Bi~'himica el Bioi*hysica Acta. 1135 (Itm2) 51 f~l
51
~9 1992 Ebeviet Science Publishers B.V. All rights re,erred Ulh7-4889/~2/$05.00
BBAMCR 1315u Minireview
Structure
and
physiological
role
p~otein Franz
Hofmann,
Wolfgang
of
cGMP-dependent
kinase
Dostmann,
Alexandra
and Peter
Keilbach,
Wotfgnt,g Laea_grnf
Ruth
Italil~et ]~r Phu~takof~lgiv mul T, JLi~,,hlgic ,h'r Tc~ Ilni~ h('a Uait',,rsitilt Miindl:'tL Miipwltelt ((;ermally)
( Rgcci~ed ~ Januao' 1~92)
Key~ords: Pr~l¢inkinase:~'clcGMP;En~ymesruc,,re;E~zyme e*~ua m S u l s r a e p osplorylatlan
Contents I.
Imtoducllon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. Tgpes and g e n ~ o f ¢ ( ; M P kin~se
51
...............................................
5]
]u. Slructu,-e at cGMPMcpendent protein kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Overall struclure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Amina-termia-d cktrnain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (7. e{;M P~inding d c ~ i n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Catab'lic detain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Peptid¢ re.oogniUon. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Regular km of edlalyUc act rzily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
53 53 53 54 54 56 56
IV. Ph~iMogitml tu rcli~ms of cG MP kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A~ In vi~ dLslribution of cGMP kina~ im~,:ym~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. lNitential phs~kdugi~dl functions uf c~MP k i n ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
56 56 57
Ackno~ledgemen I
58
Referents
.
.
.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1. I n t r o d u c t i o n Cyclic G M P is a key regulatory molecule of specific cellular functions such as t h e visual transducfion process in r o d o u t e r s e g m e n t , i n t e g r a t i o n o f neuro-excitability to e x c i t a t o ~ n e u r o t r a n s m i t t e r s , relaxation o f
Abbreviations: cGMP k m ~ , OGMP~lel~2ndgnl prolei.~ kina~¢: cAMP k i n ~ . cAMPMe.l~endenl protein kin~e; PKL protein kioase inhibitor Ixptide: CHO. Chines hamster o~-arial: KA. activaUon conSlant; mL amino acid. Correspondence: Franz IIofmann. In~titut mr Pbarmakolo~ie und Tuxikntngie, TO M0nt:hea, Biedersleiner Stra~se 29, D-~000 Mfinchen 40, G~nnany.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
58
s m o o t h muscle, intestinal secretion o f w a t e r a n d salt, a n d reabsocption o f sodium a n d w a t e r in t h e distal tubulus o f the n e p h r o n . O v e r 0 r e d u c t i o n o f c G M P in specific cells h a s been associated with n e u r o n a l d e g e n eration, c n d o t o x i n shock, diarrhoea, a n d loss o f s o d i u m a n d water. This diversity o f t h e cellular effects of c G M P are n o t m e d i a t e d by a unique protein b u t by several distinct receptors: a e G M P - g a t e d cation c h a n nel, e G M P - s t i m u l a t e d a n d i n h i b i t e d p h o s p h o d i esterases a n d c G M P - d e p e n d e n t protein kinases ( c G M P kinasc). This article focuses o n r e c e n t d e v e l o p m e n t s in o u r u n d e r s t a n d i n g o f the structure a n d function o f c G M P kinase. T h e o l d e r results o n e G M P kinase a n d related areas have b e e n s u m m a r i z e d excellently by Lincoln a n d C o r b i n [1], L i n c o l n [2] a n d W a l t e r [3].
52
TABLE I S ~ r y of utent~fwdoGMP k u m ~ n.r., ~ t ~poffed: ~ KA, ~ivaz~m ~ s t a n l for CGMP in a protein P~n~ assa~_ S~cies
TL~SUC
Fu,aty
Molecular m~.~ (~D~)
pmc Wrti~l partial Im~tiai partial pu~e
naike (kDa) 8~ l~l /,2 7~ I¢-tn 162
P~c~*n Tetrahy~ J:_-~_~m Silk ~ o ~ Silk ~otm ~t~r/~ swan
~3~a
Bm~ne Bovine ~tvilt~ Bovir~ Pat
I~ng I~mt aorta aorta intestine,
pu~ pine pule rmre pure
I~ IB 11
Bt~ine Bovine Human Dr~oMd/a Drosot~dta [kos~li[a
trachea trachea p~acea~a
cloned eapte~ed cloned cloned cloned cloud
1o I~ I~ DGI OG2-TI DG 2-T2
c$g~ pufr~e
I1. Types a n d genes e f ¢ G M P k i n a s e c G M P kinase is p.resent in a variety o f eukaryntes r a n g i n g f r o m t h e unicellular o r g a n i s m Paramecium to h u m a n s [4-19] (Table I). M a m m a l s express in most tissues a h o m o d i m e r i c e n z y m e (type !) with a native m o l e c u l a r mass o f 153 to 156 k D a a n d in the intestinal brush b ~ d e r a m o n o m e r i c e n z y m e (type I I ) w i t h a native m o l e c u l a r mas~ o r 86 kDa. T h e h u m a n type 1 g a n e is localized to t h e p l L 2 - q l l . 2 region o f c h r o m o s o m e 10 [20]. T w o c G M P kina.~ g e n e s ( D G I a n d D G 2 ) a n d several splice variant~ have b e e n cloned f r o m Drosophila melanogaster [19]. T h e c G M P - b i n d i n g d o m a i n s a n d t h e catalytic d o m a i n as derived f r o m the d e d u c e d a m i ~ o acid sequence of these g e n e s are very h o m o l o g o u s to t h e m a m m a l i a n ry~e 1 enzyme. T h e structure t,f th~ native proteins is n o t k n o w n since c G M P kinase b~s n o t b e e n purified f r o m Drosophila. T h e e n z y m e s purified f r o m Paramecium [4], TeIrahymena [5] a n d Dico'ostdium discoideum [6] are a p p m e n t l y m o n o m e r i c c o n t a i n i n g a single polypcptide chain. T h e classification o f these e n z y m e s is unclear. It is even possible t h a t they are proteolytic products of a dimcric e n z y m e , A m o n o m e r i c c G M P activatable type I c G M P kinase h a s b e e n purified f r o m bovine aorta [21]. This e n z y m e was a proteolytic p r o d u c t o f the h o m e d i m e r i c isozyme I/~ as s h o w n by p e p t i d e sequencing [14] a n d cloning [16]. T h e r e f o r e this suggests t h a t t h e .type I c G M P kinases a r e d i m e r i c e n z y m e s w h e r e a s t h e type II e n z y m e m a y be monorneric. T h e occurrence o f m o n o m e r i e c G M P [4-6] a n d c A M P [22] kinases in
d.~n~tured
Subunit number
77
re.homer n~nomer ~mcr ~ e r
K a ~
Rcfctcncc
(riM)
i£~3 1 18
4 5 6 7
82
dimer
18
9
150 150 178 170 86
74 82 78 80 85
dlrm:r dimer dimer di~r monomer
40 ~ 2e~0 4~} n.r.
10.il 12 13,14 13,14 15
153 156
76,118 77803 778o3 > 7a(~0 > 7b(~} > 7b t]~ffl
di~ dimer
I[lfl 12£0
16,17 ~6,17 18 19 19 19
-
unicellular organi~Tns supports t h e idea t h a t dimerization o f t h e kinases occurred later in evolution. S e q u e n c e c o m p a r i s o n s inificatc t h a t t h e c G M P kina.¢,e g e n e evolved ns t h e result of a fu.~ion l',etween g e n e s f o r cyclic n u d c o t i d c b i n d i n g a n d catalytic protcins [I 1]. T h i s fusion occurred early in evolution, since the c G M P - k i n a s c s f r o m simple o r g a n i s m s have cyclic nueleotide b i n d i n g a n d catalytic activities o n t h e same polypeptide chain. A n i n t e r n a l g a n e duplication prod u c i n g t h e t w o c G M P b i n d i n g sites o n e a c h subunit o f t h e m a m m a l i a n e G M P - k i n n s e runs1 also have occurred very early in es'oinfiou, since t w o c-GMP-bioding sites a r e p r e s e n t in c G M P kinnses o f lower organisms. C l o n i n g a n d purification o f the m a m m a l i a n s m o o t h mubcle c G M P kinase led to t h e identification o f t w o isoforms o f t h e type I e n z y m e [13,14,16]. designated l a a n d lfi_ T h e d e d u c e d a m i n o acid sequence o f the l a isoz~me is identical to t h a t r e p o r t e d for c G M P kinase purified f r o m bovine l u n g [11]. T h e c D N A clone f o r ti,2 I f l isozym¢ differs f r o m isozyme la only at t h e N - t e r m i n u s ( a m i n o acids 1 - 1 0 4 "1, a n d contains t h e sequence of a p c p t i d e o b t a i n e d f r o m t h e I/~ isozyme purified f r o m aorid [21]. T h e sequence o f isozyme I ~ f r o m bovine trachea was c o n f i r m e d by t h e cloning o f c G M P kinasc f r o m h u m a n p l a c e n t a [18]. T h e h u m a n
* Amino acid residtle~ for cGMP kinases are numi~red by" ignoring the a m i ~ t c ~ i n a l ~ t h l o n i ~ ~n~-pnnding to the st,an ~don in eDNA sequences, Thib i~ in aO~eldant~ ~ilh protElo s~qucncing data lit].
53 placental enzyme contains only two amino acids exchanges at positions 279 and 289 when compared to the bm4ne lung and tracheal lt~ and 1~0 enzymes replacing a lysine by a threonine and an asparagine by a serine. The bovine tracheal and human placental I/~ sequences suggest that the l~ and !fl isozymcs contain identical cGMP-binding sites and catalytic centres. The aminntermini of the cloned isozymes l a and I/~ are different and show u I~v degree of similarity to each other and to the corre~po,ding parts of the regulatory ,[.hzsnil~, of cAMP kinase. The highest degree of similarity is found in the region which contains at Thr-58 the major autophosphm3,1ation site of cGMP-kinase In. The l a and I~ form of eGMP-kinase may be derived from the same gene by alternative splicing as suggested by alignment of the l a and 1~ seque.ccs to Drosophila gene DG2-TI product which exhibits an exon/intron junction at the DNA position corresponding to amino acid 89 and 104 of the I~ and I~ cGMP-kinase, respectively [16,18.19,23]. The studies in Drosophila suggest that from the two genes DGI and 13132 at least six different mRNA are transcribed differing mainly in their amino terminal region [19]. IlL Structure d cGMP-dependen! protein kinase
HI-A. Ocerall ~tn~c'lffre cGMP kinase a~ purified from bovine lung is a 153-kDa homodimer c~ntaining two identical subunits of 76 418 Da. The primal" sequence of the subunits has been divided into several functional domains [11] (Fig_ 1). The amino-terminal domain A contains the
D G ~ 67%,~ 1
em
~
~5
~
mo~_
75%~,59%] m~r
pwrrmLcml~x
~
IWWCG'CPC~V
P ~ t | KItQGV SIIES- C
LG|GG~G
~
¢mo.lt~tG. ~
~
V
"~¢rr-cGrv~n/
xw Fig- I. The sequence of bo~qnelungt'GMpkinase(BI,) is almpared ~ith Ihe deduced aminoacidsequences ut the cloned bovine tracheal c(~MPkin~se I~(nll~) aM Ihe rn~itn Droxvlgulugene products DGI and DG2-TI ~DG2hPer~mage ~ff idenn~l amint~acids within the hnrnoh~glmsdl~ins are mdl~lcd and the sequullc¢>of Ctlil~lvcd sil~ ale aligned.The inv~dant th~eoniner~ducs ~ilhin Ihe ¢GMP binding I~chets 1~) and the b'sine residue re) imallved in ATP binding ;Ire indic~ged.
dimerizatinn site, the autoinhibitory site including the autophosphorylaled amino acid residues and a hinge region, which connects the amino-terminus with the two in-tandem cGMP binding domains B and C followed by the catalytic domain D containing the MgATP and pcptide-bioding sites. It is not known whether the actual structure follows this sebeme, but the division of the protein into separate functional domains piovides a convenient model to h~ndie its complexity.
HI-B. Am#lo-tennhtal domabJ The two isoforms of the eGMP kinase, termed l a and I/3, differ only with respect to their amlno-termini [16,181. Both isoform:~ have a leueine/isoleucinc-zipper motif within the first 40 amino acids [24,251. IH-NMR studies of a synthetic peptidc composed of aa-l-39 of the 1~ isozyme shows that the pcptide adopts an or-helical structure to form a homodimer with a parallel arrangement ~f both peptide sequences in solution [24]. The dimerization of the subunits ,,ia the hydrophobie leucine/isoleucine-zipper motif is likely. This motif is not present in the homologous region of the regulatory subunits of cAMP kinase and seems to be, therefore, unique for cGMP kinase. The antino-terminus plays further an important role in the regulation of the catalytic activity of cGMP kinnse. Tryptic cleavage at Arg-77 of the l a isozyme removes the dimerization site, the autoinhibitory region and most of the hinge region and produces a constitutively active monomeric 65-kDa cGMP kinase with intact eGMP-binding domains [26]. In contrast, endogenous proteolysis of the I/3 isoz_Yme at Lye61 (this corresponds to a clip before Gin-43 of the l a isozyme) produces a monomerie 7O-kDa fragment which is still activated by cGMP [21]. Both obset-¢ations imply that the catalytic centre of each subunit is under the control of the amino-terminus of the same snbunit and that the autoinhibitory sequence should be between Gin-43 and Arg-77. The sequence between Pro-55 and Pro-67 of the l a isozyme contains the major atttopho~phorylatod amino acid at Thr-58 [27]. Phospho~lation of Thr-58 is slow in the absence and presence of cGMP, i.e., in the non-activatod and completely activated enzyme, but proceeds rapidly in an enzyme partially activated by cAMP [28,29]. It is likely that partial activation allows dissociation of the inhibitow sequence frnm the catab~tic centre and its replacement by the sequences around the autophospho~lated amino acids Set-50, Thr-58, Set-72 aml Thr-84. The sequence S'~R-A-Q-G-I-S-A-E-P "r in the lt~ i.sozs~e - respectively 74K-R-Q-A-I-S-A-E-ps: in the 1/] isozyme which is positioned between the autophosphorylatcd residues, contains the basic motif of a kinase autoinhibitory sequence [30]. This sequence is conserved between the In. the I/~ isozyme and the Drosophila
54 cGMP kinase genes D G I and DG2--fL (Fig. 1). It is therefore l~ely that this sequence ocLaupies the peI~ tide-binding site ef the enzyme in the intact but not the activated, enzyme. However, additional residues are needed for b2gh affinity binding, since the corresponding peptides of the I a and IB isoz,ylne are poor inh~itars of cGMP kinase [30]. An important fea~.ure of the different amino-terminal sequences of both isozymes is their ability to affect the concentn~t~on of c(iMP needed to activate the kinase [17]. The l a and I ~ isozyme have K A values for cGMP of 0A and 1.2 v-M, respectively. This dramatic shift of the K A is caused by a 10-fold decrease of the affinity of cGMP binding site B [domain C). The Io~er affinity is caused by a decrease in the cooperativity between binding sites A and B (domains B and C) [17]. III-C. cGMP-birulb,.g domains The present knowledge about the structure of the two homologcous cGMP-binding domains is basically derived from kinetic studies [31-33], analogue mapping [34-36] and from a model based on the 2.5 A crystal structure of the catabolite gene activator protein (CAP) from E. colt [37.38]. This protein contains a single cAMP-binding site which is approx. 20% homologous to the cyelic-nuc|eotide-binding sites of the cAMP and cGMP kinase [l 1]. The basic features of cAMP binding to the CAP receptor involve a glutamic acid resiclue that binds to the 2'OH-group of the ribose moiety via a hydrogen bond and an arginine that chelates the phosphate diester via a salt bridge [37]_ Both residues are conserved in all cAMP- and cGMP-binding si~es of cAMP kiuase regulatory subonits and cGMP kinases [38]. In cGMP kJnase l a they correspond to G3m-167 and Arg-176 in site A and Gin-291 and Arg-300 in site B.
Compari,gon of the sequence of the eGMP kinase I~ with that of the type 1~8 regulatory subun~ts of cAMP [dlla~,e suggests that the cGMP-binding site A {also termed low-affinity binding site, rapidly exchanging site, site 2) and B (high-affinity, sinw!y exchanging site, site 1) are located ~ithin amino acids 111-227 (domain B) and amino acids 228-343 (domain C), respectively [11,38]. In contrast to the cAMP binding sites, the cGMP-binding sites do not tolerate large substituents at the 2-NH_. position or at the 6-keto function of the guanine base. cGMP kinase requires an unmodified 2-NH 2 group of cGMP for tight binding [35]. Four analogues of cGMP in vchich the 2-NH2 position was replaced with various substituents showed over 100-fold lower affimty for either of the two binding sites [35]. A model of the cGMP-binding site A based on the cAMP-binding site of the CAP protein [38] suggests that the 2-NH 2 group forms a hydrogen bond with Thr-177 and Thr-301 of domains B and C o r cGMP
kinase, respectively. Comparison of the cGMP-binding sittm with the cAMP-binding sites shows that all cAMP-binding sites contain an Ala instead of a Thr at this position. Mutation of Pda-334 to a T h r in binding site B of the regulatory subonit l a of cAMP ldnase increased the affinity of this site For cGMP over lVgb fold [39]. A double mutation of Ala-210 in binding site A and Ala-334 in binding site B of the regulatory subuni! decreased the K x of the mutated cAMP kinasc for cGMP do-fold [dO[. Mutation of the Thr-560 or Thr-537 of the cGMP binding site of rod or olfactory t3clic noeleotidc-gatcd channels t9 an Ala decreases the sensitivity of these channels for cGMP do-fold without affecting grcady their sensitivity to cAMP [41]. These mutations support the idea that Thr-tT"/ and Thr-301 of cGMP kinase are important for high affiniry and specific binding of cGMP. Analogue studies [urther show that binding sites A and B tolerate different substitutions. Several charged amino acids including Gin-270, Asp-271, Arg-281, Asp287, Asp-337, Lys-341 are present in the vicinity of the cGMP binding pocket B, whereas binding site A is more hydrophobic. This difference in hydrophohicity of the two cGMP binding sites is expected to be responsible for the altered affinities for cGMP analogues. Substitutions at C-8 of the guanine b,xse, which include charged residues, are well tolerated by the binding site B and usually increase the affinity for this site [35,36]. The increased affinity may be caused by the formation of salt bridges. III-D. Catalyiic domain The catalytic domain of cGMP ki~ase is highly conserved from mammal to fly. All cysteine residues starting with Cys-312 are present in all cloned cGMP kinases, suggesting an essential role for these residues. The cGMP kinase is the closest homologue to cAMP kinase. In particular, the catalytic domains of both enzyrnec show a remarkable sequence homology of approx. 70% [11]. In general, residues thought to be essential for MgATP binding ( ~ G V C s G F G ~Tj) and catalytic function (around Thr-516) are invariant, whereas residues thought to be important for oeptidc binding (4~RDLKPENLam a n d / o r S 4 6 L M Y . . . DMI TM)differ be~veen the two enzymes [42]. With the availabgity oi" the zrystal structure of the catalytic subunit of cAMP kina.ge [43,44) it has become feasible to correlate :kis sequence conservation with both fupetinn and struct,nre. Residues 40-350 of the mammalian cAMP catalytic subunit were aligned using a homologyprogramme with residues 356-670 of the I,~ cGMP kinasc (Dostmann, W . Knighton. D.IL, Taylor, $.5. and Sowadski, J., unpublished data). The N-terminal residt!~ ,el cAMP kinase bear no similarity to residues 306-3.'4 of cGMP kinase and were not included in the
55 t5
4o
=
2SS
~ao
Fig. 2. Residues 40-350 of the mammalian cAMP kina~c catalYtiC suhunit (cA-PK} a~c aligned ~itk residues 356-670 of the bovine lung t~ac la ,=GMpk i ~ (cG-PK). The N-terminal residues of the cAMP klnase show no similargt]; to residues 306-354 of cGMP kinase and are nm included in the alignment, ttlSett5 and gups arc marked m boxed sequences Box~ 1-3 indi=te an imerbon of one tv,o residues, whereas box 4 denotes a ~ingle amino acid deletion.
alignment. Three small inserts and one gap were identified (Fig. 2). T h e t h r e c M i m e n s i o n a l m o d e l o f c G M P k i n a s e catalytic d o m a i n b a s e d o n t h e X-ray c o o r d i n a t e s o f c A M p k i n a ~ ~ t a l y t [ e s u b u n i t s hows t h a t all inserts a n d t h e g a p a r e l ocat ed at t h e s ur f a c e o f t h e m o l e c u l e a n d a r e e i t h e r p a r t o f a loop o r / ~ - t u r n s (Fig. 3). T h e m o d e l shows t h a t t h e catalytic d o m a i n s o f c A M P kin o s e a n d c G M P kipas¢ a r c s t r u c t u r a l l y rots' similar. All r es id u es t h o u g h t t o be critical for p h o s p h o r y l t r a n s f e r ar e invariant. O n l y t h~ gl~xine rich loop s t a r t i n g at Gly-365 c o n t a i n s o n e e x t r a O l y f o r a Ser in c G M P
Fig. 3. Ribbon dra~vingof the cGMP klnase catal~.lic dommn raodal based on the X-ray coordinales of the cAMP kinas¢ catalytic sufiunit [43. 44]. ['-our inserts and gaps (see Fig. 2) are shmvn in white: they are l~med at [he surface of the molecule at~d are either purl of kw,p rcgion~ or i~-turns. The specific inl)ibito~-of cAMP kinase. PKI4aaS24). is marked in yellow, the [o~er lobe of the pr:ltein in blue and me upr~er lobe in red, The N-terminal part i~ associated wilh the upper lobe and the C-t~rminal part with Ihe [nwer lobe. The modal ~va~ subjected to a molecular dynamics simulation and minimiTed by steegns! de~ent.
kinase, t h u s c h a n g i n g t h e steric e n v i r o n m e n t to accomr a o d a t e a the'canine resi due r a t h e r t h a n a serine as p h o s p h a t e - a c c e p t i n g residue. T h e r e q u i r e m e n t s for hi gh-affi ni t y p e p t i d e b i n d i n g ha~e n o t y e t b e e n resolved. T h e crystal s t r u c t u r e o f t h e c A M P k in a se catalytic s u b u n i t was o b t a i n e d f r o m a crystal c o n t a i n i n g
TABLE II SeqUe/IC * o f :an~e io rltro od~stmte a,M b I bitoo f.3~tide~ f a r cGMP kit as'
(X) rcfer~ to 2-amin~buryrie acid attd (-) represents an identical amino acid residue. Origin
sequence
[S21] PKI2([4 22) pKI4~I4 -22) PK1414-24)
~RTGRRN$1a~Oe
1-12B429-35) [A34] H2B~29-35 ) r a i l H2B-(2q-35) [Sb=] cGk ]a455-63) cGK I aq53-f13) IX ~s]cGK l a453-:.3) [Ab2] cGK Ia455-67~ I~tuvate kinas~ P ~ v a t e kinase
. . . . .......
KM( , a M ) 0.45~: 0.12
~-a~ide
A-H0
RKRSRKE
. . . . . st---A--PRTTRAqSIemido
IG. . . . . . . 6..... X---6. . . . . . . A-SAEP LRRASLG .... g--
LandgraL W. and Hofmann. F.. u npublished dal~.
Ki (ttM) 9.2 =[:1.9 15
21 28
Vm~~ [ p m o l / m i n per me)
51 4.4 2o.{;
~6 +5 55 578
± 11 _+25
2.2+_0.2 0.07 700 2100
130
Reference
ILO±0.3
47 47 49 50 50
30 4.5
8~
40
56 the enzyme and the heat stable inhibitor peptide fragment (aa5-24) at the peptide-binding site [43,44]. The binding of the inhibitor peptide induces a conformational change of the cAMP kinase catalytic subunit [45]. This iuh~itor peptidc does not bind with high affinity to eGMP ~Jnase, presumably because it can not induce the same conformatinnal change as in the cAMP kinas¢. Thus it is likely that the peptide binding site of cGMP kinase differs from that of the cAMP kinase.
III-E. Peptide reco~ition The primary sequence requirement of the catalytic site of cGMP kinase follows the rules established for cAMP kinase [46]. The phosphate accepting serine or threonin¢ is preceded by two or three positively charged amino acids (Table II). A positively charged amino acid fo]lowing directly the phosphate-accepting amino acid decreases the affinity for cAMP kinase but is tolerated without change in K m by cGMP kinase [47]
51
~9 1992 Ebeviet Science Publishers B.V. All rights re,erred Ulh7-4889/~2/$05.00
BBAMCR 1315u Minireview
Structure
and
physiological
role
p~otein Franz
Hofmann,
Wolfgang
of
cGMP-dependent
kinase
Dostmann,
Alexandra
and Peter
Keilbach,
Wotfgnt,g Laea_grnf
Ruth
Italil~et ]~r Phu~takof~lgiv mul T, JLi~,,hlgic ,h'r Tc~ Ilni~ h('a Uait',,rsitilt Miindl:'tL Miipwltelt ((;ermally)
( Rgcci~ed ~ Januao' 1~92)
Key~ords: Pr~l¢inkinase:~'clcGMP;En~ymesruc,,re;E~zyme e*~ua m S u l s r a e p osplorylatlan
Contents I.
Imtoducllon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. Tgpes and g e n ~ o f ¢ ( ; M P kin~se
51
...............................................
5]
]u. Slructu,-e at cGMPMcpendent protein kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Overall struclure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Amina-termia-d cktrnain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (7. e{;M P~inding d c ~ i n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Catab'lic detain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Peptid¢ re.oogniUon. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Regular km of edlalyUc act rzily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
53 53 53 54 54 56 56
IV. Ph~iMogitml tu rcli~ms of cG MP kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A~ In vi~ dLslribution of cGMP kina~ im~,:ym~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. lNitential phs~kdugi~dl functions uf c~MP k i n ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
56 56 57
Ackno~ledgemen I
58
Referents
.
.
.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1. I n t r o d u c t i o n Cyclic G M P is a key regulatory molecule of specific cellular functions such as t h e visual transducfion process in r o d o u t e r s e g m e n t , i n t e g r a t i o n o f neuro-excitability to e x c i t a t o ~ n e u r o t r a n s m i t t e r s , relaxation o f
Abbreviations: cGMP k m ~ , OGMP~lel~2ndgnl prolei.~ kina~¢: cAMP k i n ~ . cAMPMe.l~endenl protein kin~e; PKL protein kioase inhibitor Ixptide: CHO. Chines hamster o~-arial: KA. activaUon conSlant; mL amino acid. Correspondence: Franz IIofmann. In~titut mr Pbarmakolo~ie und Tuxikntngie, TO M0nt:hea, Biedersleiner Stra~se 29, D-~000 Mfinchen 40, G~nnany.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
58
s m o o t h muscle, intestinal secretion o f w a t e r a n d salt, a n d reabsocption o f sodium a n d w a t e r in t h e distal tubulus o f the n e p h r o n . O v e r 0 r e d u c t i o n o f c G M P in specific cells h a s been associated with n e u r o n a l d e g e n eration, c n d o t o x i n shock, diarrhoea, a n d loss o f s o d i u m a n d water. This diversity o f t h e cellular effects of c G M P are n o t m e d i a t e d by a unique protein b u t by several distinct receptors: a e G M P - g a t e d cation c h a n nel, e G M P - s t i m u l a t e d a n d i n h i b i t e d p h o s p h o d i esterases a n d c G M P - d e p e n d e n t protein kinases ( c G M P kinasc). This article focuses o n r e c e n t d e v e l o p m e n t s in o u r u n d e r s t a n d i n g o f the structure a n d function o f c G M P kinase. T h e o l d e r results o n e G M P kinase a n d related areas have b e e n s u m m a r i z e d excellently by Lincoln a n d C o r b i n [1], L i n c o l n [2] a n d W a l t e r [3].
52
TABLE I S ~ r y of utent~fwdoGMP k u m ~ n.r., ~ t ~poffed: ~ KA, ~ivaz~m ~ s t a n l for CGMP in a protein P~n~ assa~_ S~cies
TL~SUC
Fu,aty
Molecular m~.~ (~D~)
pmc Wrti~l partial Im~tiai partial pu~e
naike (kDa) 8~ l~l /,2 7~ I¢-tn 162
P~c~*n Tetrahy~ J:_-~_~m Silk ~ o ~ Silk ~otm ~t~r/~ swan
~3~a
Bm~ne Bovine ~tvilt~ Bovir~ Pat
I~ng I~mt aorta aorta intestine,
pu~ pine pule rmre pure
I~ IB 11
Bt~ine Bovine Human Dr~oMd/a Drosot~dta [kos~li[a
trachea trachea p~acea~a
cloned eapte~ed cloned cloned cloned cloud
1o I~ I~ DGI OG2-TI DG 2-T2
c$g~ pufr~e
I1. Types a n d genes e f ¢ G M P k i n a s e c G M P kinase is p.resent in a variety o f eukaryntes r a n g i n g f r o m t h e unicellular o r g a n i s m Paramecium to h u m a n s [4-19] (Table I). M a m m a l s express in most tissues a h o m o d i m e r i c e n z y m e (type !) with a native m o l e c u l a r mass o f 153 to 156 k D a a n d in the intestinal brush b ~ d e r a m o n o m e r i c e n z y m e (type I I ) w i t h a native m o l e c u l a r mas~ o r 86 kDa. T h e h u m a n type 1 g a n e is localized to t h e p l L 2 - q l l . 2 region o f c h r o m o s o m e 10 [20]. T w o c G M P kina.~ g e n e s ( D G I a n d D G 2 ) a n d several splice variant~ have b e e n cloned f r o m Drosophila melanogaster [19]. T h e c G M P - b i n d i n g d o m a i n s a n d t h e catalytic d o m a i n as derived f r o m the d e d u c e d a m i ~ o acid sequence of these g e n e s are very h o m o l o g o u s to t h e m a m m a l i a n ry~e 1 enzyme. T h e structure t,f th~ native proteins is n o t k n o w n since c G M P kinase b~s n o t b e e n purified f r o m Drosophila. T h e e n z y m e s purified f r o m Paramecium [4], TeIrahymena [5] a n d Dico'ostdium discoideum [6] are a p p m e n t l y m o n o m e r i c c o n t a i n i n g a single polypcptide chain. T h e classification o f these e n z y m e s is unclear. It is even possible t h a t they are proteolytic products of a dimcric e n z y m e , A m o n o m e r i c c G M P activatable type I c G M P kinase h a s b e e n purified f r o m bovine aorta [21]. This e n z y m e was a proteolytic p r o d u c t o f the h o m e d i m e r i c isozyme I/~ as s h o w n by p e p t i d e sequencing [14] a n d cloning [16]. T h e r e f o r e this suggests t h a t t h e .type I c G M P kinases a r e d i m e r i c e n z y m e s w h e r e a s t h e type II e n z y m e m a y be monorneric. T h e occurrence o f m o n o m e r i e c G M P [4-6] a n d c A M P [22] kinases in
d.~n~tured
Subunit number
77
re.homer n~nomer ~mcr ~ e r
K a ~
Rcfctcncc
(riM)
i£~3 1 18
4 5 6 7
82
dimer
18
9
150 150 178 170 86
74 82 78 80 85
dlrm:r dimer dimer di~r monomer
40 ~ 2e~0 4~} n.r.
10.il 12 13,14 13,14 15
153 156
76,118 77803 778o3 > 7a(~0 > 7b(~} > 7b t]~ffl
di~ dimer
I[lfl 12£0
16,17 ~6,17 18 19 19 19
-
unicellular organi~Tns supports t h e idea t h a t dimerization o f t h e kinases occurred later in evolution. S e q u e n c e c o m p a r i s o n s inificatc t h a t t h e c G M P kina.¢,e g e n e evolved ns t h e result of a fu.~ion l',etween g e n e s f o r cyclic n u d c o t i d c b i n d i n g a n d catalytic protcins [I 1]. T h i s fusion occurred early in evolution, since the c G M P - k i n a s c s f r o m simple o r g a n i s m s have cyclic nueleotide b i n d i n g a n d catalytic activities o n t h e same polypeptide chain. A n i n t e r n a l g a n e duplication prod u c i n g t h e t w o c G M P b i n d i n g sites o n e a c h subunit o f t h e m a m m a l i a n e G M P - k i n n s e runs1 also have occurred very early in es'oinfiou, since t w o c-GMP-bioding sites a r e p r e s e n t in c G M P kinnses o f lower organisms. C l o n i n g a n d purification o f the m a m m a l i a n s m o o t h mubcle c G M P kinase led to t h e identification o f t w o isoforms o f t h e type I e n z y m e [13,14,16]. designated l a a n d lfi_ T h e d e d u c e d a m i n o acid sequence o f the l a isoz~me is identical to t h a t r e p o r t e d for c G M P kinase purified f r o m bovine l u n g [11]. T h e c D N A clone f o r ti,2 I f l isozym¢ differs f r o m isozyme la only at t h e N - t e r m i n u s ( a m i n o acids 1 - 1 0 4 "1, a n d contains t h e sequence of a p c p t i d e o b t a i n e d f r o m t h e I/~ isozyme purified f r o m aorid [21]. T h e sequence o f isozyme I ~ f r o m bovine trachea was c o n f i r m e d by t h e cloning o f c G M P kinasc f r o m h u m a n p l a c e n t a [18]. T h e h u m a n
* Amino acid residtle~ for cGMP kinases are numi~red by" ignoring the a m i ~ t c ~ i n a l ~ t h l o n i ~ ~n~-pnnding to the st,an ~don in eDNA sequences, Thib i~ in aO~eldant~ ~ilh protElo s~qucncing data lit].
53 placental enzyme contains only two amino acids exchanges at positions 279 and 289 when compared to the bm4ne lung and tracheal lt~ and 1~0 enzymes replacing a lysine by a threonine and an asparagine by a serine. The bovine tracheal and human placental I/~ sequences suggest that the l~ and !fl isozymcs contain identical cGMP-binding sites and catalytic centres. The aminntermini of the cloned isozymes l a and I/~ are different and show u I~v degree of similarity to each other and to the corre~po,ding parts of the regulatory ,[.hzsnil~, of cAMP kinase. The highest degree of similarity is found in the region which contains at Thr-58 the major autophosphm3,1ation site of cGMP-kinase In. The l a and I~ form of eGMP-kinase may be derived from the same gene by alternative splicing as suggested by alignment of the l a and 1~ seque.ccs to Drosophila gene DG2-TI product which exhibits an exon/intron junction at the DNA position corresponding to amino acid 89 and 104 of the I~ and I~ cGMP-kinase, respectively [16,18.19,23]. The studies in Drosophila suggest that from the two genes DGI and 13132 at least six different mRNA are transcribed differing mainly in their amino terminal region [19]. IlL Structure d cGMP-dependen! protein kinase
HI-A. Ocerall ~tn~c'lffre cGMP kinase a~ purified from bovine lung is a 153-kDa homodimer c~ntaining two identical subunits of 76 418 Da. The primal" sequence of the subunits has been divided into several functional domains [11] (Fig_ 1). The amino-terminal domain A contains the
D G ~ 67%,~ 1
em
~
~5
~
mo~_
75%~,59%] m~r
pwrrmLcml~x
~
IWWCG'CPC~V
P ~ t | KItQGV SIIES- C
LG|GG~G
~
¢mo.lt~tG. ~
~
V
"~¢rr-cGrv~n/
xw Fig- I. The sequence of bo~qnelungt'GMpkinase(BI,) is almpared ~ith Ihe deduced aminoacidsequences ut the cloned bovine tracheal c(~MPkin~se I~(nll~) aM Ihe rn~itn Droxvlgulugene products DGI and DG2-TI ~DG2hPer~mage ~ff idenn~l amint~acids within the hnrnoh~glmsdl~ins are mdl~lcd and the sequullc¢>of Ctlil~lvcd sil~ ale aligned.The inv~dant th~eoniner~ducs ~ilhin Ihe ¢GMP binding I~chets 1~) and the b'sine residue re) imallved in ATP binding ;Ire indic~ged.
dimerizatinn site, the autoinhibitory site including the autophosphorylaled amino acid residues and a hinge region, which connects the amino-terminus with the two in-tandem cGMP binding domains B and C followed by the catalytic domain D containing the MgATP and pcptide-bioding sites. It is not known whether the actual structure follows this sebeme, but the division of the protein into separate functional domains piovides a convenient model to h~ndie its complexity.
HI-B. Am#lo-tennhtal domabJ The two isoforms of the eGMP kinase, termed l a and I/3, differ only with respect to their amlno-termini [16,181. Both isoform:~ have a leueine/isoleucinc-zipper motif within the first 40 amino acids [24,251. IH-NMR studies of a synthetic peptidc composed of aa-l-39 of the 1~ isozyme shows that the pcptide adopts an or-helical structure to form a homodimer with a parallel arrangement ~f both peptide sequences in solution [24]. The dimerization of the subunits ,,ia the hydrophobie leucine/isoleucine-zipper motif is likely. This motif is not present in the homologous region of the regulatory subunits of cAMP kinase and seems to be, therefore, unique for cGMP kinase. The antino-terminus plays further an important role in the regulation of the catalytic activity of cGMP kinnse. Tryptic cleavage at Arg-77 of the l a isozyme removes the dimerization site, the autoinhibitory region and most of the hinge region and produces a constitutively active monomeric 65-kDa cGMP kinase with intact eGMP-binding domains [26]. In contrast, endogenous proteolysis of the I/3 isoz_Yme at Lye61 (this corresponds to a clip before Gin-43 of the l a isozyme) produces a monomerie 7O-kDa fragment which is still activated by cGMP [21]. Both obset-¢ations imply that the catalytic centre of each subunit is under the control of the amino-terminus of the same snbunit and that the autoinhibitory sequence should be between Gin-43 and Arg-77. The sequence between Pro-55 and Pro-67 of the l a isozyme contains the major atttopho~phorylatod amino acid at Thr-58 [27]. Phospho~lation of Thr-58 is slow in the absence and presence of cGMP, i.e., in the non-activatod and completely activated enzyme, but proceeds rapidly in an enzyme partially activated by cAMP [28,29]. It is likely that partial activation allows dissociation of the inhibitow sequence frnm the catab~tic centre and its replacement by the sequences around the autophospho~lated amino acids Set-50, Thr-58, Set-72 aml Thr-84. The sequence S'~R-A-Q-G-I-S-A-E-P "r in the lt~ i.sozs~e - respectively 74K-R-Q-A-I-S-A-E-ps: in the 1/] isozyme which is positioned between the autophosphorylatcd residues, contains the basic motif of a kinase autoinhibitory sequence [30]. This sequence is conserved between the In. the I/~ isozyme and the Drosophila
54 cGMP kinase genes D G I and DG2--fL (Fig. 1). It is therefore l~ely that this sequence ocLaupies the peI~ tide-binding site ef the enzyme in the intact but not the activated, enzyme. However, additional residues are needed for b2gh affinity binding, since the corresponding peptides of the I a and IB isoz,ylne are poor inh~itars of cGMP kinase [30]. An important fea~.ure of the different amino-terminal sequences of both isozymes is their ability to affect the concentn~t~on of c(iMP needed to activate the kinase [17]. The l a and I ~ isozyme have K A values for cGMP of 0A and 1.2 v-M, respectively. This dramatic shift of the K A is caused by a 10-fold decrease of the affinity of cGMP binding site B [domain C). The Io~er affinity is caused by a decrease in the cooperativity between binding sites A and B (domains B and C) [17]. III-C. cGMP-birulb,.g domains The present knowledge about the structure of the two homologcous cGMP-binding domains is basically derived from kinetic studies [31-33], analogue mapping [34-36] and from a model based on the 2.5 A crystal structure of the catabolite gene activator protein (CAP) from E. colt [37.38]. This protein contains a single cAMP-binding site which is approx. 20% homologous to the cyelic-nuc|eotide-binding sites of the cAMP and cGMP kinase [l 1]. The basic features of cAMP binding to the CAP receptor involve a glutamic acid resiclue that binds to the 2'OH-group of the ribose moiety via a hydrogen bond and an arginine that chelates the phosphate diester via a salt bridge [37]_ Both residues are conserved in all cAMP- and cGMP-binding si~es of cAMP kiuase regulatory subonits and cGMP kinases [38]. In cGMP kJnase l a they correspond to G3m-167 and Arg-176 in site A and Gin-291 and Arg-300 in site B.
Compari,gon of the sequence of the eGMP kinase I~ with that of the type 1~8 regulatory subun~ts of cAMP [dlla~,e suggests that the cGMP-binding site A {also termed low-affinity binding site, rapidly exchanging site, site 2) and B (high-affinity, sinw!y exchanging site, site 1) are located ~ithin amino acids 111-227 (domain B) and amino acids 228-343 (domain C), respectively [11,38]. In contrast to the cAMP binding sites, the cGMP-binding sites do not tolerate large substituents at the 2-NH_. position or at the 6-keto function of the guanine base. cGMP kinase requires an unmodified 2-NH 2 group of cGMP for tight binding [35]. Four analogues of cGMP in vchich the 2-NH2 position was replaced with various substituents showed over 100-fold lower affimty for either of the two binding sites [35]. A model of the cGMP-binding site A based on the cAMP-binding site of the CAP protein [38] suggests that the 2-NH 2 group forms a hydrogen bond with Thr-177 and Thr-301 of domains B and C o r cGMP
kinase, respectively. Comparison of the cGMP-binding sittm with the cAMP-binding sites shows that all cAMP-binding sites contain an Ala instead of a Thr at this position. Mutation of Pda-334 to a T h r in binding site B of the regulatory subonit l a of cAMP ldnase increased the affinity of this site For cGMP over lVgb fold [39]. A double mutation of Ala-210 in binding site A and Ala-334 in binding site B of the regulatory subuni! decreased the K x of the mutated cAMP kinasc for cGMP do-fold [dO[. Mutation of the Thr-560 or Thr-537 of the cGMP binding site of rod or olfactory t3clic noeleotidc-gatcd channels t9 an Ala decreases the sensitivity of these channels for cGMP do-fold without affecting grcady their sensitivity to cAMP [41]. These mutations support the idea that Thr-tT"/ and Thr-301 of cGMP kinase are important for high affiniry and specific binding of cGMP. Analogue studies [urther show that binding sites A and B tolerate different substitutions. Several charged amino acids including Gin-270, Asp-271, Arg-281, Asp287, Asp-337, Lys-341 are present in the vicinity of the cGMP binding pocket B, whereas binding site A is more hydrophobic. This difference in hydrophohicity of the two cGMP binding sites is expected to be responsible for the altered affinities for cGMP analogues. Substitutions at C-8 of the guanine b,xse, which include charged residues, are well tolerated by the binding site B and usually increase the affinity for this site [35,36]. The increased affinity may be caused by the formation of salt bridges. III-D. Catalyiic domain The catalytic domain of cGMP ki~ase is highly conserved from mammal to fly. All cysteine residues starting with Cys-312 are present in all cloned cGMP kinases, suggesting an essential role for these residues. The cGMP kinase is the closest homologue to cAMP kinase. In particular, the catalytic domains of both enzyrnec show a remarkable sequence homology of approx. 70% [11]. In general, residues thought to be essential for MgATP binding ( ~ G V C s G F G ~Tj) and catalytic function (around Thr-516) are invariant, whereas residues thought to be important for oeptidc binding (4~RDLKPENLam a n d / o r S 4 6 L M Y . . . DMI TM)differ be~veen the two enzymes [42]. With the availabgity oi" the zrystal structure of the catalytic subunit of cAMP kina.ge [43,44) it has become feasible to correlate :kis sequence conservation with both fupetinn and struct,nre. Residues 40-350 of the mammalian cAMP catalytic subunit were aligned using a homologyprogramme with residues 356-670 of the I,~ cGMP kinasc (Dostmann, W . Knighton. D.IL, Taylor, $.5. and Sowadski, J., unpublished data). The N-terminal residt!~ ,el cAMP kinase bear no similarity to residues 306-3.'4 of cGMP kinase and were not included in the
55 t5
4o
=
2SS
~ao
Fig. 2. Residues 40-350 of the mammalian cAMP kina~c catalYtiC suhunit (cA-PK} a~c aligned ~itk residues 356-670 of the bovine lung t~ac la ,=GMpk i ~ (cG-PK). The N-terminal residues of the cAMP klnase show no similargt]; to residues 306-354 of cGMP kinase and are nm included in the alignment, ttlSett5 and gups arc marked m boxed sequences Box~ 1-3 indi=te an imerbon of one tv,o residues, whereas box 4 denotes a ~ingle amino acid deletion.
alignment. Three small inserts and one gap were identified (Fig. 2). T h e t h r e c M i m e n s i o n a l m o d e l o f c G M P k i n a s e catalytic d o m a i n b a s e d o n t h e X-ray c o o r d i n a t e s o f c A M p k i n a ~ ~ t a l y t [ e s u b u n i t s hows t h a t all inserts a n d t h e g a p a r e l ocat ed at t h e s ur f a c e o f t h e m o l e c u l e a n d a r e e i t h e r p a r t o f a loop o r / ~ - t u r n s (Fig. 3). T h e m o d e l shows t h a t t h e catalytic d o m a i n s o f c A M P kin o s e a n d c G M P kipas¢ a r c s t r u c t u r a l l y rots' similar. All r es id u es t h o u g h t t o be critical for p h o s p h o r y l t r a n s f e r ar e invariant. O n l y t h~ gl~xine rich loop s t a r t i n g at Gly-365 c o n t a i n s o n e e x t r a O l y f o r a Ser in c G M P
Fig. 3. Ribbon dra~vingof the cGMP klnase catal~.lic dommn raodal based on the X-ray coordinales of the cAMP kinas¢ catalytic sufiunit [43. 44]. ['-our inserts and gaps (see Fig. 2) are shmvn in white: they are l~med at [he surface of the molecule at~d are either purl of kw,p rcgion~ or i~-turns. The specific inl)ibito~-of cAMP kinase. PKI4aaS24). is marked in yellow, the [o~er lobe of the pr:ltein in blue and me upr~er lobe in red, The N-terminal part i~ associated wilh the upper lobe and the C-t~rminal part with Ihe [nwer lobe. The modal ~va~ subjected to a molecular dynamics simulation and minimiTed by steegns! de~ent.
kinase, t h u s c h a n g i n g t h e steric e n v i r o n m e n t to accomr a o d a t e a the'canine resi due r a t h e r t h a n a serine as p h o s p h a t e - a c c e p t i n g residue. T h e r e q u i r e m e n t s for hi gh-affi ni t y p e p t i d e b i n d i n g ha~e n o t y e t b e e n resolved. T h e crystal s t r u c t u r e o f t h e c A M P k in a se catalytic s u b u n i t was o b t a i n e d f r o m a crystal c o n t a i n i n g
TABLE II SeqUe/IC * o f :an~e io rltro od~stmte a,M b I bitoo f.3~tide~ f a r cGMP kit as'
(X) rcfer~ to 2-amin~buryrie acid attd (-) represents an identical amino acid residue. Origin
sequence
[S21] PKI2([4 22) pKI4~I4 -22) PK1414-24)
~RTGRRN$1a~Oe
1-12B429-35) [A34] H2B~29-35 ) r a i l H2B-(2q-35) [Sb=] cGk ]a455-63) cGK I aq53-f13) IX ~s]cGK l a453-:.3) [Ab2] cGK Ia455-67~ I~tuvate kinas~ P ~ v a t e kinase
. . . . .......
KM( , a M ) 0.45~: 0.12
~-a~ide
A-H0
RKRSRKE
. . . . . st---A--PRTTRAqSIemido
IG. . . . . . . 6..... X---6. . . . . . . A-SAEP LRRASLG .... g--
LandgraL W. and Hofmann. F.. u npublished dal~.
Ki (ttM) 9.2 =[:1.9 15
21 28
Vm~~ [ p m o l / m i n per me)
51 4.4 2o.{;
~6 +5 55 578
± 11 _+25
2.2+_0.2 0.07 700 2100
130
Reference
ILO±0.3
47 47 49 50 50
30 4.5
8~
40
56 the enzyme and the heat stable inhibitor peptide fragment (aa5-24) at the peptide-binding site [43,44]. The binding of the inhibitor peptide induces a conformational change of the cAMP kinase catalytic subunit [45]. This iuh~itor peptidc does not bind with high affinity to eGMP ~Jnase, presumably because it can not induce the same conformatinnal change as in the cAMP kinas¢. Thus it is likely that the peptide binding site of cGMP kinase differs from that of the cAMP kinase.
III-E. Peptide reco~ition The primary sequence requirement of the catalytic site of cGMP kinase follows the rules established for cAMP kinase [46]. The phosphate accepting serine or threonin¢ is preceded by two or three positively charged amino acids (Table II). A positively charged amino acid fo]lowing directly the phosphate-accepting amino acid decreases the affinity for cAMP kinase but is tolerated without change in K m by cGMP kinase [47]
