Abstract
Choanoflagellates are single-celled eukaryotes with complex signaling pathways. They are considered the closest non-metazoan ancestors to mammals and other metazoans and form multicellular-like states called rosettes. The choanoflagellate Monosiga brevicollis contains over 150 PDZ domains, an important peptide-binding domain in all three domains of life (Archaea, Bacteria, and Eukarya). Therefore, an understanding of PDZ domain signaling pathways in choanoflagellates may provide insight into the origins of multicellularity. PDZ domains recognize the C-terminus of target proteins and regulate signaling and trafficking pathways, as well as cellular adhesion. Here, we developed a computational software suite, Domain Analysis and Motif Matcher (DAMM), that analyzes peptide-binding cleft sequence identity as compared with human PDZ domains and that can be used in combination with literature searches of known human PDZ-interacting sequences to predict target specificity in choanoflagellate PDZ domains. We used this program, protein biochemistry, fluorescence polarization, and structural analyses to characterize the specificity of A9UPE9_MONBE, a M. brevicollis PDZ domain-containing protein with no homology to any metazoan protein, finding that its PDZ domain is most similar to those of the DLG family. We then identified two endogenous sequences that bind A9UPE9 PDZ with <100 μM affinity, a value commonly considered the threshold for cellular PDZ–peptide interactions. Taken together, this approach can be used to predict cellular targets of previously uncharacterized PDZ domains in choanoflagellates and other organisms. Our data contribute to investigations into choanoflagellate signaling and how it informs metazoan evolution.
Highlights
PDZ domains are small peptide-binding domains named for the proteins where they were first discovered: PSD-95, Dlg1,and ZO-1 (PDZ) [1,2,3,4]
Examples include: regulation of the trafficking of receptors throughout the cell, e.g., the cystic fibrosis transmembrane conductance regulator (CFTR), signaling pathways mediated by G-protein coupled receptors (GPCRs), and interactions with proteins involved in tight
Molecules 2021, 26, 6034 cystic fibrosis transmembrane conductance regulator (CFTR), signaling pathways mediated by G-protein coupled receptors (GPCRs), and interactions with proteins involved in jtuignhcttijounnsctaionndswanitdhiwnitthheinptohsetspyonsatspytnicadpteincsditeinessiotifesneouf rnoenusro[1n0s–[2170]–. 2D7]u.eDtuoetthoeitrhecierncteranlrtroallerionleceinllucelallrulparropcerossceess,sePsD, PZDdZodmoaminasinasreartehtehrearpaepuetuictitcatragregtestsininaannuummbbeerrooffhhuummaann ddiisseeaasseess [[2288––3322]]
Summary
PDZ domains are small peptide-binding domains named for the proteins where they were first discovered: PSD-95, Dlg1,and ZO-1 (PDZ) [1,2,3,4] These are scaffolding domains that recognize the extreme C-terminus of target proteins, with the bulk of the PDZ–protein interaction involving up to 6 residues on the target [5,6,7]. Examples include: regulation of the trafficking of receptors throughout the cell, e.g., the cystic fibrosis transmembrane conductance regulator (CFTR), signaling pathways mediated by G-protein coupled receptors (GPCRs), and interactions with proteins involved in tight. FFoorr eexxaammppllee,, pprrootteeiinnss iinn tthhee SSAARRSS--CCooVV--22 pprrootteeoommee,, wwhhiicchh ccaauusseess CCOOVVIIDD--1199 ddiisseeaassee,, iinntteerraacctt wwiitthh aanndd bbiinndd sseevveerraall PPDDZZ ddoommaaiinnssdduurriinnggiinnffeeccttioionn[3[399––4422].].TThheetrtraaffiffcikckininggoof fananggioitoetnesnisni-nc-ocnovnevretrintigngeneznyzmyme 2e (2A(CAEC2E)2r)erceecpetpotro, rw, whihcihchisistatragregteetdedbbyySSAARRSS-C-CooVV-2-,2,coconntataininssaaPPDDZZ--bbininddiinnggmmoottiiffaattiittss CC--tteerrmmiinnuuss ((sseeqquueennccee:: DDVVQQTTSSFF,, mmoottiiff rreessiidduueess aarree uunnddeerrlliinneedd)) aanndd iiss aallssoo rreegguullaatteedd bbyy PPDDZZ ddoommaaiinnss [[4433]]
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