the! binding! pocket! highlighting! the! β5! and! capping! residue! interactions!
with! cGMP! (black! sticks)! are! shown! on! the! right.! In! the! apo! structure,! the! side! chain! of!
Arg297!is!extended!in!a!conformation!partially!occupying!the!empty!cGMP!binding!pocket.!!!
The!second!significant!difference!of!the!CNB!domains!in!PKG!I!and!PKA!I!is!
found!at!the!PBC!loop.!A!threonine!residue!at!the!PBC!loop!that!is!only!conserved!in!
PKG! I! provides! hydrogen! bonds! to! both! the! cyclic! phosphate! and! the! guanine! of!
cGMP!(Figure!4.5).!Previous!studies!revealed!that!the!threonine!residues!in!the!CNB!
domains! are! important! for! the! cGMP5dependent! activation! of! PKG! I! (Reed! et! al.,!
1996l! Smith! et! al.,! 2000)! and! that! these! residues! contribute! to! cGMP! selectivity!
(Reed! et! al.,! 1996l! Shabb! et! al.,! 1991).! In! this! thesis! study,! we! confirmed! that!
Thr317Ala! in! the! truncated! CNB5B! (2195369)! diminishes! cNT! binding.! Thr317Ala!
showed! that! the! mutation! not! only! reduced! cGMP! affinity! significantly,! but! also!
slightly!reduced!cAMP!affinity!(Table!4.3).!This!suggests!that!removing!two!hydrogen!
bonds! mediated! by! Thr317! affects! both! cGMP! and! cAMP! binding! (Figures! 4.2! and!
4.5,! and! Table! 4.3).! However,! the! effect! of! the! mutation! is! predominant! in! cGMP!
binding! over! cAMP! binding,! revealing! that! the! threonine! residue! may! also! play! a!
minor!role!in!cGMP!selectivity!in!PKG!I.!!
4.2$cGMP$binding$drives$conformational$changes$in$CNB$domains$$
4.2.1$cGMP=induced$conformational$changes$at$the$N=terminal$helices$in$CNB=A$is$
crucial$for$releasing$the$AI$sequence$from$the$catalytic$core$
!
Although!I!determined!the!three!CNB5A!structures!with!cGMP,!with!cAMP,!and!
without!any!cNT,!the!overall!structures!of!all!three!ligand!bound!states!of!the!CNB5A!
domain!are!almost!identical!(Kim!et!al.,!2011)!(Figure!4.1).!Soon!after,!I!realized!that!
the!reason!why!we!could!not!observe!any!structural!difference!between!the!apo!and!
the!cNT!bound!structures!was!due!to!a!truncation!artifact.!In!the!inhibited!state,!the!AI!
sequence!and!the!CNB5A!domain!docks!to!the!C5domain.!Structural!comparison!with!
the! apo! PKA! RIα! of! the! tetrameric! holoenzyme! reveals! CNB5A! undergoes! large!
conformational! changes! upon! cNT! binding,! especially! at! the! N3A! motif,! suggesting!
that! CNB5A! plays! a! role! in! dissociation! of! the! AI! sequence! from! the! catalytic! core!
upon! cGMP! binding! (Figure! 4.7).! ! However,! CNB5A! constructs! that! I! used! for! this!
structural! study! were! missing! the! AI! sequence! (amino! acid! No.! 75580)! and! the! C5 domain! contacts.! Interestingly,! based! on! the! NMR! studies! of! the! PKA! RI! CNB5A!
domain,!the!flexible!linker!including!the!AI!sequence!(amino!acid!No.!915118)!alone!
still!contributes!to!the!conformation!of!the!CNB5A!domain!without!interaction!with!the!
C5domain!(Akimoto!et!al.,!2013).!This!suggests!that!our!PKG!Iβ!CNB5A!construct!for!
structural! study! may! be! too! short! to! monitor! the! conformational! changes! of! CNB5A!
upon! cGMP! binding.! Thus,! to! capture! the! true! apo! state! of! CNB5A! of! PKG! I,! the!
CNB5A!domain!should!be!tethered!to!the!AI!sequence!and!bound!to!the!C5domain.!
Figure!4.7:!Structural!comparison!with!CNB;A!domain!of!PKA!(Kim!et!al.,!2011).!(A)$A$view$showing$the$different$conformations$in$the$
N6terminal$helical$bundles$(N3A$motif)$and$the$PBCs.$The$PKG$Iβ$CNB6A:cGMP$complex$(B6formH$activated)$is$colored$in$red$and$the$PKA$
Iα$ CNB6A:Holo$ (H6formH$ inactivated)$ and$ the$ PKA$ Iα$ CNB6A:cAMP(B6formH$ activated)$ are$ in$ yellow$ and$ cyan,$ respectively.$ (B)$ A$ view$
highlighting$different$conformations$in$the$C6terminal$helices.$(C)$The$helical$subdomains$of$PKG$Iβ$CNB6A:cGMP$(middle),$PKA$Iα$CNB6 A:cAMP$(left),$and$PKA$Iα$CNB6A:Holo$(right).!
4.2.2$cGMP)induced$conformational$changes$at$the$C)terminal$helices$in$CNB)B$act$
a$molecular$switch$for$activation$
!
While&we&failed&to&observe&any&conformational&changes&in&the&truncated&CNB:
A,& the& structures& of& the& CNB:B& domain& reveal& that& the& CNB:B& domain& alters& its&
conformation& upon& cGMP& binding& (Figure& 3.19).& Significant& structural& changes&
occurred& throughout& all& the& helical& regions,& especially& in& the& C:terminal& helices& (αB&
and&αC&helices)&(Figure&3.19&and&Figure&4.8).&Tilting&of&the&αPBC&helix&upon&cGMP&
binding&initiates&the&conformational&changes&of&the&αB&helix&through&“hinge&residues”&
(Berman& et& al.,& 2005S& Rehmann& et& al.,& 2007)& (Figure& 4.8).& This& change& allows&
repositioning& of& the& αC& helix,& which& shields& the& cGMP:binding& pocket& through& a&
distinctive& hydrophobic& capping& (Tyr351).& By& the& consequence& of& the& αC& helix&
movement,& the& N3A& motif& also& rearranges& its& position.& Since& the& CNB:B& domain& is&
located&between&the&CNB:A&domain&and&the&C:domain&through&the&N3A&motif&and&the&
αC&helix,&respectively,&the&conformational&changes&at&the&N:&and&C:&terminal&helices&
transmit&to&these&two&domains&and&also&affect&their&conformation.&&
The& most& drastic& conformational& change& is& found& at& the& αC& helix,& which&
directly&affect&the&conformation&of&the&N:terminal&of&the&C:domain&(Figure&4.8).&At&the&
end& of& the& αC& helix,& an& aromatic& residue& Tyr351& caps& the& guanine& moiety& of& the&
bound&cGMP&through&a& :stacking&interaction.&This&capping&interaction&is&identical&to&
that&previously&found&in&the&PKA&RIα&CNB:B&domain&(Figure&4.5.B)&(Su&et&al.,&1995S&
Wu& et& al.,& 2004).& In& PKA& I,& mutating& Tyr371& to& alanine& significantly& reduces& cAMP:
Figure!4.8:! Structural!comparisons!between!the!apo9! and!cGMP9bound! PKG! Iβ!CNB9B!
(Huang! et! al.,! 2014).!The& PBC& and& the& C:terminal& helices& undergo& large& conformational&
changes&upon&cGMP&binding.&The&change&in&the& PBC&is&transduced& to&the& αB&helix& through&
“hinge& residues”,& L310& and& F336,& which& are& shown& in& white& sticks& with& transparent& surface.&
The& capping& residue& Y351& is& also& shown& in& the& same.& Two& black& spheres& indicate& the& Cα&
atoms&of&two&consecutive&glycines.&&!
dependent& activation& of& PKA& Iα& (Kim& et& al.,& 2007),& suggesting& that& the& capping&
interaction&is&important&for&PKA&activation.&Because&the&homologous&residue&in&PKG&
Iβ& is& Tyr351,& we& hypothesized& Tyr351& is& important& for& PKG& Ι& activation& through&
stabilization& of& the& activated& conformation& of& the& CNB:B& domain.& To& test& this&
hypothesis,& we& mutated& this& residue& (Tyr351Ala)& and& tested& its& effects& in& kinase&
activation& and& cGMP& binding& (measurements& were& done& by& Dipl.& –Biol.& Robin&
Lorenz).& The& PKG& Iβ& full& length& Tyr351Ala& mutant& significantly& reduces& PKG& Iβ&
activation,&showing&a&30:fold&increase&in&its&activation&constant&for&cGMP&(Ka:cGMP)&
(Figure&4.9&and&Table&4.4).&Moreover,&Tyr351Ala&mutation&in&the&CNB:B&domain&also&
significantly& reduced& cGMP& affinity& from& 215& ±& 13& nM& to& 6& ±& 0.2& μM& (Table& 4.2).&
These& results& suggest& that& elimination& of& the& cGMP& capping& interaction& of& Tyr351&
may& destabilize& the& activated& conformation& of& the& αC& helix,& which,& in& turn,& affects&
both&cGMP&binding&and&kinase&activation.&
!
Figure! 4.9:! Role! of! CNB9B! in! PKG! I! activation! (Huang! et! al.,! 2014).!(A)&cGMP&contact&
residues&in&the&CNB:B.&(B)&Role&of&the&cGMP&contact&residues&in&kinase&activation.&KacGMP&
values& were& measured& using& a& microfluidic& mobility& shift& assay& (Data& were& provided& by&
Robin&Lorenz).!
Table! 4.4:! Summary! of! activation! constants! of! cGMP! contact! residues! in! CNB9B.!
Determined!Ka!for!cyclic!nucleotides!with!SEM!(Huang!et!al.,!2014).!
&
HsPKG&Iβ&(5:686)& Ka&±&SEM&
cGMP& &&&&&cAMP&
Wild&type&(n=2)& 133&±&32&nM& 6&±&2&μM&
Leu286Ala&(n=2)& 360&±&65&nM& 3.3&±&1.2&μM&
Arg297Ala&(n=2)& 619&±&170&nM& 5&±&0.5&μM&
Thr317Ala&(n=2)& 159&±&29&nM& 3.6&±&0.9&μM&
Tyr351Ala&(n=2)& 430&±&40nM& 5.5&±&0.6&μM&
This& conformational& change& in& the& αC& helix& has& been& commonly& found& in&
other&CNB&domains&and&acts&as&an&allosteric&switch&for&activation&of&various&proteins&
(Huang& et& al.,& 2014).& As& mentioned& before,& the& αC& helix& movement& with& the& : stacking& capping& in& PKA& RIα& is& crucial& for& the& kinase& activation& by& controlling&
dissociation&of&the&C:subunit&upon&cAMP&binding&(Kim&et&al.,&2007S&Su&et&al.,&1995S&
Wu& et& al.,& 2004).& Furthermore,& recent& studies& on& the& CNB& domain& containing& HCN&
and&CNG&channels&revealed&that&the&conformational&change&in&the&αC&helices&of&their&
CNB&domains&upon&cNT&binding&trigger&the&rearrangement&of&the&N:terminal&helical&
bundle,& allowing& the& channels& to& open& (Puljung& and& Zagotta,& 2013S& Taraska& et& al.,&
2009).&An&additional&example&is&found&in&the&KCNH&family&of&voltage:gated&potassium&
channels,&which&contain&a&cyclic&nucleotide:binding&homology&domain&(Brelidze&et&al.,&
2012S&Brelidze&et&al.,&2013S&Marques:Carvalho&et&al.,&2012).&Rather&than&the&binding&
of&cNT,&the&opening&of&the&KCNH&channels&is&controlled&by&a&short&β&strand&at&the&end&
of& the& αC& helix,& which& acts& as& an& “intrinsic& ligand”,& mimicking& cNT& binding& in& CNB:
domains.& Mutations& of& the& intrinsic& ligand& residues& destabilize& the& activated&
conformation& of& the& αC& helix,& which& results& in& diminishing& its& capability& to& open& the&
channels& upon& depolarization/hyperpolarization.& All& of& these& examples& suggest& that&
the&αC:helix&of&CNB&domain&plays&a&key&role&in®ulating&protein&activity.&&
Consistent&with&these&observations,&our&structural&and&biochemical&data&also&
suggest&that&repositioning&of&the&αC&helix&in&CNB:B&is&crucial&for&PKG&activation&by&
controlling& the& interaction& between& the& R:& and& C:domains& (Figures& 3.19& and& 4.6).&
Because& the& αC& helix& interconnects& the& R:& and& C:domains,& we& reasoned& that& the&
rearrangement& of& the& αC& helix& in& the& CNB:B& domain& upon& cGMP& binding& possibly&
helps&the&dissociation&of&the&C:domain&from&the&R:domain.&In&the&absence&of&cGMP,&
PKG& remains& in& its& inhibited& state& with& an& interaction& of& the& R:& and& C:domains.& In&
contrast,& in& the& presence& of& cGMP,& PKG& undergoes& a& large& conformational& change&
and& releases& the& C:domain& from& the& R:domain,& allowing& activation.& Since& the& αC&
helix& in& CNB:B& connects& the& R:& and& C:domains,& we& suggest& that& the& structural&
rearrangement&at&the&C:terminal&helices&of&CNB:B&contribute&to&forming&the&activated&
conformation&of&PKG&and&acts&as&an&“allosteric&switch”,&which&directly&pulls&out&the&C:
domain& from& the& R:domain& in& response& to& cGMP& binding& and& thus& allows& PKG& I&
activation.&&
&