The WW domain is a new protein module with two highly conserved tryptophans that binds proline-rich peptide motifs in vitro. It is present in a number of signalling and regulatory proteins, often in several copies. Here we investigate the solution structure of the WW domain of human YAP65 (for Yes kinase-associated protein) in complex with proline-rich peptides containing the core motif PPxY. The structure of the domain with the bound peptide GTPPPPYTVG is a slightly curved, three-stranded, antiparallel beta-sheet. Two prolines pack against the first tryptophan, forming a hydrophobic buckle on the convex side of the sheet. The concave side has three exposed hydrophobic residues (tyrosine, tryptophan and leucine) which form the binding site for the ligand. A non-conserved isoleucine in the amino-terminal flanking region covers a hydrophobic patch and stabilizes the WW domain of human YAP65 in vitro. The structure of the WW domain differs from that of the SH3 domain and reveals a new design for a protein module that uses stacked aromatic surface residues to arrange a binding site for proline-rich peptides.
Phosphatidylinositol bisphosphate has been found to bind specifically to pleckstrin homology (PH) domains that are commonly present in signalling proteins but also found in cytoskeleton. We have studied the complexes of the beta‐spectrin PH domain and soluble inositol phosphates using both circular dichroism and nuclear magnetic resonance spectroscopy, and X‐ray crystallography. The specific binding site is located in the centre of a positively charged surface patch of the domain. The presence of 4,5‐bisphosphate group on the inositol ring is critical for binding. In the crystal structure that has been determined at 2.0 A resolution, inositol‐1,4,5‐trisphosphate is bound with salt bridges and hydrogen bonds through these phosphate groups whereas the 1‐phosphate group is mostly solvent‐exposed and the inositol ring has virtually no interactions with the protein. We propose a model in which PH domains are involved in reversible anchoring of proteins to membranes via their specific binding to phosphoinositides. They could also participate in a response to a second messenger such as inositol trisphosphate, organizing cross‐roads in cellular signalling.
Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidic picolitre droplet compartments. Using bait substrates, new hydrolases for sulfate monoesters and phosphotriesters were identified, mostly based on promiscuous activities presumed not to be under selection pressure. Spanning three protein superfamilies, these break new ground in sequence space: promiscuity now connects enzymes with only distantly related sequences. Most hits could not have been predicted by sequence analysis, because the desired activities have never been ascribed to similar sequences, showing how this approach complements bioinformatic harvesting of metagenomic sequencing data. Functional screening of a library of unprecedented size with excellent assay sensitivity has been instrumental in identifying rare genes constituting catalytically versatile hubs in sequence space as potential starting points for the acquisition of new functions.
Bruton's tyrosine kinase (Btk) is an enzyme which is involved in maturation of B cells. It is a target for mutations causing X‐linked agammaglobulinaemia (XLA) in man. We have determined the structure of the N‐terminal part of Btk by X‐ray crystallography at 1.6 Å resolution. This part of the kinase contains a pleckstrin homology (PH) domain and a Btk motif. The structure of the PH domain is similar to those published previously: a seven‐stranded bent β‐sheet with a C‐terminal α‐helix. Individual point mutations within the Btk PH domain which cause XLA can be classified as either structural or functional in the light of the three‐dimensional structure and biochemical data. All functional mutations cluster into the positively charged end of the molecule around the predicted binding site for phosphatidylinositol lipids. It is likely that these mutations inactivate the Btk pathway in cell signalling by reducing its affinity for inositol phosphates, which causes a failure in translocation of the kinase to the cell membrane. A small number of signalling proteins contain a Btk motif that always follows a PH domain in the sequence. This small module has a novel fold which is held together by a zinc ion bound by three conserved cysteines and a histidine. The Btk motif packs against the second half of the β‐sheet of the PH domain, forming a close contact with it. Our structure opens up new ways to study the role of the PH domain and Btk motif in the cellular function of Btk and the molecular basis of its dysfunction in XLA patients.
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