The dimerized pentraxin-like domain of the adhesion G protein-coupled receptor 112 (ADGRG4) suggests function in sensing mechanical forces.

Adhesion G protein-coupled receptors (aGPCRs) feature large extracellular regions with modular domains that often resemble protein classes of various function. The pentraxin (PTX) domain, which is predicted by sequence homology within the extracellular region of four different aGPCR members, is well known to form pentamers and other oligomers. Oligomerization of GPCRs is frequently reported and mainly driven by interactions of the seven-transmembrane region and N or C termini. While the functional importance of dimers is well-established for some class C GPCRs, relatively little is known about aGPCR multimerization. Here, we showcase the example of ADGRG4, an orphan aGPCR that possesses a PTX-like domain at its very N-terminal tip, followed by an extremely long stalk containing serine-threonine repeats. Using X-ray crystallography and biophysical methods, we determined the structure of this unusual PTX-like domain and provide experimental evidence for a homodimer equilibrium of this domain which is Ca2+-independent and driven by intermolecular contacts that differ vastly from the known soluble PTXs. The formation of this dimer seems to be conserved in mammalian ADGRG4 indicating functional relevance. Our data alongside of theoretical considerations lead to the hypothesis that ADGRG4 acts as an in vivo sensor for shear forces in enterochromaffin and Paneth cells of the small intestine.

PTK7 is a positive allosteric modulator of GPR133 signaling in glioblastoma.

The adhesion G-protein-coupled receptor GPR133 (ADGRD1) supports growth of the brain malignancy glioblastoma. How the extracellular interactome of GPR133 in glioblastoma modulates signaling remains unknown. Here, we use affinity proteomics to identify the transmembrane protein PTK7 as an extracellular binding partner of GPR133 in glioblastoma. PTK7 binds the autoproteolytically generated N-terminal fragment of GPR133 and its expression in trans increases GPR133 signaling. This effect requires the intramolecular cleavage of GPR133 and PTK7's anchoring in the plasma membrane. PTK7's allosteric action on GPR133 signaling is additive with but topographically distinct from orthosteric activation by soluble peptide mimicking the endogenous tethered Stachel agonist. GPR133 and PTK7 are expressed in adjacent cells in glioblastoma, where their knockdown phenocopies each other. We propose that this ligand-receptor interaction is relevant to the pathogenesis of glioblastoma and possibly other physiological processes in healthy tissues.

Structural identification of DnaK binding sites within bovine and sheep bactenecin Bac7.

Bacterial resistance against common antibiotics is an increasing health problem. New pharmaceuticals for the treatment of infections caused by resistant pathogens are needed. Small proline-rich antimicrobial peptides (PrAMPs) from insects are known to bind intracellularly to the conventional substrate binding cleft of the E. coli Hsp70 chaperone DnaK. Furthermore, bactenecins from mammals, members of the cathelicidin family, also contain potential DnaK binding sites. Crystal structures of bovine and sheep Bac7 in complex with the DnaK substrate binding domain show that the peptides bind in the forward binding mode with a leucine positioned in the central hydrophobic pocket. In most structures, proline and arginine residues preceding leucine occupy the hydrophobic DnaK binding sites -1 and -2. Within bovine Bac7, four potential DnaK binding sites were identified.

Structural studies on the forward and reverse binding modes of peptides to the chaperone DnaK.

Hsp70 chaperones have been implicated in assisting protein folding of newly synthesized polypeptide chains, refolding of misfolded proteins, and protein trafficking. For these functions, the chaperones need to exhibit a significant promiscuity in binding to different sequences of hydrophobic peptide stretches. To characterize the structural basis of sequence specificity and flexibility of the Escherichia coli Hsp70 chaperone DnaK, we have analyzed crystal structures of the substrate binding domain of the protein in complex with artificially designed peptides as well as small proline-rich antimicrobial peptides. The latter peptides from mammals and insects were identified to target DnaK after cell penetration. Interestingly, the complex crystal structures reveal two different peptide binding modes. The peptides can bind either in a forward or in a reverse direction to the conventional substrate binding cleft of DnaK in an extended conformation. Superposition of the two binding modes shows a remarkable similarity in the side chain orientations and hydrogen bonding pattern despite the reversed peptide orientation. The DnaK chaperone has evolved to bind peptides in both orientations in the substrate binding cleft with comparable energy without rearrangements of the protein. Optimal hydrophobic interactions with binding pockets -2 to 0 appear to be the main determinant for the orientation and sequence position of peptide binding.