Structural Biochemistry/Conformational changes in Adenylyl Cyclases control regulation of signal transduction

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Adenylyl cyclases (ACs) are proteins that transduce a large variety of extracellular signals into intracellular responses, and therefore are important in signal transduction. ACs function to control the rate of conversion of ATP (substrate) into the second messenger cAMP (cyclic Adenine monophosphate), which in turn activates effector cells. In eukaryotes, activated effector cells proceed through a mechanism, depending on pathway, resulting in intracellular signal amplification.

There are six classes of ACs, one of which is the Class III AC. Class II AC possesses a characteristic a dimeric tertiary structure. The dimer has important functional significance, because it forms catalytic pockets at the dimer interface. Previous research suggests that regulation of Class III AC activity is achieved by shifts of the three-dimensional spatial orientation of the two monomers towards each other. Specifically, research has been done on the homologs, mammalian class III ACs and bacterial Class III ACs.

Mammalian Class III Adenylyl Cyclases[edit]

Mammalian class III ACs have two catalytic domains, called C1 and C2. The C1 and C2 domains are 25-30% identical, and they have low affinity for each other. C1 and C2 interact to form two distinct binding pockets at the interface. One of the pockets binds ATP and catalyzes the cyclization of ATP into cAMP. The cyclization is directed by four amino acid residues that stabilize the involved species. Two negatively charged aspartic acid residues on C1 bind to Mg2+ ion to stabilize the triphosphate present in the ATP substrate. The positively charged residues arginine and asparagine contributed by the C2 domain stabilize the transition state by neutralizing an excess negative charge at the alpha-phosphoryl. The other binding pocket is a docking site for the activator molecule, forskolin.

Forskolin

Evidence of Mammalian AC Regulation via Reorientation[edit]

Evidence for AC regulation by reorientation in mammalian ACs is seen in conformational changes induces by the binding of G proteins, which are transducers, to both C1 and C2 at non-catalytic sites. Regulation by reorientation occurs in mammalian ACs when the AC is activated by the G-protein, Gs-alpha. When the stimulatory Gs-alpha binds to C1 and C2, a conformational change of a 7° rotation leads to a closure of the catalytic site, which enhances the catalysis of ATP into cAMP occurs. When the inhibitory Gi-alpha binds opposite the G2alpha, it counteracts the rotation induced by Gs-alpha, thus effectively reducing the efficiency of ATP catalysis. Hence, G-proteins activate (Gs-alpha) and inhibit (Gi-alpha) the catalyzation by causing changes in the dimer interface conformation.

Bacterial Class III Adenylyl Cyclases[edit]

Bacterial class III ACs are homodimers. Note that this makes mammalian ACs and bacterial ACs orthologs. A few examples of bacterial AC isoforms and their reorientation mechanisms are presented below:

Class III AC Rv1264[edit]

The mycobacterial AC Rv1264 is a homodimer whose conformation is regulated by pH. At pH = 6, it is in its activated state for the ATP substrate. At neutral pH, Rv1264 is in its inhibited state, in which alpha helix is extended disrupting the catalytic pocket. In the inhibited state of Rv1264, the monomers are pulled close to the regulatory platform such that massive reorientation involving the rotation of each catalytic domain by 55° and a translation by 6 angstroms. The lysine and aspartate residue on Rv1264 are essential for the substrate to bind.

Class III AC Rv1900c[edit]

Mycobacterial AC Rv1900c is regulated by the binding of the ATP substrate itself In the absence of the substrate, the catalytic domain of Rv1900c is slightly asymmetric. When the bacterial ATP-analog binds, the dimer closes and the asymmetry is heightened, rotating the monomers by 16.6° and a translation of 11.4 angstrom of one monomer. Owing to is dimeric tertiary structure, there are two binding pockets, but ATP-analog binds to only one, while the other is non-functional.

Class III AC CyaC[edit]

CyaC is activated when bicarbonate binds to the catalytic domain which causes a shift in a single alpha-helix while overall orientation of the monomers in the homodimer remains unchanged. This is an example of an activation mechanism in which a small regulator moiety (the bicarbonate) activates the cyclase by only minor structural rearrangements. As the CyaC is activated, the catalytic pocket folds.

A CyaC adenylyl cyclase. Note the identical subunits in the dimer.

Conclusion[edit]

Dimer interface of ACs regulate catalysis of ATP into cAMP, and therefore regulate signal transduction pathways that involve ATP. The dimer interface regulates cyclization of ATP into cAMP via conformational changes in the tertiary structure of the adenylyl cyclase arising from various biochemical mechanisms and factors. These conformational changes in structure of the dimer arise from the binding of G-proteins that stimulate or inhibit the catalyzation reaction in mammalian adenylyl cyclases. In bacterial adenylyl cyclases, biochemical conditions and factors such as pH, presence of a particular moiety (e.g. bicarbonate), and the binding of the substrate itself (ATP) may change the conformation of the tertiary structure, which in turn regulates the activity of the catalytic site by opening or closing it.

References[edit]

Linder JU, Schultz JE. (2008) Versatility of signal transduction encoded in dimeric adenylyl cyclases.