Structural insights into the allosteric activation mechanism of cGMP-dependent protein kinase I

Structural(insights(into(the(allosteric(activation(

mechanism(of(cGMP6dependent(protein(kinase(I

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Dissertation*zur*Erlangung*des*akademischen*Grades*eines*

Doktors*der*Naturwissenschaften*(Dr.*rer.*nat.)

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Angefertigt*im* Fachbereich*10*–*Mathematik*und*Naturwissenschaften,* Institut*für*Biologie,*Abteilung*Biochemie,* Universität*Kassel*

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vorgelegt!von*

Jeong!Joo!Kim!

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Kassel!im!November!2015

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* * * * * * * * * * * * * * * Parts*of*the*work*described*were*put*together*in*manuscripts*that*have*been*either* published*or*have*been*submitted*for*publication:* * Jeong*Joo*Kim,*Darren*E.*Casteel,*Gilbert*Huang,*Taek*Hun*Kwon,*Ronnie*Kuo*Ren,* Peter*Zwart,*Jeffrey*J.*Headd,*Nicholas*Gene*Brown,*DarUChone*Chow,*Timothy* Palzkill,*Choel*Kim*(2011)* Co6crystal(structures(of(PKG(Iβ((926227)(with(cGMP(and(cAMP(reveal(the( molecular(details(of(cyclic6nucleotide(binding* PLoS*One.*6(4),*e18413.* * Gilbert*Y.*Huang*,*Jeong*Joo*Kim*,*Albert*S.*Reger,*Robin*Lorenz,*EuiUWhan*Moon,* Chi*Zhao,*Darren*E.*Casteel,*Daniela*Bertinetti,*Bryan*VanSchouwen,*Rajeevan* Selvaratnam,*James*W.*Pflugrath,*Banumathi*Sankaran,*Giuseppe*Melacini,* Friedrich*W.*Herberg,*Choel*Kim*(2014)** Structural(basis(for(cyclic6nucleotide(selectivity(and(cGMP6selective(activation* Structure,*22(1),*116U124** **CoUcontributed*author* * Jeong*Joo*Kim*,*Gilbert*Y.*Huang*,*Robert*Rieger,*Antonius*Koller,*DarUChone* Chow,*Choel*Kim*(2015)** A(protocol(for(expression(and(purification(of(cyclic6nucleotide(free(protein(in( Escherichia)coli) Cyclic*nucleotide*signaling,*Edited*by*Xiaodong*Cheng,*CRC*press,*191U201** **CoUcontributed*author* * Jeong*Joo*Kim,*Robin*Lorenz,*Stefan*T.*Arold,*Albert*S.*Reger,*Banumathi* Sankaran,*Darren*E.*Casteel,*Friedrich*W.*Herberg,*and*Choel*Kim*(2016)* Crystal(structure(of(PKG(I:cGMP(complex(reveals(a(cGMP6mediated(dimeric( interface(that(facilitates(cGMP6induced(activation( Structure,*in*press

( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( Promotionskommission:( ( * Date*of*oral*defense:*25.*January*2016* * * * * * * 1.*Gutachter:* Prof.*Dr.*Friedrich*W.*Herberg* Abteilung*Biochemie,*Universität*Kassel* * 2.*Gutachter:* Prof.*Dr.*Choel*Kim* Department*of*Pharmacology,*Baylor*College*of*Medicine* * 3.*Prüfer:* Prof.*Dr.*Markus*Maniak* Abteilung*Zellbiologie,*Universität*Kassel* * 4.*Prüferin:* Prof.*Dr.*Monika*Stengl* Abteilung*Tierphysiologie,*Universität*Kassel*

Abstract!

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Cyclic*GMPUdependent*protein*kinase*(PKG)*is*a*key*transducer*in*the*NOU cGMP* signaling* pathway.* In* this* line,* PKG* has* been* considered* an* important* drug* target* for* treating* hypertensive* cardiovascular* and* pulmonary* diseases.* However,* the*investigation*of*PKG’s*allosteric*activation*mechanism*has*been*hampered*by*a* lack* of* structural* information.* One* of* the* fundamental* questions* on* the* cGMPU dependent* activation* of* PKG* is* how* the* enzyme* can* distinguish* cGMP* over* cAMP* and* selectively* respond* to* cGMP.* To* ensure* proper* signaling,* PKG* must* have* developed*unique*features*to*ensure*its*activation*upon*the*right*activation*signal.** In*this*thesis,*the*cGMPUselective*activation*mechanism*of*PKG*was*studied* through*determining*crystal*structures*of*three*truncated*constructs*of*the*regulatory* domain*[CNBUA*(92U227),*CNBUB*(271U369),*and*CNBUA/B*(92U351)]*of*PKG*Iβ*in*the* absence*or*presence*of*cyclic*nucleotides.* Herein,*two*individual*CNB*domain*structures*with*biochemical*data*revealed* that* the* CUterminal* CNB* domain* (CNBUB)* is* responsible* for* cGMP* selectivity,* while* the*NUterminal*CNBUdomain*(CNBUA)*has*a*higher*binding*affinity*for*both*cGMP*and* cAMP* without* showing* any* selectivity.* Based* on* these* crystal* structures,* mutagenesis* studies* were* performed* in* which* the* critical* residues* for* cyclic* nucleotide*selectivity*and*activation*were*identified.*Furthermore,*we*discovered*that* the* conformational* changes* of* the* CUterminal* helix* of* the* CNBUB* that* bridges* between* the* regulatory* and* catalytic* domains* including* the* hydrophobic* capping* interaction*are*crucial*for*PKG*activation.**

In* addition,* to* observe* the* global* conformation* of* the* activated* RUdomain,* I* solved* a* coUcrystal* structure* of* the* CNBUA/B* with* cGMP.* Although* a* monomeric* construct* was* crystallized,* the* structure* displays* a* dimer.* Strikingly,* the* CNBUA* domain* and* its* bound* cGMP* provide* a* key* interface* for* this* dimeric* interaction.* Using* small* angle* XUray* scattering* (SAXS),* the* existence* of* the* cGMPUmediated* dimeric* interface* within* the* CNB* domains* was* confirmed.* Furthermore,* measuring* cGMPUbinding*affinities*(EC50)*of*the*dimeric*interface*mutants*as*well*as*determining*

activation* constants* (Ka)* revealed* that* the* interface* formation* is* important* for* PKG*

activation.*To*conclude,*this*thesis*study*provides*a*new*mechanistic*insight*in*PKG* activation*along*with*a*newly*found*interface*that*can*be*targeted*for*designing*PKGU specific*activity*modulators.*****

Zusammenfassung!

Die*cGMPUabhängige*Proteinkinase*(PKG)*ist*ein*zentraler*Effektor*des*NOUcGMPU Signalweges*und*gilt*daher*als*wichtiger*Ansatzpunkt*zur*Therapie*kardiovaskulärer* und*pulmonaler*Erkrankungen.*Der*allosterische*Aktivierungsmechanismus*der*PKG* durch*cGMP*ist*bisher*aufgrund*begrenzter*struktureller*Daten*nur*wenig*untersucht.* Dabei* liegt* eine* grundlegende* Frage* darin,* wie* PKG* zwischen* cGMP* und* cAMP* unterscheiden* kann.* PKG* muss* selektiv* auf* cGMP* reagieren,* um* die* Identität* des* Signalweges*zu*erhalten.*

In* dieser* Arbeit* wurde* der* cGMPUselektive* Aktivierungsmechanismus* der* PKG* anhand* von* Kristallstrukturen* dreier* verkürzter* Konstrukte* der* regulatorischen* Domäne*[CNBUA*(92U227),*CNBUB*(217U369)*und*CNBUA/B*(92U351)]*der*PKG*I *mit* und*ohne*zyklische*Nukleotide*untersucht.*

Anhand* der* Strukturen* sowie* biochemischer* Daten* der* zwei* isolierten* CNBU Domänen* zeigte* sich,* dass* die* CUterminale* CNBUDomäne* (CNBUB)* cGMPU Selektivität* vermittelt,* während* die* NUterminale* CNBUDomäne* (CNBUA)* cGMP* und* cAMP* hochaffin* bindet* und* keine* Selektivität* aufweist.* Ausgehend* von* diesen* Kristallstrukturen* wurden* Mutagenesestudien* durchgeführt* und* die* kritischen* Aminosäuren* für* ZyklonukleotidUSelektivität* und* Aktivierung* identifiziert.* Darüber* hinaus*konnte*gezeigt*werden,*dass*Konformationsänderungen*einer*Helix*der*CNBU B*( C*Helix),*essentiell*für*die*Aktivierung*der*PKG*sind.*Diese*Helix*beinhaltet*ein* für*das*„Capping“*des*Zyklonukleotids*notwendiges*Sequenzmotiv.*

Des* Weiteren* löste* ich* eine* KoUKristallstruktur* (+cGMP)* eines* Konstruktes* mit* beiden*CNBUDomänen,*um*die*gesamte*RUDomäne*in*ihrer*aktivierten*Konformation* zu*analysieren.*Obwohl*ein*monomeres*Protein*(CNBUA/Br*92U351)*zur*Kristallisation* eingesetzt* wurde,* zeigt* die* gelöste* Struktur* ein* Dimer* aus* zwei* CNBUA/BU Domänenkonstrukten.* Interessanterweise* dienen* die* CNBUA* und* das* gebundene* cGMP*als*Schnittstelle*für*die*Interaktion*zwischen*beiden*Monomeren.*Auch*mittels* KleinwinkelURöntgenstreuung*(SAXS)*konnte*die*cGMPUvermittelte*Dimerisierung*der* CNBUDomänen* bestätigt* werden.* Die* Bedeutung* dieser* Dimerisierungsschnittstelle* für* die* Aktivierung* der* PKG* konnte* anhand* von* BindungsU* und* Aktivierungsstudien* demonstriert* werden.* Ein* struktureller* Vergleich* zwischen* PKG* und* PKA* verdeutlicht,* dass* sich* beide* Kinasen* in* ihrer* aktiven* Konformation* drastisch* unterscheiden.* Zusammenfassend* liefert* diese* Arbeit* einen* neuen* Einblick* in* den* PKGUAktivierungsmechanismus.* Die* hier* beschriebene* Dimerisierungsschnittstelle* der* PKG* könnte* eine* Grundlage* für* die* Entwicklung* PKGUspezifischer* Modulatoren* darstellen.

List!of!Abbreviations!

A* absorption* Å** angstrom,*1*x*10U10*m** AI* autoUinhibitory* APS* ammonium*persulfate* ATP* adenosineU5’Utriphosphate* BH2* dihydrobiopterin* BH4* tetrahydrobiopterin* BKca* largeUconductance*calciumUactivated*potassium*channel* BSA* bovine*serum*albumin* CUdomain* catalytic*domain* CaM* calciumUmodulated*proteinr*calmodulin* cAMP* 3’U5’Ucyclic*adenosine*monophosphate* CAP* catabolite*activator*protein* CC* coiledUcoil* cGMP* 3’U5’Ucyclic*guanosine*monophosphate* CHAPS* 3U[(3UCholamidopropyl)dimethylammonio]U1Upropanesulfonate* CNB* cyclic*nucleotide*binding*domain* CNBUA* NUterminal*cyclic*nucleotide*binding*domain* CNBUA/B* tandem*cyclic*nucleotide*binding*domains* CNBUB* CUterminal*cyclic*nucleotide*binding*domain* CNG* cyclic*nucleotideUgated*channel* cNT* cyclic*nucleotide** DTT* 1,*4Udithiothreitol. EC50* half*maximal*effective*concentration* EDRF* endothelialUderived*relaxing*factor* EDTA* ethylenediaminetetraacetic*acid* EPAC* exchange*protein*directly*activated*by*cAMP* FP* fluorescence*polarization* GC* guanylyl*cyclase* GMP* guanosine*monophosphate* GPCR* G*proteinUcoupled*receptor* GTP* guanosine*triphosphate* HUNOX* hemeUnitric*oxide/oxygen*binding* HCN* hyperpolarizationUactivated*cyclic*nucleotideUgated*

HDXMS** hydrogen/deuterium*exchange*mass*spectrometry** HEK* human*embryonic*kidney* HEPES* 2U[4U(2Uhydroxyethyl)piperazinU1Uyl]ethanesulfonic*acid* IP3* inositol*triphosphate* IPTG* isopropyl*βUDU1Uthiogalactopyranoside*(IPTG)* ITC* isothermal*titration*calorimetry* Ka* activation*constant* KD* equilibrium*constant* kDa* kilodalton*

LUVDCC* LUtype*voltageUdependent*Ca2+_*channel*

LB** lysogeny*broth* LPS* lipopolysaccharide* LZ* leucine*zipper* MAD* multi*wavelength*anomalous*dispersion* MIR* multiple*isomorphous*replacement* MLC* myosin*light*chain* MLCP* myosin*light*chain*phosphatase* MOPS* (3U(NUmorpholino)propanesulfonic*acid)* mPol** millipolarization* MR* molecular*replacement* MW* molecular*weight* MWCO* molecular*weight*cut*off* NADPH* nicotinamine*adenine*dinucleotide*phosphate* NiUNTA* nickelUnitriloacetic*acid* NO* nitric*oxide* NOS* nitric*oxide*synthases* NPs* naturetic*peptides* OD* optical*density* PAGE* polyacrylamide*gel*electrophoresis* PAS* perUarntUsim* PBC** phosphate*binding*cassette* PDE* phosphodiesterase* PEG* polyethylene*glycol* PKA* cyclic*AMPUdependent*protein*kinase* PKC* protein*kinase*C* PKG* cyclic*GMPUdependent*protein*kinase*

PMSF* phenylmethylsulfonyl*fluoride* psi* poundUforce*per*square*inch* RUdomain* regulatory*domain* RGS* regulator*of*G*protein*signaling* RhoA* Ras*homologue*gene*family*member*A* RMSD* rootUmeanUsquare*deviation* ROCK* RhoUassociated*protein*kinase* ROS* reactive*oxygen*species* SAD* single*wavelength*anomalous*diffraction* SAXS* small*angle*XUray*scattering* SDS* sodium*dodecyl*sulfate:* SEM* standard*error*of*mean* SIR* single*isomorphs*replacement* SOC* super*optimal*broth*with*catabolite*repression* SPR* surface*plasmon*resonance* TAAD* thoracic*aortic*aneurysms*and*dissections* Tris* 2UaAminoU2UhydroxymethylUpropaneU1,3Udiol* UV* ultraviolet* VSMC* vascular*smooth*muscle*cell* w/v* weight*per*volume* WT* wild*type* * *

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List(of(Figures(

Figure*1.1:*NOU*and*ANPUcGMP*Pathways*...*13

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Figure*1.2:*Dysfunction*of*cGMP*signaling*involved*enzymes*in*cardiovascular*diseases*....*14

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Figure*1.3:*PDE*family*classifications*...*19

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Figure*1.4*Domain*organization*of*PKG*I*and*II*...*21

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Figure*1.5:*Roles*of*PKG*in*smooth*muscle*cell*relaxation*...*23

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Figure*1.6:*Comparisons*of*the*three*LZ*domains*of*PKG*...*25

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Figure*1.7:*Crystal*structure*of*CNB*domain*and*amino*sequence*alignment*of*PBCs*...*27

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Figure*1.8.*Crystal*structure*of*PKA*catalytic*subunit*and*amino*sequence*alignment*with* PKG*catalytic*domain*...*28

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Figure*1.9:*Amino*acid*sequence*alignment*between*the*PBC*of*PKGs*and*PKAs*...*29

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Figure*2.1:*A*phase*diagram*of*macromolecules*crystallization*...*45

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Figure*2.2:*Schematic*illustration*of*vapor*diffusion*method*with*its*phase*diagram.*...*46

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Figure*2.3:*Schematic*illustration*of*micro*batch*method*with*its*phase*diagram*...*47

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Figure*2.4:*Schematic*summary*of*XUray*crystallography*...*49

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Figure*2.5:*Illustration*of*Bragg’s*raw*...*50

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Figure*2.6:*Schematic*diagram*of*small*angle*XUray*scattering*and*scattering*images*from* SAXS*and*crystallography*...*56

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Figure*2.7:*Schematic*representation*of*fluorescence*polarization*based*binding*assay*...*59

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Figure*2.8:*Summary*of*microfluidic*mobilityUshift*assay*...*61

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Figure*3.1:*HisUaffinity*purification*profiles*of*PKG*Iβ*CNBUA*(92U227)*...*64

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Figure*3.2:*Purification*profiles*of*anion*exchange*chromatography*and*size*exclusion* chromatography*of*PKG*Iβ*CNBUA*(92U227)*...*65

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Figure*3.3:*Crystallization*of*CNBUA*of*PKG*Iβ*with*cGMP,*cAMP,*or*no*ligand*...*66

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Figure*3.4:*Overall*structure*of*the*PKG*Iβ*CNBUA:cGMP*complex*...*68

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Figure*3.5:*cGMPUbound*structure*of*CNBUA*...*69

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Figure*3.6:*Cyclic*nucleotide*binding*pockets*of*CNBUA*in*PKG*Iβ*and*PKA*RIα*...*70

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Figure*3.7:*cAMPUbound*structure*of*CNBUA*...*71

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Figure*3.8:*Partial*apo*structure*of*CNBUA*...*73

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Figure*3.9:*ITC*characterization*of*cyclic*nucleotide*binding*in*CNBUA*(92U227)*of*PKG*Iβ*...*74

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Figure*3.10:*IonUexchange*(Mono*Q*10/100*GL)*profiles*of*PKG*Iβ*92U363*samples*and*their* cAMP*contamination*(%)*...*76

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Figure*3.11:*Representative*cAMP*elution*traces*for*two*samples*...*76

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Figure*3.12:*Cloning*and*expression*of*CNBUB*(219U369)*of*PKG*Iβ*...*77

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Figure*3.13:*CNBUB*purification*profiles*of*HisUaffinity*chromatography*and*sizeUexclusion* chromatography*...*78

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Figure*3.14:*Cyclic*nucleotide*affinity*measurement*of*PKG*Iβ*CNBUB*(219U369)*...*79

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Figure*3.15:*Crystals*of*CNBUB*with*cGMP*...*80

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Figure*3.16:*Overall*structure*of*CNBUB:cGMP*complex*...*80

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Figure*3.17:*Overall*structure*of*apo*CNBUB*...*81

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Figure*3.18:*Detailed*interactions*between*cGMP*and*the*binding*pocket*of*CNBUB*...*82

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Figure*3.19:*Conformational*changes*in*CNBUB*upon*cGMP*binding*...*84

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Figure*3.20:*HisUaffinity*purification*profiles*of*PKG*Iβ*CNBUA/B*domain*(92U351)*...*86

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Figure*3.21:*CNBUA/B*purification*profiles*of*anion*exchange*chromatography*and*size* exclusion*chromatography*...*87

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Figure*3.22:*Crystal*and*diffraction*images*of*CNBUA/B*(92U369)*with*cGMP*...*88

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Figure*3.23:*Optimized*CNBUA/B*(92U351)*crystals*with*cGMP*...*89

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Figure*3.24:*A*diffraction*image*of*the*CNBUA/B*(92U351):cGMP*complex*...*90

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Figure*3.25:*Asymmetric*unit*of*PKG*Iβ*CNBUA/B:cGMP*complex*and*omit*maps*density*for* bound*cGMPs*...*92

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Figure*3.26:*Overall*structure*of*the*CNBUA/B:cGMP*complex*...*94

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Figure*3.27:*Structural*comparison*with*the*PKG*Iα*78U355:cAMP*complex*...*95

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Figure*3.28:*Structural*comparison*of*CNBUA*and*CNBUB*...*97

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Figure*3.29:*Detailed*interactions*at*the*CNBUA/B*cGMPUmediated*dimer*interface*...*99

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Figure*3.30:*Role*of*the*dimeric*interface*in*kinase*activation*...*101

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Figure*3.31:*Small*angle*scattering*data*of*the*dimeric*RUdomain*with*their*models*bound*to* cyclic*nucleotides*...*103

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Figure*4.1:*Structural*comparisons*of*four*different*ligand*bound*states*of*PKG*Iβ*CNBUA*.*105

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Figure*4.2:*Cyclic*nucleotide*binding*site*of*CNBUA*with*different*cyclic*nucleotide*binding* states*...*107

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Figure*4.3:*cGMP*and*cAMP*...*107

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Figure*4.4:*Structural*comparison*between*the*CNBUA:cGMP*and*the*CNBUB:cGMP* complexes*...*109

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Figure*4.5:*Cyclic*nucleotide*interacting*residues*in*CNBUdomain*of*PKG*I*and*PKA*RI*...*111

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Figure*4.6:*Structural*comparisons*between*the*apoU*and*cGMPUbound*CNBUB*of*PKG*Iβ*.*111

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Figure*4.7:*Structural*comparison*with*CNBUA*domain*of*PKA*...*114

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Figure*4.8:*Structural*comparisons*between*the*apoU*and*cGMPUbound*PKG*Iβ*CNBUB*...*115

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Figure*4.9:*Role*of*CNBUB*in*PKG*I*activation*...*116

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Figure*4.10:*Activated*conformation*of*CNBUA/B*in*PKA*RIα*...*123

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Figure*4.11:*Activated*conformation*of*the*regulatory*domain*of*PKG*Iβ*...*123

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Figure*4.12:*Model*of*PKG*I*Activation*...*124

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List(of(Tables(

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Table*1.1:*Substrate*specificities*and*kinetics*properties*of*cGMPUhydrolyzing*PDE5*in* cardiac*muscle*cells*...*20

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Table*1.2*Substrates*of*PKG*I*in*smooth*muscle.*...*24

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Table*2.1:*CNB*domain*constructs*for*crystallization*...*38

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Table*2.2:*PCR*amplification*of*target*DNAs*...*39

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Table*2.3:*Restriction*enzyme*treatment*for*plasmid*and*insert*DNAs*...*40

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Table*2.4:*Ligation*mixture*for*PKG*constructs.*...*40

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Table*2.5:*Restriction*enzyme*treatment*for*insert*verification.*...*41

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Table*2.6:*Basic*characteristics*of*PKG*Iβ*constructs.*...*44

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Table*3.1:*Data*and*refinement*statistics*of*the*CNBUA:cNT*complexes*...*67

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Table*3.2.*Data*and*refinement*statistics*of*the*CNBUA/B:cGMP*complex*...*91

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Table*3.3:*cGMP*binding*affinities*of*human*PKG*Iβ*RUdimer*wild*type*and*mutants*...*101

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Table*3.4:*SAXS*data*collection*and*scattering*derived*parameters*...*104

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Table*4.1:*Summary*of*ITC*measurements*of*human*PKG*Iβ*CNBUA*(92U227)*with*cGMP*and* cAMP*...*106

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Table*4.2:*Affinity*measurements*of*PKG*Iβ*CNBUA*and*CNBUB*...*108

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Table*4.3:*Cyclic*nucleotide*binding*affinities*of*PKG*Iβ*CNBUB*(219U329)*wild*type*and* mutants*...*110

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Table*4.4:*Summary*of*activation*constants*of*cGMP*contact*residues*in*CNBUB*...*116

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Table!of!Contents!

Abstract(...(1* Zusammenfassung(...(2* List(of(Abbreviations(...(3* List(of(Figures(...(6* List(of(Tables(...(8* Table(of(Contents(...(9* 1(Introduction(...(11* 1.1(Cyclic(GMP6dependent(protein(kinase:(a(possible(option(for(treatment(of( hypertensive(cardiovascular(disease(...(11* 1.2(Brief(understanding(of(key(enzymes(in(the(NO6cGMP(signaling(pathway (...(13* 1.2.1*Nitric*oxide*synthesis:*Nitric*oxide*synthases*(NOSs)*...*14* 1.2.2*cGMP*synthesis:*Guanylyl*cyclase*(GCs)*...*16* 1.2.3*cGMP*degradation:*Phosphodiesterases*(PDEs)*...*18* 1.2.4*cGMP*signal*transducer:*Cyclic*GMPUdependent*protein*kinases*(PKGs) *...*21* 1.3(Detailed(understanding(of(functional(domains(of(PKG(...(25* 1.3.1*Leucine*zipper*(LZ)*domain*...*25* 1.3.2*AutoUinhibitory*(AI)*sequence*...*26* 1.3.3*Cyclic*nucleotide*binding*(CNB)*domain*...*26* 1.3.4*Catalytic*domain*...*28* 1.4(Cyclic(nucleotide(selectivity(of(PKG(I(...(29* 1.5(cGMP6dependent(activation(mechanism(of(PKGs(...(29* 1.6(Objective(...(30* 2(Materials(and(Methods(...(32* 2.1(Materials(...(32* 2.1.1*Plasmids*...*32* 2.1.2*Primers*...*32* 2.1.3*Enzymes*...*33* 2.1.4*Cell*lines*...*33* 2.1.5*Chemicals*...*33* 2.1.6*Crystallization*screens*...*35* 2.1.7*DNA*and*protein*molecular*weight*standards*...*35* 2.1.8*Devices*and*supplies*...*35* 2.1.9*Kits*...*37* 2.1.10*Software*for*XUray*crystallography*...*37* 2.2(Methods(...(38* 2.2.1*Crystallization*construct*design*...*38* 2.2.2*SubUcloning*...*38*

2.2.3*Protein*expression*and*purification*...*42* 2.2.4*XUray*crystallography*...*44* 2.2.5*Small*angle*XUray*scattering*...*55* 2.2.6*cGMPUbinding*affinity*measurements*...*58* 2.2.7*Kinase*activation*assay*...*60* 3(Results(...(63* 3.1(Cyclic(nucleotide(selectivity(of(human(PKG(Iβ(...(63* 3.1.1*CoUcrystal*structures*of*PKG*Iβ*CNBUA*(92U227)*with*and*without*cyclic* nucleotide*...*63* 3.1.2*Preparing*the*cAMP*free*samples*using*adenylyl*cyclase*deficient*E.coli.. *...*75* 3.1.3*CoUcrystal*structures*of*PKG*Iβ*CNBUB*(219U369)*with*and*without*cGMP *...*77* 3.2(Allosteric(activation(mechanism(of(PKG(I(...(85* 3.2.1*Crystallization*and*structure*determination*of*PKG*Iβ*CNBUA/B*(92U351)*85* 3.2.2*Overall*structure*of*the*tandem*CNB*domain*with*cGMP*...*93* 3.2.3.*CNBUA*and*CNBUB*differ*from*each*other*...*96* 3.2.4*cGMP*binding*induces*dimer*formation*between*two*CNBUA/B*domains*97* 3.2.5*cGMPUinduced*dimeric*interface*promotes*PKG*activation*...*100* 3.2.6*SAXS*data*validate*the*structure*of*the*cGMPUmediated*dimer*...*102* 4(Discussion(...(105* 4.1(Two(CNB(domains(in(PKG(I(show(different(cyclic(nucleotide(selectivity (...(105* 4.1.1*CNBUA*has*no*cyclic*nucleotide*selectivity*...*105* 4.1.2*CNBUB*is*responsible*for*cGMP*selectivity*...*108* 4.1.3*Distinct*cyclic*nucleotide*interacting*residues*in*PKG*I*and*PKA*I*explain* their*cNT*preference*...*110* 4.2(cGMP(binding(drives(conformational(changes(in(CNB(domains(...(112* 4.2.1*cGMPUinduced*conformational*changes*at*the*NUterminal*helices*in*CNBUA* is*crucial*for*releasing*the*AI*sequence*from*the*catalytic*core*...*112* 4.2.2*cGMPUinduced*conformational*changes*at*the*CUterminal*helices*in*CNBUB* act*a*molecular*switch*for*activation*...*115* 4.2.3*Global*changes*in*tandem*CNBUA/B*domain*result*in*formation*of*a*novel* interface*specific*for*PKG*I*...*118* 5(Conclusions(...(125* 6(Future(directions(...(128* Bibliography(...(130* Acknowledgments(...(148* ( ( ( (

1!Introduction!

!

1.1!Cyclic!GMPAdependent!protein!kinase:!a!possible!option!for!treatment!of!

hypertensive!cardiovascular!disease!!!

(

Cardiovascular* disease* (CVD)* is* the* number* one* leading* cause* of* death* in* the*world*based*on*the*report*by*the*WHO*(WHO,*2011a,*b).*It*kills*over*17.5*million* people*worldwide*annually.*Most*developed*countries*suffer*from*high*prevalence*of* CVD*as*well*as*subsequent*increases*of*the*healthUcare*costs.*Among*many*known* risk*factors,*high*blood*pressure*(chronic*hypertension)*is*one*of*the*main*causes*of* developing* CVD* (Messerli* et* al.,* 2007r* Oparil* and* Schmieder,* 2015r* Tamargo* and* LopezUSendon,* 2011).* LongUterm* exposure* to* high* blood* pressure* in* the* heart* and* blood* vessel* causes* serious* damages* by* abnormally* thickening* or* weakening* their* muscle* tissues,* which* possibly* promote* several* diseases* that* may* lead* to* sudden* heart*attacks*or*stroke.**

There*are*a*total*of*69*antihypertensive*drugs*in*15*different*classes*available* in*the*United*States*for*treating*chronic*hypertension*(Oparil*and*Schmieder,*2015).* Most* of* these* drugs* aim* at* relaxing* the* blood* vessels* by* inhibiting* adrenergic* receptors* that* are* related* to* vascular* smooth* muscle* contraction.* While* multiple* signaling*pathways*are*involved*in*modulating*vascular*smooth*muscle*tone,*the*NOU cGMP* signaling* pathway* draws* a* significant* attention* for* its* pharmacological* potentials,*because*activation*of*the*NOUcGMP*pathway*in*vascular*smooth*muscle* cells*and*cardiomyocytes*results*in*immediate*vasorelaxation*(Evgenov*et*al.,*2006r* Maurice*et*al.,*2014r*Potter,*2011br*Schlossmann*and*Schinner,*2012).**

The* relation* between* heart* disease* and* the* NOUcGMP* pathway* was* discovered*in*the*late*1990s*U*more*than*100*years*later*after*the*Scottish*physician* Dr.*William*Murrell*observed*that*nitroglycerin*(glyceryl*trinitrate)*relieved*chest*pain* (angina*pectoris)*from*heart*failure*patients*(Murrell,*1879).**While*nitrate*compounds* have*been*used*widely*for*treating*heart*failure*without*knowing*its*clear*molecular* mechanism,*in*1977,*Dr.*Ferid*Murad*demonstrated*that*nitric*oxide*generated*from* sodium*nitrate*directly*activates*soluble*guanylyl*cyclase*(sGC)*(Katsuki*et*al.,*1977),* leading* to* significant* increases* of* cyclic* guanosine* 3’,* 5’* monophosphate* (cGMP)* production* in* several* tissues* including* lung,* heart,* kidney,* and* cerebral* cortex* (Arnold* et* al.,* 1977).* Meanwhile,* an* interesting* paper* came* out* in* Nature* by* Dr.* Robert* Furchgott,* which* suggested* that* a* mysterious* substance* produced* from*

endothelial*cells*relaxes*blood*vessels*upon*acetylcholine*stimulation.*He*named*the* substance* endothelialUderived* relaxing* factor* (EDRF)* (Furchgott* and* Zawadzki,* 1980).* Subsequently,* Drs.* Luis* Ignarro* and* Salvador* Moncada* discovered* that* the* mysterious* endogenous* substance* was* actually* nitric* oxide* (Ignarro* et* al.,* 1987ar* Ignarro*et*al.,*1987br*Palmer*et*al.,*1987),(and*the*several*following*studies*indicated* that* nitric* oxide* was* naturally* synthesized* in* the* endothelial* cell* by* nitric* oxide* synthases*(NOS),*using*LUarginine*(Knowles*et*al.,*1989r*Palmer*et*al.,*1988).*

The* NOUcGMP* signaling* pathway* is* tightly* regulated* by* production* and* degradation*of*cGMP*(Beavo*and*Brunton,*2002r*Schlossmann*and*Hofmann,*2005).* Upon* stimulating* signals* such* as* nitric* oxide* (NO)* and* natriuretic* peptides* (NPs),* guanylyl* cyclases* produce* cyclic* guanosine* 3’,5’* monophosphate* (cGMP)* which* activates* its* cellular* receptors,* such* as* cGMPUdependent* protein* kinase* (PKG),* cyclic* nucleotide* phosphodiesterases* (PDEs),* and* cyclic* nucleotide* gated* channels* (CNGs).*While*PKG*acts*as*a*transducer*that*delivers*the*activation*signal*of*cGMP* to*downstream*effectors,*resulting*in*vasorelaxation*(Francis*et*al.,*2010r*Hofmann*et* al.,* 2009r* Schlossmann* and* Hofmann,* 2005),* PDEs* degrade* cGMP,* leading* to* a* negative*feedback*of*the*signaling*pathway,*eventually*vanishing*the*signal*(Bender* and* Beavo,* 2006r* Francis* et* al.,* 2010r* Maurice* et* al.,* 2014).* Therefore,* pharmaceutical* companies* mainly* focus* on* developing* therapeutics* that* can* maintain*high*cellular*cGMP*level*by*inhibiting*PDEs*activity*or*by*enhancing*NOS*or* GC* activity.* A* few* commercial* drugs* are* now* available* which* include* riociguat* (Adempas,*Bayer),*a*soluble*guanylyl*cyclase*(sGC)*activator*(Ghofrani*et*al.,*2013ar* Ghofrani*et*al.,*2013b)*and*sildenafil*(Viagra,*Pfizer),*a*PDE5*inhibitor*(Goldstein*et* al.,* 1998).* In* contrast,* there* are* no* drugs* that* directly* target* PKG,* even* though* directly* enhancing* PKG* activity* (PKGUspecific* activators)* is* expected* to* result* in* similar* or* even* more* specific* effects,* compared* to* targeting* upstream* signaling* proteins.* Either*the*sGC*activator*or*the*PDE5*inhibitor*is*not*a*capable*treatment*for* patients*who*have*dysfunctional*PKG.*Recently,*Dr.*Milewicz’s*group*discovered*that* families*that*suffered*from*Thoracic*Aortic*Aneurysms*and*Dissections*(TAAD)*share* a*single*amino*acid*mutation*(Arg177Gln)*in*the*regulatory*domain*of*PKG*I*(Guo*et* al.,*2013).*This*result*suggests*that*those*drugs*cannot*be*efficient*for*these*familiar* TAAD*patients.*However,*developing*therapeutics*that*target*PKG*has*been*greatly* hampered* due* to* lack* of* highUresolution* structural* information* of* PKG* and* poor* understanding*of*its*activation*mechanism.*Therefore,*in*this*thesis*study,*I*aimed*to* solve*an*activated*conformation*of*the*regulatory*domain*of*PKG*I*and*to*understand* its*cGMPUdependent*activation*mechanism.*

1.2!Brief!understanding!of!key!enzymes!in!the!NOAcGMP!signaling!pathway!!

(

The* NOUcGMP* signaling* cascade* is* a* tightly* regulated* process* through* several*key*functional*enzymes*(Figure*1.1)*(Russwurm*et*al.,*2013r*Takimoto,*2012r* Tsai* and* Kass,* 2009).* Responding* to* stimulation* of* the* shear* stress* on* the* endothelium* within* the* arterioles* or* stimulation* of* acetylcholine,* endothelial* cells* placed* near* vascular* smooth* muscle* cell* (VSMC)* start* producing* NO* (Tsai* and* Kass,* 2009).* NO* is* produced* by* nitric* oxide* synthases* (NOS),* which* convert* LU arginine* into* NO* and* LUcitrulline.* The* gaseous* NO* is* diffused* into* the* plasma*

membrane* of* VSMC* and* activates* soluble* guanylyl* cyclase* (sGC),* generating* cGMP.* Meanwhile,* particulate* guanylyl* cyclase* (pGC)* associated* to* the* cell* membrane* is* activated* by* hormonal* natriuretic* peptides* (NPs)* and* also* produces* cGMP.*The*elevation*of*cGMP*results*in*activation*of*PKG,*which*is*a*key*transducer* in* this* signaling* pathway.* Upon* cGMP* binding,* PKG* becomes* activated* and* phosphorylate* downstream* substrate* proteins* that* lead* to* numerous* physiological* responses,* including* vasodilation,* platelet* aggregation,* and* cardiac* remodeling* (Francis*et*al.,*2010r*Hofmann*et*al.,*2009r*Schlossmann*and*Hofmann,*2005).*The* cellular*cGMP*level*is*negatively*controlled*by*PDEs*that*hydrolyze*cGMP*to*GMP.* Since* the* NOUcGMP* pathway* is* tightly* regulated* by* harmonization* of* all* protein* components*in*the*pathway,*defects*in*any*enzyme*can*cause*serious*health*issues* in* cardiovascular* tissue,* as* well* as* neuronal* and* renal* tissues.* Therefore,*

(

Figure(1.1:(NO6(and(ANP6cGMP(Pathways.(Adapted*from*(Tamargo*and*LopezUSendon,*

understanding* each* enzyme* function* on* a* molecular* level* is* an* important* step* for* developing*treatments*that*target*the*pathway*(Figure*1.2).*

!

1.2.1!Nitric!oxide!synthesis:!Nitric!oxide!synthases!(NOSs)!

( In*mammals,*there*are*three*families*of*NOS*encoded*by*distinctive*genes :* neuronal* (nNOS* or* NOSU1),* inducible* (iNOS* or* NOSU2),* and* endothelial* (eNOS* or* NOSU3)*(Alderton*et*al.,*2001r*Carnicer*et*al.,*2013r*Rafikov*et*al.,*2011).*They*are* all* expressed* in* cardiac* endothelium* and* share* 50U60%* amino* acid* sequence* homology.*NOSU1*was*found*in*neuronal*tissue*and*is*important*for*neurotransmition* in* nerve* system (Bredt* et* al.,* 1991).* NOSU3* is* mainly* found* in* vascular* endothelial* cells*and*important*for*vascular*homeostasis (Sessa*et*al.,*1992).*Both*NOSU1*and* NOSU3* are* constitutively* expressed* in* the* cardiovascular* system* (Carnicer* et* al.,* 2013).*NOSU2*is*involved*in*defense*mechanism*against*pathogens*(Charles*et*al.,* 1993),* and* its* expression* is* induced* within* macrophages* by* bacterial* lipopolysaccharide* (LPS)* and* cytokines,* unlike* the* other* two* NOS* (Alderton* et* al.,* 2001).* The* NOS* consists* two* functional* domains,* which* are* the* NUterminal* oxygenase* domain* and* the* CUterminal* reductase* domain* (Alderton* et* al.,* 2001r*

*

Figure( 1.2:( Dysfunction( of( cGMP( signaling( involved( enzymes( in( cardiovascular( diseases.( Although* therapeutics* that* targets* four* different* enzymes* among* these* 5*

essential* enzymes* is* available,* a* single* drug* that* targets* PKG* has* not* been* developed* yet.*Modified*from*(Tsai*and*Kass,*2009).(

Stuehr,* 1997).* The* oxidase* domain* contains* binding* sites* for* the* substrate* LU arginine,* the* cofactor* tetrahydrobiopterin* (BH4),* and* a* heme* active* site.* The* reductase* domain* consists* of* the* flavin* cofactors* (flavin* mononucleotide* and* flavin* adenine* dinucleotide)* and* nicotinamine* adenine* dinucleotide* phosphate* (NADPH)* binding*sites.*NOS*functions*as*a*homodimer,*and*its*activity*is*highly*dependent*on* Ca2+Ucalmodulin*(CaM)*binding,*except*the*CaM*independent*NOSU2.*

All*three*isoforms*of*NOS*have*been*detected*in*cardiac*and*vascular*muscle* and*endothelial*cells (Tsai*and*Kass,*2009),*but*NOSU1*and*NOSU3*mostly*play*roles* in*cardiac*functions*through*PKG*dependent*or*PKG*independent*manners*(Alderton* et* al.,* 2001r* Carnicer* et* al.,* 2013r* Rafikov* et* al.,* 2011).* Either* NOSU1* or* NOSU3* deletion* in* mouse* models* showed* worse* recovery* after* cardiac* or* vascular* injury,* compared*to*that*in*the*wild*type*mouse*models*(Carnicer*et*al.,*2013r*Kuhlencordt* et* al.,* 2006r* Moroi* et* al.,* 1998).* While* deletion* of* either* NOSU1* or* NOSU3* causes* minor* vascular* defects,* the* NOS* triple* knockout* mice* showed* spontaneous* myocardial*infarction*and*sudden*death,*suggesting*that*the*function*of*each*isoform* could*be*compensated*by*others*(Carnicer*et*al.,*2013r*Nakata*et*al.,*2008).**

One* of* the* main* aspects* of* NOS* dysfunction* is* producing* O2−* upon* shifting*

the*cofactor*BH4*to*BH2*(Beckman*and*Koppenol,*1996r*Carnicer*et*al.,*2013).*BH4* is*crucial*for*maintaining*the*functional*NOS*dimer*and*LUarginine*binding*(Rafikov*et* al.,*2011)r*hence,*without*BH4,*NOS*is*not*able*to*maintain*its*proper*function*(NOS* uncoupling)* and* produces* O2,*instead* of*producing* NO.* This* reactive* superoxide*

species* can* be* converted* into* peroxynitrate* (ONOO−),* together* with* NOr* thus,* it* disrupts* NOUcGMP* signaling* dependent* vasorelaxation* by* reducing* NO* bioavailability* or* by* changing* the* redox* state* of* sGC.* In* addition,* this* reactive* peroxynitrate* also* causes* cell* damage* –* and* eventually* cell* death* –* through* lipid* peroxidation,*inactivation*of*enzymes*and*proteins*by*oxidation*and*nitration,*matrix* metalloproteinases* (MMP)* and* poly* (ADPUribose)* polymerase* (PARP)* activation* (apoptosis*and*necrosis,*respectively),*and*DNAUstrand*break*(Carnicer*et*al.,*2013r* Evgenov*et*al.,*2006).**

To* enhance* NO* production,* several* treatment* options* have* been* studied* in* various*approaches*(Carnicer*et*al.,*2013r*Evgenov*et*al.,*2006).*These*approaches* are* mainly* categorized* into* 1)* providing* more* organic* nitrates,* 2)* increasing* NOS* translation*or*enhancing*NOS*activity*(NOS*activator),*3)*promoting*NOS*recoupling* (NOS* recouplersr* BH4),* 4)* enhancing* cGMP* signaling,* 5)* inhibiting* arginase* inhibitors,* and* 6)* modulating* NOUROS* (reactive* oxygen* species)* interaction* (antioxidants).*Although*these*all*appeared*as*promising*approaches,*there*are*only*

a*few*options*available*as*commercial*therapeuticsr*however,*most*of*them*have*not* been*thoroughly*studied.*(Carnicer*et*al.,*2013r*Evgenov*et*al.,*2006).**

**

1.2.2!cGMP!synthesis:!Guanylyl!cyclase!(GCs)!

*

Guanylyl* (Guanylate)* cyclases* (GCs)* are* widely* distributed* signalU transduction* enzymes* that* convert* GTP* to* cGMP* upon* various* cellular* stimuli* including* NO* or* natriuretic* peptides* stimulation (Derbyshire* and* Marletta,* 2012r* Evgenov*et*al.,*2006).*There*are*two*types*of*GCs:*a*soluble*GC*(sGC)*that*is*found* in* a* cytosol,* and* a* transUmembrane* particulate* GC* (pGC)* that* is* found* in* cell* membrane.* Both* GCs* produce* cGMPr* however,* their* activations* are* initiated* upon* different* signaling* molecules.* While* the* pGC* is* activated* by* the* interaction* of* natriuretic*peptides*at*its*outer*membrane*receptor,*the*activation*of*sCG*is*regulated* by* gaseous* ligands,* NO* (Derbyshire* and* Marletta,* 2012r* Evgenov* et* al.,* 2006).* Since*GCs*play*the*main*role*in*producing*second*messenger*cGMP,*dysfunctions*of* the* enzymes* lead* to* various* maladies* in* cardiovascular* and* neuronal* systems (Erdmann* et* al.,* 2013b).* The* distinctive* properties* of* each* enzyme* are* described* below*in*detail.*(

*

1.2.2.1$Soluble$guanylyl$cyclase$(sGC)!

**

Soluble* GC* is* a* heterodimer,* comprised* with* two* homologous* subunits,* a* larger* αUsubunit* (690* amino* acids)* and* a* smaller* βUsubunit* (619* amino* acids)* (Derbyshire* and* Marletta,* 2012r* Evgenov* et* al.,* 2006).* There* are* two* isoforms* of* each* α* and* β* subunits* (α1* and* α2,* β1* and* β2).* Although* theoretically* four* dimeric* isoforms*of*sGC*can*exist,*only*two*isoforms,*α1β1*and*α2β1*dimers*exist*as*active* forms.* Interestingly,* the* α1β2* and* α2β2* dimers* are* inactive,* but*homodimeric*β2* is* active*without*αUsubunit*(Koglin*et*al.,*2001).*While*the*most*wellUstudied*α1β1*dimer* (cytosolic)*is*found*in*most*tissues,*including*vascular*smooth*muscle,*α2β1*dimer*is* found*in*neuronal*cells (Burette*et*al.,*2002r*Mergia*et*al.,*2003),*and*is*associated* with* the* plasma* membrane* through* proteinUprotein* interactions* through* the* CU terminus* of* the* α2* protein* (Derbyshire* and* Marletta,* 2012r* Russwurm* et* al.,* 2001r* Zabel* et* al.,* 2002).* All* four* αU* and* βUisoforms* include* a* NUterminal* hemeUnitric* oxide/oxygen* binding* (HUNOX)* domain,* a* Per/Arnt/Sim* (PAS)* domain,* a* coiledUcoil* (CC)* domain,* and* a* CUterminal* catalytic* (CAT)* domain* (Derbyshire* and* Marletta,* 2012r*Evgenov*et*al.,*2006).*The*HUNOX*senses*NO*binding*(Pellicena*et*al.,*2004),*

the* PAS,* and* CC* domains* are* important* for* proteinUprotein* interaction* and* sGC* dimerization* (Ma* et* al.,* 2010r* Moglich* et* al.,* 2009),* and* the* CAT* domain* is* responsible* for* cyclase* activity* that* converts* GTP* to* cGMP (Rauch* et* al.,* 2008r* Winger* et* al.,* 2008).* While* the* NUterminal* HUNOX* domain* of* the* βUsubunit* that* contains*a*heme*cofactor*is*responsible*for*the*NO*sensing,*the*NUNOX*domain*in*αU subunit* does* not* contain* a* heme* (degenerated* HUNOX),* and* the* function* of* this* domain*remains*unknown*(Derbyshire*and*Marletta,*2012r*Evgenov*et*al.,*2006).*

The* heme* cofactor* is* located* in* the* hemeUbinding* domain* where* three* anchoring*residues,*tyrosine*(Try135),*serine*(Ser137),*and*arginine*(Arg139)*form*a* conserved* hemeUbinding* motif* (YUxUSUxUR)* (rat’s* sGC* amino* acid* No.)* (Pellicena* et* al.,* 2004r* Schmidt* et* al.,* 2004).* Moreover,* a* conserved* histidine* (His105)* residue* acts*as*an*axial*ligand*for*the*heme*group,*interacting*with*the*ferrous*cation*(Fe2+)* (Herzik* et* al.,* 2014r* Pellicena* et* al.,* 2004r* Wedel* et* al.,* 1994).* This* histidineUiron* bond* causes* a* distortion* of* the* heme* planarity.* Breaking* of* the* HisUiron*bond* upon* NO* binding* allows* relaxation* of* heme* toward* planarity,* leading* to* a* conformational* change* that* ultimately* activates* cyclase* at* the* CUterminus.* Although* a* plethora* of* crystal*structures*are*available*on*each*functional*domain*of*sGC,*a*crystal*structure* of*the*fullUlength*enzyme*is*still*not*available.*Therefore,*an*understanding*of*how*the* NO* sensing* at* the* HUNOX* domain* transmits* to* the* CAT* domain* has* remained* elusive.* Recent* evidence* suggested* that* the* NUNOX* and* the* CAT* domains* directly* interact*and*modulate*the*sGC*activity*(Busker*et*al.,*2014r*Underbakke*et*al.,*2014).* In*addition,*a*single*particle*cryoUelectron*microscopy*(EM)*study*demonstrated*that* the* enzyme* is* very* flexible* and* accommodates* a* wild* range* of* conformations* through* a* long* helical* linker* (CCUdomain)* between* the* PAS* and* the* CAT* domains,* suggesting*direct*interaction*between*the*HUNOX*and*the*CAT*domains*(Campbell*et* al.,*2014).**

There*have*been*lots*of*trials*to*develop*sGC*stimulators*and*activators*as*a* treatment* of* hypertensive* cardiovascular* diseases.* Recently,* Bayer* AG* commercialized*the*hemeUdependent*sGC*stimulator*called*‘Adempas’*(riociguat)*to* treat* both* pulmonary* hypertension* and* chronic* thromboembolic* pulmonary* hypertension*(CTEPH)*(Ghofrani*et*al.,*2013ar*Ghofrani*et*al.,*2013b).*Riociguat*acts* in* a* dual* mode,* which* not* only* stimulates* sCG* activation* with* an* NOUindependent* manner* (differentUbinding* site),* but* also* increases* the* NO* sensitivity* of* sGC* by* stabilizing*the*nitrosylUheme*complex.*In*addition,*there*is*a*hemeUindependent*sGC* activator,*cinaciguat,*which*acts*to*either*NOUinsensible*oxidized*(ferric*ionr*Fe3+_)*or*

hemeUdeficient*form*of*sGC*and*is*currently*undergoing*clinical*trials*for*heart*failure* (Erdmann*et*al.,*2013a).**

1.2.2.2$Particulate$guanylyl$cyclase$(pGC);$guanylyl$cyclase$receptor!

(

In* contrast* to* sGC,* pGC* is* a* homodimeric* membrane* receptor* that* is* activated*by*natriuretic*peptide*hormones,*such*as*atrial*natriuretic*peptide*(ANP),*BU type* natriuretic* peptide* (BNP),* CUtype* natriuretic* peptide* (CNP),* and* intestinal* peptide* gyanylin* (Kuhn,* 2009r* Potter,* 2011a,* b).* Seven* transmembrane* GC* receptors* (pGCUA* to* pGCUG)* are* found* in* mammals,* and* they* all* have* domain* organizations*in*common*(Kuhn,*2009r*Potter,*2011a,*b).*The*membrane*bound*GCs* consist* of* five* distinct* domains:* an* extracellular* ligand* binding* domain,* a* juxtatransmembrane* domain,* an* intracellular* kinase* homology* domain,* a* dimerization* domain,* and* a* catalytic* domain.* The* extracellular* domains* of* different* GCs*recognize*their*specific*ligand*peptides*and*transduce*the*signal*to*the*catalytic* domain* to* activate* its* catalytic* activity,* converting* GTP* to* cGMP* and* two* phosphates.* However,* molecular* understanding* of* the* activation* mechanism* of* pGCs*is*still*elusive.*A*few*crystal*structures*of*the*dimeric*extracellular*domain*were* solved* (Ogawa* et* al.,* 2004r* van* den* Akker* et* al.,* 2000),* which* revealed* that* ANP* binds*in*the*cleavage*between*two*monomers*of*pGCUA*with*a*ratio*of*1:2**(Ogawa* et*al.,*2004).*Recent*study*of*pGCUA*and*UB*revealed*that*the*cyclase*domain*forms* an* asymmetrical* dimer* upon* binding* ATP,* and* the* ATP* binding* at* the* cyclase* domain*facilitates*the*production*of*cGMP*(Robinson*and*Potter,*2012).*Expression* of*the*pGCs*widely*distribute*to*various*tissues,*and*displacement*of*pGCs*result*in* many*diseases*in*cardiovascular,*skeletal,*intestinal,*or*visual*systems*(Kuhn,*2009).* Two* synthetic* peptide* drugs* that* target* pCGUA–carperitide* and* nesiritide–are* available* for* treating* congestive* heart* failure.* In* addition,* linacotide,* which* targets* pGCUC,*is*also*available*for*the*treatment*of*irritable*bowel*syndrome*(Potter,*2011a,* b).**

(

1.2.3!cGMP!degradation:!Phosphodiesterases!(PDEs)!

*

While* activation* of* the* NOUcGMP* pathway* is* initiated* by* the* production* of* cGMP* by* two* GCs,* the* lowering* of* the* cellular* cGMP* level* is* regulated* by* cyclic* nucleotide* phosphodiesterases* (PDEs).* Cyclic* nucleotide* PDEs* are* the* enzymes* that*hydrolyze*the*3’*cyclic*phosphate*bond*of*cGMP*and*cAMP,*generating*5’UGMP* or*5’UAMP*(Bender*and*Beavo,*2006r*Francis*et*al.,*2011r*Ke*et*al.,*2011r*Maurice*et* al.,*2014).*There*are*11*PDE*families*encoded*by*at*least*21*genes,*and*more*than* 100* PDEs* were* identified* within* mammals.* These* PDEs* are* involved* in* numerous*

cellular*processes*with*different*tissue*specific*patterns.*Because*of*this*complexity,* the*specific*cellular*roles*of*different*isotypes*remain*unclear.*All*PDEs*consist*of*an* NUterminal* regulatory* domain* and* a* CUterminal* catalytic* domain.* Several* crystal* structures* of* the* catalytic* domain* of* PDEs* revealed* that* the* catalytic* domains* are* wellUconserved* among* all* PDE* families,* having* three* subdomains* comprised* with* more* than* 16* helices* (Huai* et* al.,* 2003r* Ke* et* al.,* 2011r* Xu* et* al.,* 2000).* A* hydrophobic* active* pocket* consists* of* two* binding* sites,* which* include* a* cyclic* nucleotide* binding* site* and* a* divalent* metal* binding* sites,* containing* a* signature* motif,* HD(X2)H(X4)N* (Ke* et* al.,* 2011r* Maurice* et* al.,* 2014).* Although* all* catalytic*

domains* are* very* similar,* the* regulatory* regions* of* PDEs* are* very* diverse* in* their* lengths* and* their* domain* structures.* These* differences* are* directly* related* to* subcellular* localization,* and* ligand* specificity* of* the* different* families* of* PDEs* (Bender*and*Beavo,*2006r*Francis*et*al.,*2011r*Maurice*et*al.,*2014).****

Among* 11* PDE* families,* 8* PDE* families* are* involved* in* cGMP* hydrolysis* (Figure* 1.3).* Particularly,* PDE5s,* PDE6s,* and* PDE9s* specifically* hydrolyze* cGMP,* while* 4* PDE* families* including* PDE1s,* PDE2s,* PDE3s,* and* PDE5s* are* known* to* play*main*roles*in*cardiac*muscle*cells*by*modulating*cGMP*signaling*(Bender*and* Beavo,*2006r*Francis*et*al.,*2011r*Maurice*et*al.,*2014)*(Table*1.1).*PDE1*is*known* as* Ca2+_{/calmodulin* (CaM)Udependent* phosphodiesterases* and* expresses* in*}

cardiomyocytes* fraction,* implying* its* role* in* cardiac* hypertrophy* and* remodeling* (Johnson*et*al.,*2012r*Miller*et*al.,*2009).*PDE1s*hydrolyze*both*cGMP*and*cAMPr* however*their*preferences*differ*within*the*isotypes*(Bender*and*Beavo,*2006).*PDE2* also*hydrolyze*both*cGMP*and*cAMP*and*is*known*as*“the*cGMPUstimulated*PDEs”,*

(

Figure(1.3:(PDE(family(classifications((Bender*and*Beavo,*2006).(The*11*PDE*families*

are* categorized* into* three* groups* upon* their* substrate* specificity.* They* can* be* also* categorized*depending*on*their*allosteric*regulation*characteristics.((

because* cGMP* binding* at* its* NUterminal* GAF* domains* enhances* PDE2* activity* (Pandit* et* al.,* 2009).* * PDE3* binds* cGMP* and* cAMP* with* similar* binding* affinityr* however,*it*hydrolyzes*cAMP*with*higher*Vmax

(Bender*and*Beavo,*2006).*Moreover,*

PDE3*activity*(cAMP*hydrolysis)*is*suppressed*by*cGMP*in.vivor*therefore,*PDE3*is* called*“the*cGMPUinhibited*PDE”*(Bender*and*Beavo,*2006).*Because*PDE3*plays*a* role*in*cardiac*and*vascular*myocyte*contractility,*it*has*been*considered*as*a*drug* target*for*treating*congestive*heart*failure*(Maurice*et*al.,*2014).**

*

Among* all* PDE* families,* PDE5s* is* the* most* well* characterized* PDE* family* that* modulates* NOUcGMP* effects* in* vascular* smooth* muscle,* platelets,* and* the* urinary* tract* (Bender* and* Beavo,* 2006r* Francis* et* al.,* 2011r* Maurice* et* al.,* 2014).* PDE5* families* hydrolyze* cGMP* more* preferably* than* cAMP* by* at* least* 100Ufold.* Moreover,* their* activity* is* tightly* regulated* by* cGMP* binding* at* the* NUterminal* GAF* domainsr*therefore,*they*are*considered*“cGMPUspecific*PDEs”*(Bender*and*Beavo,* 2006r*Francis*et*al.,*2011).*PDE5*contains*two*GAF*domains*(GAFUA*and*GAFUB)*in* its*regulatory*domain,*and*cGMPUbinding*at*the*highUaffinity*GAFUA*domain*(KD*<*40*

nM)* leads* to* 10Ufold* higher* activation* of* PDE5* (Rybalkin* et* al.,* 2003).* PDE5* activation* is* also* facilitated* by* PKG* IUmediated* phosphorylation* at* Ser92* near* the* GAFUA* domain* (Corbin* et* al.,* 2000r* Rybalkin* et* al.,* 2003).* The* PKG* IUmediated* phosphorylation* enhances* PDE5* catalytic* activity* significantly* in. vitro,* suggesting* that* the* PKGUdependent* PDE5* phosphorylation* is* a* negative* feedback* mechanism* of*cGMP*signaling*(Corbin*et*al.,*2000).**Many*studies*suggested*that*elevations*of* PDE5* expression* and* activity* are* associated* with* cardiac* hypertrophy* and* heart* failure,* and* inhibition* of* PDE5* activity* in* cardiac* tissues* result* in* antihypertrophic* effect (Takimoto* et* al.,* 2005r* Tsai* and* Kass,* 2009r* Zhang* et* al.,* 2008).* * For* the* reason,* PDE5* has* long* been* considered* as* a* drug* target* for* heart* failure.* PDE5U specific* inhibitors* sildenafil* (marked* by* Pfizer* as* Viagra)* and* other* similar* drugs,*

Table(1.1:(Substrate(specificities(and(kinetics(properties(of(cGMP6hydrolyzing(PDE5( in(cardiac(muscle(cells.*Modified*from*(Francis*et*al.,*2010).( * Isoenzyme* Substrate* Preference* Km* Vmax* cGMP* cAMP* cGMP* cAMP* * μM. μmol/min/mg.

PDE1A* cGMP>cAMP* 3U4* 73U120* 50U300* 70U750* PDE1B* cGMP>cAMP* 1U6* 10U24* 30* 10* PDE1C* cGMP=cAMP* 1U2* 0.3U1* N.D.* N.D.*

PDE2A* cGMP=cAMP* 10* 30* 123* 120*

PDE3A* cGMP<cAMP* 0.02U0.2* 0.2* 0.3* 3U6*

PDE3B* cGMP<cAMP* 0.3* 0.4* 2* 9*

such* as* tadalafil* (Cialis* by* Lilly* and* ICOS)* and* vardenafil* (Levitra* by* Bayer* and* GSK),* have* been* used* to* treat* erectile* dysfunction* and,* more* recently,* pulmonary* arterial*hypertension*(Francis*et*al.,*2011r*Ghofrani*et*al.,*2006r*Maurice*et*al.,*2014).* These* inhibitors* act* as* competitive* binders* of* cGMP* at* the* PDE5* active* site* and* protect* cGMP* from* degradation* (Sung* et* al.,* 2003),* which* results* in* increasing* cellular* cGMP* level.* Many* other* PDE* inhibitors* of* cGMPUhydrolyzing* PDEs* have* been* also* developed,* and* some* of* them* are* in* stages* of* clinical* trials* for* treating* cardiovascular*dysfunctions*(Francis*et*al.,*2010r*Maurice*et*al.,*2014).***

*

1.2.4!cGMP!signal!transducer:!Cyclic!GMPAdependent!protein!kinases!(PKGs)!

(

Signals* of* the* second* messenger* cGMP* are* transduced* to* the* downstream* effector*proteins*by*the*cGMPUdependent*protein*kinase*(PKG) (Beavo*and*Brunton,* 2002r*Francis*et*al.,*2010r*Hofmann*et*al.,*2009r*Schlossmann*and*Hofmann,*2005).* PKG* is* one* of* the* main* subcellular* receptors* of* cGMP* and* belongs* to* the* AGC* (PKA,* PKG,* and* PKC)* family* of* serine/threonine* kinases.* Three* different* isoforms* have*been*identified*in*mammals:*PKG*Iα,*PKG*Iβ,*and*PKG*II.*All*three*isoforms*are* present*as*homodimers*(Gamm*et*al.,*1995).*PKG*Iα*and*Iβ*are*alternative*splicing* variants*of*the*prkg1*gene,.and*they*only*differ*in*approximately*100*amino*acids*at* their* NUterminus* (Wernet* et* al.,* 1989).* PKG* II* is* a* gene* product* of* prkg2. and* is* membraneUbound* via* its* myristoyl* group* (Gly2)* at* the* NUterminus* (Uhler,* 1993).* Although*they*are*different*gene*products,*type*I*and*II*PKGs*share*a*large*degree*of* similarity* in* their* amino* acid* sequences* and* domain* organizations* (Gamm* et* al.,* 1995)* (Figure* 1.4).* PKG* consists* of* two* distinct* functional* domains,* an* NUterminal* regulatory*(R)Udomain*and*a*CUterminal*catalytic*(C)Udomain.*The*RUdomain*includes* three*functional*domains,*a*leucine*zipper*(LZ)*domain,*an*autoUinhibitory*sequence,* and*two*consecutive*cGMPUbinding*domains*(Gamm*et*al.,*1995r*Takio*et*al.,*1984).* The*CUdomain*consists*of*a*small*lobe*that*is*responsible*for*Mg2+*and*ATP*binding* and*a*large*lobe*that*is*responsible*for*substrate*binding.**

Figure( 1.4( Domain( organization( of( PKG( I( and( II.( ( PKG* I* and* PKG* II* have* the* similar*

PKG*I*is*predominantly*expressed*in*smooth*muscles,*platelets,*cerebellum,* hippocampus,* dorsal* root* ganglia,* neuromuscular* endplate,* and* the* kidney* as* well* as*in*cardiac*muscle,*vascular*endothelium,*granulocytes,*chondrocytes,*osteoclasts,* and*brain*nuclei*(Francis*et*al.,*2010r*Hofmann*et*al.,*2009).*Both*α*and*β*isoforms* of*PKG*I*are*often*expressed*in*these*tissues*and*exist*mostly*in*cytosol.*In*contrast,* in* platelets,* the* β* form* is* dominantly* identified* and* found* in* the* membrane* fraction* (Francis* et* al.,* 2010r* Hofmann* et* al.,* 2009).* PKG* II* is* expressed* in* several* brain* nuclei,* intestinal* mucosa,* kidney,* adrenal* cortex,* and* chondrocytes* (Francis* et* al.,* 2010r*Hofmann*et*al.,*2009).*

*Roles*of*PKG*I*in*cardio*vascular*system*have*been*extensively*studied*for* its*pharmacological*importance.*Several*PKG*I*knockout*mice*studies*have*indicated* that* PKG* I* plays* a* crucial* role* in* cardiac* negative* inotropy* (Schroder* et* al.,* 2003r* Wegener* et* al.,* 2002)* and* VSMC* relaxation* (Koeppen* et* al.,* 2004r* Pfeifer* et* al.,* 1998r*Weber*et*al.,*2007).*In*VSMCs,*activation*of*PKG*I*results*in*VSMC*relaxation* by* reducing* intracellular* free* Ca2+_{* concentration,* desensitizing* contractile* protein*}

from* the* effects* of* calcium,* and* decreasing* the* contractile* state* of* smooth* muscle* (Figure*1.5*and*Table*1.2)*(Francis*et*al.,*2010r*Hofmann*et*al.,*2009).*PKG*I*mainly* relaxes* VSMC* through* phosphorylating* many* cellular* target* proteins* that* are* involved*in*many*processes,*as*listed*below (Francis*et*al.,*2010r*Schlossmann*and* Desch,*2009).**

*

1)* Preventing* myosin* contraction* through* myosin* light* chain* phosphatase* (MLCP)* activation* and* Ras* homologue* gene* family* member* A* (RhoA)/RhoU associated* protein* kinase* (ROCK)* inhibition,* which* induces* increased* dephosphorylation*of*myosin*light*chain*(MLC) (Ellerbroek*et*al.,*2003).** 2)* Reducing* or* sequestering* Ca2+_{* releases* from* the* sarcoplasmic* reticulum.*}

Phosphorylation* of* IP3R1Uassociated* cGMP* kinase* substrate* (IRAG)

(Geiselhöringer*et*al.,*2004r*Schlossmann*et*al.,*2000)*and*phospholamban* (PLB) (Koller* et* al.,* 2003r* Lalli* et* al.,* 1999)* inhibits* Ca2+* releasing* from* the* sarcoplasmic* reticulum* through* the* IP3* receptor* type1* (IP3R1)* and* the*

sarcoplasmic*reticulum*calcium/ATPase*pump*(SERCA),*respectively.*

3)* Preventing* intracellular* Ca2+_{* mobilization* by* decorating* inositol* triphosphate*}

(IP3)* production* through* inhibiting* phospholipaseUCβ3* and* thromboxane*

receptor* (TP)Uα* isoform.* PKG* I* phosphorylates* phospholipaseUCβ3* (Ser26* and*Ser1105) (Xia*et*al.,*2001)*and*TPUα*(Ser331)*(KelleyUHickie*et*al.,*2007).** 4)* Inducting* membrane* hyperpolarization* through* phosphorylation* of* the* largeU

Intracellular* potassium* and* hyperpolarization* of* the* cell* membrane,* which* eventually* decreases* Ca2+_{* influx* through* LUtype* voltageUdependent* Ca}2+_*

channel*(LUVDCC)*(Fukao*et*al.,*1999r*Hall*and*Armstrong,*2000r*Sausbier*et* al.,*2000).*

5)* Interfering* interactions* between* G* protein* and* G* proteinUcoupled* receptor* (GPCR)*through*phosphorylation*of*the*regulator*of*G*protein*signaling*(RGSr* RGS2* and* 4) (Sun* et* al.,* 2005r* Tang* et* al.,* 2003).* PhosphoURGS* activates* the* GTPase* activity* of* G* protein* and* disrupts* interaction* to* GPCR,* which* eventually* leads* to* deactivation* of* cAMP* signalingUdependent* Ca2+_*

mobilization.* * Although*PKG*I*is*involved*in*such*diverse*signaling*pathways*and*acts*as*one*of*the* key*players,*its*activation*mechanism*and*correlations*between*human*diseases*are* still*poorly*understood.** ( ( ( Figure(1.5:(Roles(of(PKG(in(smooth(muscle(cell(relaxation.(((Francis*et*al.,*2010)(

Table!1.2!Substrates!of!PKG!I!in!smooth!muscle!(Schlossmann+and+Desch,+2009).! + Substrate! MW! (kDa)! PKG! isoform! Function! Reference!

BKCa+ 130+ PKG+I+ Membrane+hyperpolarization+ (Sausbier+et+al.,+2000)+

CRP2/4+ 22.5+ PKG+I+ Mediates+cGMP/PKG+stimulation+of+SMKspecific+gene+expressionN+ mediates+PKG+dependently+VSMC+phenotype+

(Chang+et+al.,+2007N+Zhang+et+ al.,+2007)+

FHOD1+ 130+ PKG+I+ Inhibition+of+VSMC+stress+fiber+formation/migration?+ (Wang+et+al.,+2004)+

IP3+receptor+type+1+ 230+ PKG+I+ Stimulation+of+calcium+release+from+IP3Ksensitive+stores+ (Haug+et+al.,+1999N+Wagner+et+

al.,+2003)+

IRAG+ 125+ PKG+Iβ+ Reduced+calcium+release+from+IP3Ksensitive+stores+ (Geiselhöringer+et+al.,+2004N+

Schlossmann+et+al.,+2000)+ MYPT1+ 130+ PKG+Iα+ Inhibition+of+myosin+phosphatase+inhibition+by+rho+kinaseN+decreased+

calcium+sensitization+

(Wooldridge+et+al.,+2004)+ PDE5+ 100+ PKG+I+ Enhanced+cGMP+degradation+ (Rybalkin+et+al.,+2002)+ Phospholamban+ 6+ PKG+I+ Enhanced+calcium+uptake+by+the+Ca2+KATPase+Serca+ (Lalli+et+al.,+1999)+

RGSK2/4+ 24+ PKG+Iα+ Inhibition+of+IP3+generation,+reduced+GPCR+signaling+ (Sun+et+al.,+2005N+Tang+et+al.,+ 2003)+

RhoA+ 22+ PKG+I+ Reduced+MLC+phosphorylation,+Vesicle+trafficking+ (Ellerbroek+et+al.,+2003)+ SMTNL1/CHASM+ 60+ PKG+I+ Decreased+suppression+of+MLCK+activity+ (Wooldridge+et+al.,+2008)+ Telokin+ 17+ PKG+I+ Inhibition+of+MLCK+activity+ (Walker+et+al.,+2001)+ Thromboxane+

receptor+(TPα)+

55+ PKG+I+ Receptor+desensitization+ (Wikstrom+et+al.,+2008)+ TRIM39R+ 46+ PKG+I+ Cellular+homeostasis?+ (Roberts+et+al.,+2007)+ VASP+ 46/50+ PKG+I+ Regulation+of+the+actin+cytoskeleton,+vesicle+trafficking+ (Butt+et+al.,+1994N+Hauser+et+

1.3$Detailed$understanding$of$functional$domains$of$PKG$

$

1.3.1$Leucine$zipper$(LZ)$domain$

!

The$ LZ$ domain$ is$ a$ common$ structural$ motif$ that$ displays$ leucines$ or$ isoleucines$ along$ the$ zipper$ interface$ (Iα:$ five$ heptad$ repeats,$ Iβ:$ eight$ heptad$ repeats) and$ dimerizes$ through$ hydrophobic$ pairing$ of$ these$ residues.$ In$ PKG,$ it$ also$ allows$ the$ dimerization$ of$ PKG$ and$ interaction$ with$ downstream$ effector$ proteins (Francis$et$al.,$2010J$Hofmann$et$al.,$2009J$RichieNJannetta$et$al.,$2003).$All$ three$ isoforms$ of$ PKG$ have$ LZ$ domains$ at$ their$ NNtermini,$ but$ their$ amino$ acid$ sequences$are$varied.$Due$to$these$sequence$variations,$PKGs$are$known$to$interact$ with$their$substrates$in$an$isotypeNspecific$manner (Casteel$et$al.,$2008J$Sharma$et$ al.,$ 2008J$ Wilson$ et$ al.,$ 2008).$ $ Our$ group$ has$ solved$ both$ individual$ crystal$ structures$ of$ the$ LZ$ domains$ of$ PKG$ Iα$ and$ Iβ$ and$ the$ coNcrystal$ structure$ of$ the$ PKG$II$LZ$domain$with$its$interaction$partner$Rab11 (Casteel$et$al.,$2010J$Qin$et$al.,$ 2015J$Reger$et$al.,$2014).$The$surface$charge$distributions$of$the$three$LZ$domains$ are$ significantly$ different,$ suggesting$ their$ distinct$ preferences$ for$ substrate$ recognitions$ (Figure$ 1.6).$ Interestingly,$ there$ are$ increasing$ evidences$ that$ the$ LZ$ domain$ modulates$ PKG$ I$ activation$ in$ either$ cGMPNdependent$ or$ independent$ manners$ (Burgoyne$ et$ al.,$ 2007J$ RichieNJannetta$ et$ al.,$ 2003).$ However,$ the$ molecular$mechanism$underlying$these$observations$is$poorly$understood.$$

!

Figure! 1.6:! Comparisons! of! the! three! LZ! domains! of! PKG.! Left:$ PKG$ Iα$ LZ$ domain$

(PDB$code:$4R4M)$with$its$electrostatic$surface$(electrostatic$potential=−96$to$96),$Middle:$ PKG$ Iβ$ LZ$ domain$ (PDB$ code:$ 3NMD)$ with$ its$ electrostatic$ surface$ (electrostatic$ potential=−92$ to$ 92),$ Right:$ PKG$ II$ LZ$ domain$ (PDB$ code:$ 4OJK)$ with$ its$ electrostatic$ surface$(electrostatic$potential=−85$to$85)!

1.3.2$Auto@inhibitory$(AI)$sequence$

$

An$ autoNinhibitory$ (AI)$ sequence$ is$ a$ short$ motif$ that$ acts$ as$ a$ pseudoN substrate$ of$ PKGs$ and$ inhibits$ the$ kinase$ activity$ in$ absence$ of$ cGMP.$ The$ motif$ contains$ a$ signature$ sequence$ (K/RNK/RNXNG/ANI/VNSNANENP/S)J$ PKG$ Iα$

59_TRAQGIS65_{,$ PKG$ Iβ$}75_KRQAIS80,$_{and$ PKG$ II$}121_AKAGV125$_{(Francis$ et$ al.,$ 2010J$}

Francis$et$al.,$2002J$Smith$et$al.,$1996).$In$the$absence$of$cGMP,$this$AI$sequence$ inhibits$ the$ catalytic$ activity$ of$ the$ CNdomain$ by$ docking$ to$ the$ catalytic$ cleft,$ preventing$ of$ the$ access$ of$ substrate$ proteins$ to$ the$ active$ site.$ In$ contrast,$ upon$ cGMP$binding$at$the$CNB$domains,$the$autoNinhibitory$sequence$is$released$from$the$ catalytic$core,$and$then$the$active$site$becomes$accessible$to$PKG’s$substrates$for$ phosphorylation.$ In$ the$ presence$ of$ ATP,$ binding$ of$ cGMP$ leads$ to$ an$ autoN phosphorylation$at$SerN64$(type$Iα,$if$counted$from$Met1:$SerN65)$or$SerN79$(type$Iβ,$ SerN80)$in$the$AI$sequence,$and$it$sterically$disrupts$the$interaction$between$the$autoN inhibitory$sequence$and$the$CNdomainJ$therefore,$the$AI$sequence$is$discharged$and$ the$CNdomain$becomes$activated$(Busch$et$al.,$2002J$Smith$et$al.,$1996).$The$autoN phosphorylation$sites$are$all$varied$between$isotypes$of$PKGs$(PKG$Iα:$SerN50,$ThrN 58,$ SerN64,$ SerN72,$ and$ ThrN84,$ PKG$ Iβ:$ SerN63$ and$ SerN79:$ if$ counted$ from$ Met1,$ the$residue$numbers$should$be$one$higher$than$these$numbers)$(Francis$et$al.,$2010J$ Smith$et$al.,$1996).$$$$ $

1.3.3$Cyclic$nucleotide$binding$(CNB)$domain$

$ The$cyclic$nucleotideNbinding$domain$(CNB)$is$an$ancient$structural$motif$that$ recognizes$ cyclic$ nucleotides$ (Berman$ et$ al.,$ 2005J$ Rehmann$ et$ al.,$ 2007).$ In$ eukaryotes,$the$CNB$domain$has$been$found$in$many$proteins,$including$cAMP$and$ cGMPNdependent$ protein$ kinases$ (PKAs$ and$ PKGs) (Pearce$ et$ al.,$ 2010),$ cyclic$ nucleotideNgate$(CNG)$and$hyperpolarizationNactivated$cyclic$nucleotideNgated$(HCN)$ ion$ channels (Craven$ and$ Zagotta,$ 2006),$ cAMPNregulate$ guanine$ nucleotide$ exchange$ factors$ (cAMPGEFs,$ also$ known$ as$ EPACs) (Bos,$ 2006),$ bacterial$ transcriptional$ factors$ (Cordes$ et$ al.,$ 2011J$ McKay$ and$ Steitz,$ 1981J$ Won$ et$ al.,$ 2009),$ nucleotidyl$ cyclases$ (An$ et$ al.,$ 2013),$ and$ acetyltransferases$ (Lee$ et$ al.,$ 2012).$ The$ CNB$ domains$ in$ these$ proteins$ function$ as$ an$ allosteric$ switch$ that$ triggers$ the$ proteins’$ activity$ by$ changing$ its$ conformations$ upon$ binding$ to$ cyclic$ nucleotides$(Alverdi$et$al.,$2008J$Brelidze$et$al.,$2012J$Wall$et$al.,$2003).$

In$PKG,$the$CNB$domains$also$act$as$allosteric$switch$that$recognize$cyclic$ nucleotide$as$an$activation$signal$and$transduce$the$signal$to$the$rest$of$the$kinase$in$ order$ to$ drive$ kinase$ activation.$ All$ mammalian$ PKGs$ contain$ two$ CNB$ domains$ in$ their$RNdomainJ$thus,$the$native$dimer$has$four$CNB$domains$(Francis$et$al.,$2010J$ Hofmann$ et$ al.,$ 2009).$ The$ structure$ of$ the$ CNB$ domain$ was$ first$ identified$ in$ the$ E.coli$transcription$factor$Catabolite$Activator$Protein$(CAP),$and$the$overall$folds$are$ evolutionally$ well$ conserved$ in$ all$ CNB$ domains$ (Berman$ et$ al.,$ 2005J$ McKay$ and$ Steitz,$1981J$Rehmann$et$al.,$2007).$The$CNB$domain$consists$of$an$8Nstranded$βN barrel$flanked$by$αNhelices$at$both$termini$(Figure$1.7).$The$βNbarrel$provides$a$stable$ scaffold$ for$ cyclic$ nucleotide$ bindingJ$ whereas,$ the$ αNhelical$ regions,$ including$ a$ conserved$ helical$ motif$ called$ Phosphate$ Binding$ Cassette$ (PBC),$ undergo$ rearrangements$ and$ adapt$ into$ their$ activated$ conformation$ upon$ cGMP$ binding$ (Berman$et$al.,$2005J$Rehmann$et$al.,$2007).$$

Previous$ biochemical$ studies$ revealed$ that$ the$ two$ CNB$ domains$ of$ PKGs$ have$different$affinities$for$cGMP$(Corbin$and$Døskeland,$1983J$Corbin$et$al.,$1986J$ Smith$et$al.,$2000),$and$the$affinities$of$each$domain$are$varied$within$PKG$isoforms$ (Reed$ et$ al.,$ 1997J$ Taylor$ and$ Uhler,$ 2000).$ In$ PKG$ I,$ the$ CNBNA$ domain$ has$ a$ significantly$higher$cGMP$affinity,$compared$to$that$of$the$CNBNB$domain.$In$addition,$ both$domains$show$significantly$different$specificities$for$cGMP$analogues,$revealing$ that$the$two$domains$have$evolved$distinctively$(Corbin$et$al.,$1986J$Schlossmann$et$ al.,$2005).$However,$why$they$show$different$affinities$for$cGMP$and$how$this$aspect$ is$related$to$PKG$I$activation$still$remains$unclear.$$ $ !

Figure! 1.7:! Crystal! structure! of! CNB! domain! and! amino! sequence! alignment! of! PBCs.!(A)$Crystal$structure$of$the$CNB$domain$of$Catabolite$Activator$Protein$(CAP,$PDB$

1.3.4$Catalytic$domain$

!

All$ eukaryotic$ AGC$ kinases$ share$ their$ bilobal$ structures$ and$ every$ element$ of$ conserved$ catalytic$ cores$ (Endicott$ et$ al.,$ 2012J$ Pearce$ et$ al.,$ 2010J$ Taylor$ and$ Kornev,$ 2011).$ The$ catalytic$ core$ is$ comprised$ by$ an$ NNterminal$ (N,$ or$ small)$ lobe$ containing$the$ATPNbinding$site$and$a$CNterminal$(C,$or$large)$lobe$that$provides$the$ peptide$ binding$ and$ catalytic$ sites.$ The$ phosphotransferase$ reaction$ occurs$ in$ this$ core$(cleft)$in$the$presence$of$ATP$and$Mg2+_{.$A$phosphorylated$threonine$residue$–$}

Iα:$ T516$ (if$ counted$ from$ Met1$ N$ T517),$ Iβ:$ T531$ (T532),$ and$ II:$ T608$ (T609)$ –$ is$ essential$ for$ the$ catalytic$ activity$ of$ PKGs$ (Feil$ et$ al.,$ 1995).$ So$ far,$ no$ structural$ information$ is$ available$ for$ the$ CNdomain$ of$ PKGs,$ while$ numerous$ structures$ of$ catalytic$ domains$ of$ other$ kinases$ were$ already$ solved$ (Figure$ 1.8)$ (Pearce$ et$ al.,$ 2010).$A$high$sequence$similarity$(over$40%)$between$the$catalytic$domains$of$PKA$ and$ PKG$ suggests$ that$ the$ CNdomain$ includes$ a$ regulatory$ (R)$ spine$ (hydrophobic$ motifs)$ and$ a$ catalytic$ (C)$ spine$ predicted$ to$ be$ very$ similar$ to$ other$ AGC$ kinases$ including$PKA$(Figure$1.8).$$$

$ $ $

$

Figure! 1.8.! Crystal! structure! of! PKA! catalytic! subunit! and! amino! sequence! alignment! with! PKG! catalytic! domain.! (A)$Crystal$structure$of$ PKA$CNdomain$(PDB$code:$1ATP),$(B)$

1.4$Cyclic$nucleotide$selectivity$of$PKG$I$$

$

Since$ the$ two$ homologue$ kinases,$ PKG$ and$ PKA$ are$ often$ involved$ in$ completely$ opposite$ physiological$ outcomes$ (Hofmann$ et$ al.,$ 2009),$ how$ these$ kinases$ specifically$ recognize$ their$ natural$ ligands$ and$ how$ they$ segregate$ each$ respective$signaling$pathway$have$been$challenging$questions$to$answer$in$the$cyclic$ nucleotideNsignaling$field.$PKG$and$PKA$share$high$sequence$homology$and$domain$ organization$ (Pearce$ et$ al.,$ 2010).$ In$ addition,$ they$ share$ the$ allosteric$ activation$ machinery,$ which$ releases$ the$ CNdomain$ when$ cyclic$ nucleotides$ bind$ at$ the$ RN domain.$ Both$ kinases$ have$ two$ CNB$ domains$ in$ their$ RNdomain.$ Despite$ the$ high$ sequence$ homology$ between$ them,$ PKG$ and$ PKA$ showed$ approximately$ 50–200N fold$selectivity$for$cGMP$or$cAMP,$respectively (Francis$et$al.,$2010).$Primary$amino$ acid$ sequence$ alignment$ of$ the$ PBC$ regions$ of$ the$ two$ kinases$ reveals$ that$ there$ are$ a$ few$ distinctive$ amino$ acid$ residues$ conserved$ in$ either$ kinase$ (Figure$ 1.9).$ Previous$mutagenesis$studies$already$predicted$that$threonine/serine$residue$at$the$ PBC$ loop$ of$ PKG$ is$ crucial$ for$ cGMP$ selectivity$ by$ interacting$ with$ the$ 2Nposition$ amino$group$of$cGMP$(Shabb$et$al.,$1991J$Smith$et$al.,$2000J$Weber$et$al.,$1989).$As$ seen$in$the$sequence$alignment,$there$are$several$residues$in$PKG$that$possibly$play$ a$ role$ in$ cGMP$ selectivity$ (Figure$ 1.9).$ However,$ the$ cyclic$ nucleotide$ selectivity$ of$ PKG$ was$ not$ clearly$ understood,$ since$ no$ crystal$ structure$ of$ any$ CNB$ domain$ of$ PKG$with$appropriate$biochemical$data$was$available,$prior$to$this$thesis$study.$!

1.5$cGMP@dependent$activation$mechanism$of$PKGs$$

$

Based$ on$ the$ similar$ domain$ organization$ and$ high$ sequence$ similarity$ between$ PKGs$ and$ PKAs,$ current$ models$ of$ the$ activation$ of$ PKG$ are$ taken$ from$

Figure! 1.9:! Amino! acid! sequence! alignment! between! the! PBC! of! PKGs! and! PKAs.!

the$ wellNestablished$ activation$ mechanism$ of$ PKA (Pearce$ et$ al.,$ 2010).$ However,$ there$ is$ a$ distinctive$ difference$ between$ the$ two$ kinases.$ While$ the$ RN$ and$ CN domains$ in$ PKG$ are$ fused$ on$ the$ same$ amino$ acid$ chain,$ these$ domains$ exist$ as$ separate$ subunits$ in$ PKA.$ This$ suggests$ that$ the$ detailed$ molecular$ modes$ of$ activation$between$PKG$and$PKA$may$differ$from$each$other.$Many$solutionNbased$ techniques,$ such$ as$ small$ angle$ XNray$ scattering$ (SAXS)$ and$ H/D$ exchange$ mass$ spectrometry$analyses,$have$been$used$for$structural$studies$of$PKGs,$but$all$these$ methods$ have$ been$ unable$ to$ describe$ the$ molecular$ details$ of$ the$ PKG$ activation$ mechanism$(Alverdi$et$al.,$2008J$Lee$et$al.,$2011J$Wall$et$al.,$2003J$Zhao$et$al.,$1997).$$

PKG$ is$ thought$ to$ undergo$ drastic$ conformational$ changes$ upon$ cGMP$ binding,$particularly$at$its$RNdomain,$which$then$leads$to$the$release$of$the$CNdomain$ (Alverdi$ et$ al.,$ 2008J$ Lee$ et$ al.,$ 2011J$ Wall$ et$ al.,$ 2003J$ Zhao$ et$ al.,$ 1997).$ In$ the$ absence$of$cGMP,$the$CNdomain$binds$the$RNdomain,$inhibiting$the$kinase$activity.$In$ contrast,$in$the$presence$of$cGMP,$the$RNdomain$alters$its$conformation$and$leads$to$ a$ major$ structural$ rearrangement$ within$ a$ whole$ enzyme.$ This$ change$ somehow$ results$ in$ autoNphosphorylation$at$the$NNterminus$ and$causes$dissociation$of$the$CN domain$ from$ the$ RNdomain$ (Smith$ et$ al.,$ 1996).$ While$ this$ whole$ concept$ of$ PKG$ activation$ is$ derived$ from$ PKA’s$ activation,$ the$ structural$ rearrangement$ of$ PKG$ upon$ activation$ has$ never$ been$ observed$ in$ an$ atomic$ level.$ Other$ than$ this$ thesis$ work,$Osborne$et$al.$reported$a$structure$of$the$tandem$CNBNA/B$domain$of$PKG$Iα (Osborne$ et$ al.,$ 2011).$ However,$ the$ structure$ contains$ nonNnatural$ ligand$ cAMP$ from$ E.coli$ expression$ system$ in$ only$ its$ high$ cGMP$ affinity$ site.$ Therefore,$ the$ structure$does$not$represent$neither$of$an$activated$nor$an$inhibited$(apo)$state$of$the$ RNdomain.$ For$ the$ reason,$ structural$ information$ of$ the$ CNBNA/B$ domain$ with$ and$ without$ cGMP$ is$ still$ necessary$ for$ understanding$ cGMPNdependent$ allosteric$ activation$of$PKG.$

$

1.6$Objective$$

$

As$ a$ main$ regulator$ of$ smooth$ muscle$ tone,$ PKG$ has$ been$ considered$ an$ important$ therapeutic$ target$ for$ treating$ cardiovascular$ and$ pulmonary$ diseases.$ However,$there$is$no$clear$mechanistic$insight$to$cGMPNdependent$activation$of$PKG.$ Structural$studies$of$PKG$have$been$hindered$due$to$its$flexible$nature.$The$activity$ of$ PKG$ is$ regulated$ by$ the$ interaction$ between$ the$ regulatory$ (R)$ and$ catalytic$ (C)$ domains.$ In$ the$ absence$ of$ cGMP,$ the$ RNdomain$ directly$ interacts$ with$ the$ CN domain,$ blocking$ the$ catalytic$ core:$ therefore,$ the$ kinase$ activity$ is$ inhibited.$ This$

inhibition$is$relieved$upon$cGMP$binding$at$the$RNdomain.$In$the$presence$of$cGMP,$ the$RNdomain$undergoes$drastic$conformational$changes$and$releases$the$CNdomain$ to$ become$ active.$ However,$ no$ atomic$ level$ information$ is$ available$ for$ understanding$this$process$yet.$$ Through$this$thesis$work,$I$mainly$focused$on$understanding$the$role$of$the$RN domain$in$PKG$activation.$The$RNdomain$is$the$part$that$makes$PKG$unique$among$ other$kinases,$and$especially$the$tandem$CNB$domains$are$the$parts$that$sensor$the$ cellular$cGMP$signals$and$initiate$PKG$activation.$Therefore,$discovering$the$role$of$ the$RNdomain$is$an$initial$key$step$to$comprehend$the$activation$mechanism$of$PKG.$ To$do$this,$I$aimed$to$answer$the$following$questions:$ $ 1)$ How$do$the$CNB$domains$recognize$cGMP$over$cAMP?$ 2)$ Which$one$of$the$two$CNB$domains$is$more$cGMP$selective?$ 3)$ What$is$the$active$conformation$of$the$tandem$CNB$domains?$ $ To$answer$these$questions,$I$applied$biophysical$and$biochemical$techniques$ including$ XNray$ crystallography,$ small$ angle$ XNray$ scattering,$ fluorescence$ polarization$assay,$and$microfluidic$mobility$shift$assay.$The$results$discussed$in$this$ thesis$study$can$be$utilized$for$developing$a$novel$drug,$specific$for$PKG.$ $ $ $ $ $ $ $ $ $ $ $ $

2$Materials$and$Methods$

$

2.1$Materials$

2.1.1$Plasmids$

$ E.coli!expression!plasmid! $ pQTEV$ $ Gift$from$Dr.$K.$Büssow$(Helmholtz$ Centre$for$InfectionJ$Braunschweig,$ Germany)$ Human!cell!expression!plasmid! $

pCDNA$3.1$(+)$ InvitrogenTM$_{(Carlsbad,$CA$USA)$}

!

2.1.2$Primers$

! Primer!name!! DNA!sequence!(5’!to!3’)! PKG$Iβ'BamHI_1$! att$gta$gga$tcc$ATG$GGC$ACC$TTG$CGG$GAT$TTA$! PKG$Iβ'BamHI_92$! att$gta$gga$tcc$AGC$CAT$GTG$ACC$CTG$CCC$TT!

PKG$Iβ'BamHI$_219! att$gta$gga$tcc$ACA GGA CTC ATC AAG CAT ACC G $ PKG$Iβ'NotI_227$ att$gta$gcg$gcc$gc$TCA$ATA$CTC$GGT$ATG$CTT$GAT$GAG$TC! PKG$Iβ'NotI_351! att$gta$gcg$gcc$gc$TCA$ATA$TGC$TTT$ATT$AGA$AAC$ATC$ATC$C! PKG$Iβ'NotI_363! att$gta$gcg$gcc$gc$TCA$TCA$TTC$AGC$TTC$ATA$TTT$TGC$TTT$AGC! PKG$Iβ'NotI_369! att$gta$gcg$gcc$gc$TCA$TCA$GTT$GGC$GAA$GAA$AGC$CGC$TTC! PKG$Iβ' BamHI_5_pCDNA$ att$gta$gga$tcc$GAA$ATG$GGA$GAT'TAC'AAG'GAC'GAC'GAT'GAC' AAG$CGG$GAT$TTA$CAG$TAC$GCG$CTC$C$ PKG$Iβ' BamHI_55_pCDNA$ att$gta$gga$tcc$GAA$ATG$GGA$GAT'TAC'AAG'GAC'GAC'GAT'GAC' AAG$CGA$CCA$GCC$ACC$CAG$CAG$GC$ PKG$Iβ' XhoI_685_pCDNA$ att$gta$ctc$gag$TTA$TTA$GAA$GTC$TAT$ATC$CCA$TCC$TG$ PKG$Iβ'N189A_For! ggggaattggctattctttacgcctgtacccggacagcg$ PKG$Iβ'N189A_Rev! cgctgtccgggtacaggcgtaaagaatagccaattccc$ PKG$Iβ'E229A_For! catcaagcataccgagtatatggcatttttaaaaagcgttccaacat! PKG$Iβ'E229A_Rev! atgttggaacgctttttaaaaatgccatatactcggtatgcttgatg! PKG$Iβ'L286A_For! ccgagtgaagacccagtctttgctagaactttaggaaaaggag$! PKG$Iβ'L286A_Rev! ctccttttcctaaagttctagcaaagactgggtcttcactcgg! PKG$Iβ'R287A_For! caccgagtgaagacccagtctttcttgcaactttaggaaaagg$! PKG$Iβ'R287A_Rev! ccttttcctaaagttgcaagaaagactgggtcttcactcggtg! PKG$Iβ'T317A_For! agggggaagatgtgagagcagcaaacgtaattgct$! PKG$Iβ'T317A_Rev! agcaattacgtttgctgctctcacatcttccccct! PKG$Iβ'Y351A_For! gggctggatgatgtttctaataaagcagctgaagatgcagaagct! PKG$Iβ'Y351A_Rev! agcttctgcatcttcagctgctttattagaaacatcatccagccc$! $