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Beyond PKA: Evolutionary and structural insights that define a docking and dimerization domain superfamily.

ABSTRACT: Protein-interaction domains can create unique macromolecular complexes that drive evolutionary innovation. By combining bioinformatic and phylogenetic analyses with structural approaches, we have discovered that the docking and dimerization (D/D) domain of the PKA regulatory subunit is an ancient and conserved protein fold. An archetypal function of this module is to interact with A-kinase-anchoring proteins (AKAPs) that facilitate compartmentalization of this key cell-signaling enzyme. Homology searching reveals that D/D domain proteins comprise a superfamily with 18 members that function in a variety of molecular and cellular contexts. Further in silico analyses indicate that D/D domains segregate into subgroups on the basis of their similarity to type I or type II PKA regulatory subunits. The sperm autoantigenic protein 17 (SPA17) is a prototype of the type II or R2D2 subgroup that is conserved across metazoan phyla. We determined the crystal structure of an extended D/D domain from SPA17 (amino acids 1-75) at 1.72 Å resolution. This revealed a four-helix bundle-like configuration featuring terminal β-strands that can mediate higher order oligomerization. In solution, SPA17 forms both homodimers and tetramers and displays a weak affinity for AKAP18. Quantitative approaches reveal that AKAP18 binding occurs at nanomolar affinity when SPA17 heterodimerizes with the ropporin-1-like D/D protein. These findings expand the role of the D/D fold as a versatile protein-interaction element that maintains the integrity of macromolecular architectures within organelles such as motile cilia.

SUBMITTER: Dahlin HR

PROVIDER: S-EPMC8339350 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:SPOUT methyltransferases (MTases) are a large class of S-adenosyl-L-methionine-dependent enzymes that exhibit an unusual alpha/beta fold with a very deep topological knot. In 2001, when no crystal structures were available for any of these proteins, Anantharaman, Koonin, and Aravind identified homology between SpoU and TrmD MTases and defined the SPOUT superfamily. Since then, multiple crystal structures of knotted MTases have been solved and numerous new homologous sequences appeared in the databases. However, no comprehensive comparative analysis of these proteins has been carried out to classify them based on structural and evolutionary criteria and to guide functional predictions.We carried out extensive searches of databases of protein structures and sequences to collect all members of previously identified SPOUT MTases, and to identify previously unknown homologs. Based on sequence clustering, characterization of domain architecture, structure predictions and sequence/structure comparisons, we re-defined families within the SPOUT superfamily and predicted putative active sites and biochemical functions for the so far uncharacterized members. We have also delineated the common core of SPOUT MTases and inferred a multiple sequence alignment for the conserved knot region, from which we calculated the phylogenetic tree of the superfamily. We have also studied phylogenetic distribution of different families, and used this information to infer the evolutionary history of the SPOUT superfamily.We present the first phylogenetic tree of the SPOUT superfamily since it was defined, together with a new scheme for its classification, and discussion about conservation of sequence and structure in different families, and their functional implications. We identified four protein families as new members of the SPOUT superfamily. Three of these families are functionally uncharacterized (COG1772, COG1901, and COG4080), and one (COG1756 represented by Nep1p) has been already implicated in RNA metabolism, but its biochemical function has been unknown. Based on the inference of orthologous and paralogous relationships between all SPOUT families we propose that the Last Universal Common Ancestor (LUCA) of all extant organisms contained at least three SPOUT members, ancestors of contemporary RNA MTases that carry out m1G, m3U, and 2'O-ribose methylation, respectively. In this work we also speculate on the origin of the knot and propose possible 'unknotted' ancestors. The results of our analysis provide a comprehensive 'roadmap' for experimental characterization of SPOUT MTases and interpretation of functional studies in the light of sequence-structure relationships.

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Beyond PKA: Evolutionary and structural insights that define a docking and dimerization domain superfamily.

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