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KCTD family proteins typically assemble into cullin-RING E3 ligases. KCTD1 is an atypical member that functions instead as a transcriptional repressor. Mutations in KCTD1 cause developmental abnormalities and kidney fibrosis in scalp-ear-nipple syndrome. Here, we present unexpected mechanistic insights from the structure of human KCTD1. Disease-causing mutation P20S maps to an unrecognized extension of the BTB domain that contributes to both its pentameric structure and TFAP2A binding. The C-terminal domain (CTD) shares its fold and pentameric assembly with the GTP cyclohydrolase I feedback regulatory protein (GFRP) despite lacking discernible sequence similarity. Most surprisingly, the KCTD1 CTD establishes a central channel occupied by alternating sodium and iodide ions that restrict TFAP2A dissociation. The elucidation of the structure redefines the KCTD1 BTB domain fold and identifies an unexpected ion-binding site for future study of KCTD1's function in the ectoderm, neural crest, and kidney. [Display omitted] • Crystal structure of full-length human KCTD1 that forms a pentamer • The pre-BTB region is essential for TFAP2A binding and makes domain-swap interactions • C-terminal domain pentamer forms a central channel with bound sodium and iodide ions • The structure explains how mutations disrupt KCTD1 in scalp-ear-nipple syndrome Pinkas et al. determined the pentameric structure of full-length KCTD1 revealing a pre-BTB extension forming cyclic domain-swap interactions and a C-terminal domain forming a central channel occupied by sodium and iodide ions. Both features appear important for TFAP2A binding. The structure explains how mutations disrupt KCTD1 in scalp-ear-nipple syndrome. [ABSTRACT FROM AUTHOR]

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types inflammation and cell injury. The enzymatic activity of extracelular TG2 thus appears to be tightly regulated. As TG2 is know to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys230, Cys370 and Cys371 that is involved in oxidative inactivation of TG2. Within this triad, Cys230 was found to participate in disulfide bonds with both Cys230 and its neighbor, Cys231 Notably, Ca2+ was found to protect against formation of these disulfide bonds. To investigate the role of each cysteine residue, we created alanine mutants and found that Cys230 appears to promote oxidation and inactivation of TG2 facilitating formation of Cys370-Cys371 through formation the Cys230-Cys370 disulfide bond. Although vicinal disulfide pairs are found in several transglutaminase isoforms, Cys230 is unique for TG2, suggesting that this residue acts as an isoform specific redox sensor. Our findings suggest that oxidation is likely to influence the amount of active TG2 present in the extra cellular environment. [ABSTRACT FROM AUTHOR]

Receptor-interacting protein kinase 2 (RIPK2) is a key mediator of nucleotide-binding oligomerization domain (NOD) cell signaling that has been implicated in various chronic inflammatory conditions. A new class of RIPK2 kinase/NOD signaling inhibitors based on a 3,5-diphenyl-2-aminopyridine scaffold was developed. Several co-crystal structures of RIPK2•inhibitor complexes were analyzed to provide insights into inhibitor selectivity versus the structurally related activin receptor-like kinase 2 (ALK2) demonstrating that the inhibitor sits deeper in the hydrophobic binding pocket of RIPK2 perturbing the orientation of the DFG motif. In addition, the structure-activity relationship study revealed that in addition to anchoring to the hinge and DFG via the 2-aminopyridine and 3-phenylsulfonamide, respectively, appropriate occupancy of the region between the gatekeeper and the αC-helix provided by substituents in the 4- and 5-positions of the 3-phenylsulfonamide were necessary to achieve potent NOD cell signaling inhibition. For example, compound 18t (e.g. CSLP37) displayed potent biochemical RIPK2 kinase inhibition (IC 50 = 16 ± 5 nM), >20-fold selectivity versus ALK2 and potent NOD cell signaling inhibition (IC 50 = 26 ± 4 nM) in the HEKBlue assay. Finally, in vitro ADME and pharmacokinetic characterization of 18t further supports the prospects of the 3,5-diphenyl-2-aminopyridine scaffold for the generation of in vivo pharmacology probes of RIPK2 kinase and NOD cell signaling functions. Image 1 • Demonstration of 3,5-diphenyl-2-aminopyridines as potent inhibitors of receptor-interacting protein kinase 2 (RIPK2). • SAR insights into achieving RIPK2 dependent nucleotide-binding oligomerization domain (NOD) cell signaling inhibition. • SAR insights into selectivity versus structurally related activin receptor-like kinase 2 (ALK2). • In vitro ADME and in vivo pharmacokinetic characterization of a representative inhibitor illustated. [ABSTRACT FROM AUTHOR]

Abstract: Biomaterials that mimic the extracellular matrix in both modularity and crosslinking chemistry have the potential to recapitulate the instructive signals that ultimately control cell fate. Toward this goal, modular protein polymer-based hydrogels were created through genetic engineering and enzymatic crosslinking. Animal derived tissue transglutaminase (tTG) and recombinant human transglutaminase (hTG) enzymes were used for coupling two classes of protein polymers containing either lysine or glutamine, which have the recognition substrates for enzymatic crosslinking evenly spaced along the protein backbone. Utilizing tTG under physiological conditions, complete crosslinking occurred within 2 min, as determined by particle tracking microrheology. Hydrogel composition impacted the elastic storage modulus of the gel over 4-fold and also influenced microstructure and degree of swelling, but did not appreciably effect degradation by plasmin. Mouse 3T3 and primary human fibroblasts were cultured in both 2- and 3-dimensions without a decrease in cell viability and displayed spreading in 2D. The properties, which are controlled through the specific nature of the protein polymer precursors, render these gels valuable for in situ therapies. Furthermore, the modular hydrogel composition allows tailoring of mechanical and physical properties for specific tissue engineering applications. [ABSTRACT FROM AUTHOR]