GAF-Tandem Signal Transduktion in Chimeren mit der CyaB1 Adenylat Cyclase als Reporter

The human cGMP-stimulated PDE2 has a long proline-rich N-terminus while the hPDE5 had a glutamine-rich hydrophilic N-terminus ahead of the respective tandem GAF. The swapping of any of them in front of hPDE2/CyaB1 chimera had inhibitory effect. In rPDE2/CyaB1 chimera, CyaB1 N-terminus had a regulatory effect on the enzyme by either reducing the binding of cGMP to the GAF domain or reducing the signal transduction from the GAF domain to the catalytic domain. The CyaB2 GAF linker was important with regard to the length and its amino acid sequence. Shortening the length of the connecting helix reduced signalling represented by fold stimulation and increased the cAMP-EC50. The difference in the fold stimulation between the different mutants can be due to a shift in the dimerization or the differences in the exposed and embedded amino acids in the helix which affect dimerization. In the constructs with three inserted aa, the effect on fold stimulation and cAMP-EC50 was less dramatic but the mutant with 4 aa inserted was unstable which indicates the importance of this linker not only for the proper signalling but also for the stability of this domain. The cooperativity was lost in all deletion and insertion mutants. The loss of the cooperativity may be due to the interruption of signalling between GAFa and GAFb motifs. Mutations of the Met/Leu couple in the alpha1-helix or the two methionines in the connecting helix partially disrupted dimerization leading to dramatic reduction in the fold stimulation, decrease in the affinity toward cAMP and reduction in the Hill coefficient for most constructs. A quadruple mutation in the four amino acids reduced the fold stimulation more than 100 times, in addition to the need of high concentrations of protein to achieve steady state. Unlike CyaB2 GAF alpha-helices, the mutation in any of the helices preceding the GAF motifs in hPDE5 increased the basal activity and the cGMP-affinity. The quadruple mutation in leucines of both helices led to complete loss of regulation and increase in the basal activity. Mutations of the alpha1-helix to serines in PDE2 GAFs increased the fold stimulation without affecting cGMP affinity while the mutations in the connecting helix caused little reduction in the fold-stimulation with an 28-fold increase in the cGMP affinity. The quadruple mutation in both helices and the truncation of the N-terminus showed loss of regulation and a 25-fold increase in the basal activity. The disruption of dimerization of the GAF in three enzymes had different effects on regulation of each of them. This indicates that each GAF domain has a unique structure and drugs may be targeted toward it without cross-inhibition between different GAF domains.The PAS domain of CyaB1 was shown to be important for the proper and efficient signalling from the GAF domain to the catalytic domain. The exchange of the PAS domain by the hPDE5 linker showed that it is required between the GAF and catalytic for signalling. This applies also to the exchange by PAS domain of CyaB2 which was able to transduce the signal. Regarding the mechanism of signalling between the different domains, the situation was highly complicated. I expect that the N-terminus of hPDE2 was only for anchoring the enzyme to the membrane but PDE5 N-terminus had physical interaction with the GAF domain that regulates the binding of cGMP. The GAF domain tandems are responsible for dimerization and the allosteric activation by cNMP, the binding of the cyclic nucleotide to one of the GAF motifs is changing the conformation of that GAF leading to rotation of the connecting helix which will loosen the dimerization interface in the other GAF motif and cause further conformational changes in the other GAF that will be transferred to the catalytic domain by rotational mechanism that will cause the change in the conformation of the catalytic domain and affect the Km or the Vmax for that enzyme accordingly.Die cyanobakterielle Adenylatcyclase CyaB1 aus /Anabaena /wurde als Reporterenzym verwendet, um die Signal Transduktion drei verschiedener N-terminaler Domänen zu untersuchen. a) Die Funktion der Linkerbereiches der Tandem-GAF Domäne der cyanobakteriellen Adenylatcyclase, b) die Bedeutung und Rolle des N-terminalen Bereichs der Tandem GAF Domäne aus der humanen Phosphodiesterase 2 und 5 (hPDE2 und hPDE5), und c) die Bedeutung der cyanobakteriellen PAS Domäne unmittelbar N-terminal zur katalytischen Adenylatcyclase Region, die üblicherweise Teil des Reportersystems ist. a) Ein längen Linker verbindet in allen Tandem GAF Domänen beide Einzeldomänen, GAF A und B. Es stellte sich heraus, daß der Linkerbereich sehr kritisch ist für die Signalübertragung innerhalb des Chimären Moleküls, da jedwede Längenveränderung in der CyaB2 Tandem Domäne (+ oder -) die Aktivierung durch cAMP drastisch erniedrigte. Darüber beeinträchtige eine Verlängerung um vier Aminosäuren die Stabilität der Chimäre, erheblich. Verschiedene experimentelle Befunde deuten an, daß die für eine enzymatische Aktivität notwendige Dimerisierung beeinträchtigt ist. Mutation in der alpha1-Helix von GAF A der hPDE2 erhöhten den Aktivierbarkeitsfaktor durch cGMP, ohne die cGMP Affinität oder die Kooperativität des Enzyms zu beeinflussen. b) Die 227 N-terminalen Aminosäuren der PDE2 oder 150 der hPDE5 wirken hemmend auf die Adenylatcyclaseaktivität in eine Proteinchimäre aus der Tandem-GAF Domäner der hPDE2 und CyaB1. Die Entfernung des hPDE2 N-Terminus erhöhte die spezifische Aktivität der Cyclase, hatte aber keinen Einfluss auf die Affinität von cGMP oder das Ausmaß der Aktivierung. c) Es wurde die CyaB1-PAS Domäne gegen den Linkerbereich in zwei Längenvarianten ausgetauscht, der zwischen der hPDE5 Tandem GAF Domäne und der katalytischen hPDE5 Domäne. sitzt linker des Katalytische-Domänes (zwei verschiedene Länge). Dies führte zwar zu einer starken Reduktion aller Aktivitäten, zeigte jedoch grundsätzlich, daß die PAS Domäne nicht essentiell ist für die Kommunikation zwischen den GAF Tandem Domänen und der Adenylatcyclase. Die CyaB1 PAS Domäne konnte kristallisiert werden, jedoch war es nicht möglich, die Struktur aufzuklären