Search

cAMP and the cAMP binding domain (CBD) constitute a ubiquitous regulatory switch that translates an extracellular signal into a biological response. The CBD contains α- and β-subdomains with cAMP binding to a phosphate binding cassette (PBC) in the β-sandwich. The major receptors for cAMP in mammalian cells are the regulatory subunits (R-subunits) of PKA where cAMP and the catalytic subunit compete for the same CBD. The R-subunits inhibit kinase activity, whereas cAMP releases that inhibition. Here, we use NMR to map at residue resolution the cAMP-dependent interaction network of the CBD-A domain of isoform Iα of the R-subunit of PKA. Based on H/D, H/H, and N z exchange data, we propose a molecular model for the allosteric regulation of PKA by cAMP. According to our model, cAMP binding causes long-range perturbations that propagate well beyond the immediate surroundings of the PBC and involve two key relay sites located at the C terminus of β 2 (I163) and N terminus of β 3 (D170). The I163 site functions as one of the key triggers of global unfolding, whereas the D170 locus acts as an electrostatic switch that mediates the communication between the PBC and the B-helix. Removal of cAMP not only disrupts the cap for the B′ helix within the PBC, but also breaks the circuitry of cooperative interactions stemming from the PBC, thereby uncoupling the α- and β-subdomains. The proposed model defines a signaling mechanism, conserved in every genome, where allosteric binding of a small ligand disrupts a large protein–protein interface.

D. H. Maclennan, M. Abu-Abed and Chulhee Kang. Structure–Function Relationships in Ca2+ Cycling Proteins. Journal of Molecular and Cellular Cardiology (2002) 34, 897–918.

The skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1a) mediates muscle relaxation by pumping Ca(2+) from the cytosol to the ER/SR lumen. In efforts aimed at understanding the structural basis for the conformational changes accompanying the reaction cycle catalyzed by SERCA1a, we have studied the ATP-binding domain of SERCA1a in both nucleotide-bound and -free forms by NMR. Limited proteolysis analyses guided us to express a 28 kDa stably folded fragment containing the nucleotide-binding domain of SERCA1a spanning residues Thr357-Leu600. ATP binding activity was demonstrated for this fragment by a FITC competition assay. A nearly complete backbone resonance assignment of this 28 kDa ATP-binding fragment, in both the AMP-PNP-bound and -free forms, was obtained by means of heteronuclear multidimensional NMR techniques. NMR titration experiments with AMP-PNP revealed a confined nucleotide-binding site which coincides with a cytoplasmic pocket region identified in the crystal structure of apo-SERCA1a. These results are consistent with previous site-directed mutagenesis studies of SERCA1a.

The major soluble protein component of avian and reptilian eye lenses, delta crystallin, is highly homologous to the urea cycle enzyme, argininosuccinate lyase (ASL). In duck lenses there are two highly homologous delta crystallins, termed delta I and delta II, that are 94% identical in amino acid sequence. While delta II crystallin has been shown to exhibit ASL activity in vitro, delta I crystallin is inactive. The X-ray structure of a His to Asn mutant of duck delta II crystallin (H91N) has been determined to 2.5 A resolution using the molecular replacement technique. The overall fold of the protein is similar to other members of the superfamily to which this protein belongs, with the active site located in a cleft between three different monomers of the tetrameric protein. A reexamination of the kinetic properties of the H91N mutant reveals that the mutant has 10% wild-type activity. The Vmax of the mutant protein is identical to that of the wild-type protein, but a 10-fold increase in the Michaelis constant is seen, suggesting that His 91 is involved in binding the substrate. In an effort to determine the reasons for the loss of enzymatic activity in delta I crystallin, a structural comparison of the H91N mutant with the enzymatically inactive turkey delta I crystallin has been performed. This study revealed a remarkable similarity in the overall structures of the two proteins. Three regions of secondary structure do differ significantly between the two models; these include the N-terminal tail, a loop containing residues 76-91, and a cis versus trans peptide linkage at residue Thr 322. The cis to trans peptide variation appears to be an interspecies difference between turkey and duck and is therefore not directly involved in the loss of enzymatic activity. All the residues implicated in the catalytic mechanism are conserved in both the active and inactive proteins, and given the linearity of the relationship between the enzymatic activity of duck delta I/delta II heterotetramers and their delta II content (PiatigorskyHorwitz, 1996), it is evident from the structure that only one of the three domains that contributes to the active site is responsible for the loss of activity in the delta I protein. Given the structural differences found in domain 1 (N-terminal tail and 76-91 loop), we postulate that these differences are responsible for the loss of catalytic activity in the delta I crystallin protein and that the delta I protein is inactive because it no longer binds the substrate.