The APH(3')-IIIa enzyme is composed of two lobes, a 94-residue N-terminal lobe and a larger 157-residue C-terminal lobe. The two lobes are joined by a 12-residue linker region containing a short b-strand and a-helix. The N-ter-minal lobe consists of a five-stranded antiparallel b-sheet, with an a-helix located between two b-strands and a short 310 helix preceding the first b-strand. The architecture of the N-terminal lobe of APH(30)-IIIa is similar to that observed in eukaryotic protein kinases (ePKs) such as the catalytic subunits of cyclic AMP (cAMP)-dependant protein kinase (cAPK), casein kinase (CsK) and phosphorylase kinase (PhK) (Owen et al. 1995; Xu et al. 1995; Knighton et al. 1991). For structurally conserved main-chain atoms in the N-terminal lobe, the rms deviation in atomic position is only 1.8 A between APH(3')-IIIa and cAPK. This is remarkable, given the fact that the sequence identity between the two enzymes is less than 6% for these residues.
The 12-residue linker that connects the N- and C-terminal lobes of APH(3')-IIIa is also structurally similar to the equivalent region in the structures of protein kinases such as cAPK. The linker consists of a short a-helix flanked by sections of random coil. The C-terminal lobe of APH(3')-IIIa can be divided into three sections: (1) a central core composed of two a-helices and a long hairpin-shaped loop incorporating two short sections of antiparallel b-sheet, (2) an insert region composed of two a-helices connected by long loop and (3) a C-terminal region composed of two a-helices.
There are four areas in which APH(3')-IIIa differs significantly from ePKs such as cAPK (Fig. 2). First, in APH(3')-IIIa, the loop between b-strands 1 and 2 contains a one-residue insertion and adopts a different conformation than that observed in ePKs (red). While this loop is the location of the conserved GXGXXG motif in protein kinases, the motif is not observed in APH(3')-IIIa. Second, as a consequence of differences in the C-terminal lobes of APH(30)-IIIa and the protein kinases, the location of the a-helix in the linker region is shifted in APH(3')-IIIa with respect to where it is usually located in the protein kinases (yellow). Third, APH(3')-IIIa has a 60-residue insert in its C-terminal lobe (blue). Consisting of two a-helices and a 19-res-
Fig. 2 A comparison of the structures of APH(3')-IIIa (left) and cAPK (right) highlighting, in colour, the areas with significant differences. These areas include: (1) the loop between strands and p2 (red), (2) the linker region between the N- and C-terminal domains (yellow), (3) a 60-residue insert in APH(3')-IIIa (blue), (4) a smaller insertion found in ePK's (orange) and (5) the last two a-helices of the C-terminal domain (magenta). The ATP cofactor and its associated magnesium ions are depicted as green balls-and-sticks
Fig. 2 A comparison of the structures of APH(3')-IIIa (left) and cAPK (right) highlighting, in colour, the areas with significant differences. These areas include: (1) the loop between strands and p2 (red), (2) the linker region between the N- and C-terminal domains (yellow), (3) a 60-residue insert in APH(3')-IIIa (blue), (4) a smaller insertion found in ePK's (orange) and (5) the last two a-helices of the C-terminal domain (magenta). The ATP cofactor and its associated magnesium ions are depicted as green balls-and-sticks idue loop, this insertion occupies the space of a smaller section of polypeptide found in ePKs (orange). The latter has a distinctly different conformation from that seen in APH(30)-IIIa and has been identified as important in substrate selectivity and specificity (Taylor et al. 1992; Madhusudan et al. 1994). This section of the ePK sequence also corresponds to the activation segment in some protein kinases, containing sites of phosphorylation that switch the enzyme from an inactive to an active conformation (Johnson et al. 1996; Yamaguchi and Hendrickson 1996). Lastly, the positions of the two a-helices at the end of the C-terminal lobes are significantly different in the APH(3')-IIIa and ePK structures (magenta).
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