Regulatory subdomain

Unexpectedly, the R1 chimera had sensitivity to the E. coli allosteric activator, ornithine. However, the x-ray structure (26) subsequently showed that ornithine is not bound exclusively to the regulatory subdomain, but rather bridges the catalytic and regulatory subdomains. These experiments clearly demonstrated that the mammalian allosteric effectors bind to B3, a separately folded regulatory subdomain located at the carboxyl end of CPS.B. 

A deletion mutant (Figure 11) was constructed in which the carboxyl half of the regulatory domain in the R2 chimera was deleted. The resulting construct bound allosteric inhibitors nearly as well as the parent molecule, but the allosteric transitions were abolished. Thus, the amino half of the regulatory subdomain binds allosteric effectors, while the carboxyl half is crucial for transmitting the allosteric signal to the catalytic domain. This result is consistent with the x-ray structure of E. coli CPS and the CAD model that indicates that most of the interactions between B3 and B2 (or A2 in the case of the chimera) involve residues in the carboxyl half of the regulatory domain. 

These experiments suggest that the regulatory subdomain is an exchangeable ligand binding module that can control both CPS.A and CPS.B subdomains. CPS.B is not unique in its sensitivity to allosteric effectors. There has been no differentiation in structure or function that have rendered it subject to control, rather it is the locus of regulation in the native molecule as a consequence of its proximity to the regulatory domain. 

Dasgupta, M. Blumenthal, D.K. Characterization of the regulatory domain of the y-subunit of phosphorylase kinase. The two noncontiguous calmodulin-binding subdomains are also autoinhibitory. J. Biol. Chem., 270, 22283-22289 (1995)... 

CAD is a 1.5 Mda complex that catalyzes the first three steps in de novo pyrimidine biosynthesis in mammalian cells. The protein consists of six copies of a 243 kDa polypeptide that is organized into 15 domains, subdomains and linkers each with a specific catalytic or regulatory function. Most of these domains have been subcloned and expressed in E. coli where they fold into stable, fully functional proteins. While each domain functions autonomously, interdomain signaling modulates the reactions occurring on different domains to ensure that biosynthesis proceeds in a coordinated fashion. Insights into the signaling mechanism have been provided by the analysis of several hybrid and chimeric molecules constructed by combining domains and subdomains of the mammalian, yeast and E. coli proteins in novel ways. 

There is kinetic evidence (66-67) that the target of allosteric effectors is CPS.B. Since the CPS subdomains are functionally and structurally equivalent, we were curious as to whether CPS.A could be placed under allosteric control. To determine whether this occurs, a second chimeric molecule (R2) was constructed (21) in which the mammalian regulatory domain (B3) replaced the A3 subdomain in E. coli CPS.A (Figure 11). The control mechanisms (Figure 12) are nearly the same as that observed for the mammalian CAD. The chimera is inhibited by UTP and activated by PRPP although the affinity for the latter ligand is somewhat lower than in the native molecule. While chimera R1 was catalytically active, its aggregation made it a poor substrate for protein kinase A. The second chimera, R2 is monodisperse and can be readily phosphorylated. Protein kinase A abolishes UTP inhibition and reduces the affinity of the protein for PRPP, the same effect observed in CAD. 

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