Determination of the Structure of the N-terminal Splice Region of the Cyclic AMP-specific Phosphodiesterase RD1 (RNPDE4A1) by 1H NMR and Identification of the Membrane Association Domain Using Chimeric Constructs

Abstract

A 25-residue peptide representing the membrane targeting N-terminal splice region of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1) was synthesized, and its structure was determined by 1 H NMR. Two independently folding helical regions were identified, separated by a highly mobile "hinge" region. The first helical region was formed by an N-terminal amphipathic ␣-helix, and the second consisted of multiple overlapping turns and contained a distinct compact, hydrophobic, tryptophan-rich domain (residues 14 -20). Chimeric molecules, formed between the N-terminal region of RD1 and the soluble bacterial protein chloramphenicol acetyltransferase, were used in an in vitro system to determine the features within the splice region that were required for membrane association. The ability of RD1-chloramphenicol acetyltransferase chimera to become membrane-associated was not affected by deletion of any of the following regions: the apolar section (residues 2-7) of the first helical region, the polar part of this region together with the hinge region (residues 8 -13), or the polar end of the C-terminal helical region (residues 21-25). In marked contrast, deletion of the compact, hydrophobic tryptophan-rich domain (residues 14 -20) found in the second helical region obliterated membrane association. Replacement of this domain with a hydrophobic cassette of seven alanine residues also abolished membrane association, indicating that membrane-association occurred by virtue of specific hydrophobic interactions with residues within the compact, tryptophan-rich domain. The structure of this domain is well defined in the peptide, and although the region is helical, both the backbone and the distribution of side chains are somewhat distorted as compared with an ideal ␣-helix. Hydrophobic interactions, such as the "stacked" rings of residues Pro 14 and Trp 15 , stabilize this domain with the side chain of residue Leu 16 adopting a central position, interacting with the side chains of all three tryptophan residues 15, 19, and 20. These bulky side chains thus form a hydrophobic cluster. In contrast, the side chain of residue Val 17 is relatively exposed, pointing out from the opposite "face" of the peptide. Although it appears that this compact, tryptophan-rich domain is responsible for membrane association, at present the target site and hence the specific interactions involved in membrane targeting by the RD1 splice region remain unidentified.

Intracellular Membrane Association of the Aplysia cAMP Phosphodiesterase Long and Short Forms via Different Targeting Mechanisms

“…These results suggest that the molecular mechanism(s) of the membrane targeting of PDE4A1 and ApPDE4 long form mutants might be similar and through direct membrane association. The N terminus of PDE4A1 is involved in membrane association via two different motifs, namely, helix 1 and helix 2, that are separated by a hinge region (22,48). Our results showed that at least 16 N-terminal amino acids are required for sufficient membrane targeting of the long form.…”

Isoform-Selective Susceptibility of DISC1/Phosphodiesterase-4 Complexes to Dissociation by Elevated Intracellular cAMP Levels

“…35 S]-labeled PDE4A1 for various binding studies (Smith et al, 1996;. Using a cDNA encoding fl-DISC1 as a template, we identified a single radiolabeled protein product of the expected size (100 kDa) in extracts analyzed on SDS-PAGE (data not shown).…”

Helix-1 of the cAMP-specific phosphodiesterase PDE4A1 regulates its phospholipase-D-dependent redistribution in response to release of Ca2+

“…The first of these helices (Helix-1; amino acids 1-8) comprises a Nterminal amphipathic ␣-helix whereas the second (Helix-2; amino acids 14-25) is a compact helical structure that is highly hydrophobic and tryptophan-rich (Smith et al, 1996). More recently we have identified a novel microdomain within helix-2, called TAPAS-1, which allows association with membranes and lipid vesicles .…”

“…analyses of the unique N-terminal region of PDE4A1 showed it to be composed of two distinct helical structures separated by a mobile 'hinge' region (Smith et al, 1996). The first of these helices (Helix-1; amino acids 1-8) comprises a Nterminal amphipathic ␣-helix whereas the second (Helix-2; amino acids 14-25) is a compact helical structure that is highly hydrophobic and tryptophan-rich (Smith et al, 1996).…”

“…Deletion of this region has been shown to yield a fully active, entirely soluble enzyme, whereas the fusion of this unique N-terminal region to various proteins that were normally soluble rendered them membrane associated with identical distributions to the full-length PDE4A1 (Scotland and Houslay, 1995;Shakur et al, 1993;Smith et al, 1996). NMR The unique N-terminal regions of PDE4 cAMP-specific phosphodiesterases confer interaction with distinct signalling and scaffolding proteins.…”