Structural Insights of CCT Domain Interactions: identification of non-canonical CCT-binding motifs and previously unrecognized CCT-Containing Proteins

With-No-Lysine (WNK) kinases regulate renal sodium reabsorption by modulating Slc12 cotransporters. Proteins from the TSC22D family interact with WNK kinases and modulate their activity. Interactions within the WNK pathway are mediated by Conserved C-Terminal (CCT) domains, which typically bind short linear RFxV motifs.We previously showed that the TSC22D–WNK interaction is mediated by the two CCT domains present in WNKs. TSC22D proteins contain a canonical RFxV motif, as well as a non-canonical RWTC motif that binds to the CCT domain of NRBP1. Thus, we hypothesized that WNK CCTs would interact with the RFxV and/or RWTC motifs found in TSC22Ds. However, in silico structural modeling revealed an additional, highly conserved CCT-binding motif in TSC22Ds. This motif diverges markedly from canonical RFxV sequences and consists of a KxxSxF pattern. Co-immunoprecipitation experiments using mutant proteins confirmed that this motif binds selectively to the second CCT domain in WNK kinases, whereas the first CCT interacts primarily with the RFxV motif of TSC22Ds, suggesting that structural and sequence differences in CCTs and CCT-binding motifs dictate binding specificity among CCT-motif pairs. Our second objective was to identify additional mammalian proteins harboring potential CCT domains on the basis of structural homology. We used the structural comparison platforms Dali and Foldseek to screen the Alphafold3 database. Four and eight known CCT domains were used as queries for Dali and Foldseek, respectively. On average, each CCT query yielded 517 hits in Dali and 370 hits in Foldseek. These hit lists were merged and curated by retaining only proteins detected by at least two independent CCT queries and with Dali Z-scores > 5 and Foldseek probabilities ≥0.2. The proteins in the final list were then manually inspected by superposition against known CCTsto verify hallmark features of bona fide CCT domains, including the hydrophobic groove. A subset of candidates was cloned, and their ability to interact with TSC22D2—chosen because it contains three distinct motifs (KxxSxF, RFxV, RWTC)—was tested by co-immunoprecipitation. Among the candidates tested, only Ferry3 displayed detectable binding to TSC22D2. This interaction was lost when using a TSC22D2 mutant lacking its motifs, indicating that binding is motif-dependent and providing proof-of-principle that the candidate domain in Ferry3 can function as a CCT. Whether this interaction is physiologically relevant or mediates binding to other Ferry3 partners remains to be determined. Our results indicate that this interaction mechanism is largely restricted to the WNK pathway, but also suggests that a small number of proteins may have aquired it. Overall, this work defines new determinants of CCT–motif recognition, providing deeper mechanistic insight into how WNK signaling specificity is achieved. This has implications for the regulation of sodium balance and blood pressure by the kidney. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.