Marko Hyvönen

Structure of the PH domain and Btk motif from Bruton's tyrosine kinase: molecular explanations for X-linked agammaglobulinaemia.

Brutons 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.

Structure of the PH domain from Bruton's tyrosine kinase in complex with inositol 1,3,4,5-tetrakisphosphate

BACKGROUND The activity of Brutons tyrosine kinase (Btk) is important for the maturation of B cells. A variety of point mutations in this enzyme result in a severe human immunodeficiency known as X-linked agammaglobulinemia (XLA). Btk contains a pleckstrin-homology (PH) domain that specifically binds phosphatidylinositol 3,4,5-trisphosphate and, hence, responds to signalling via phosphatidylinositol 3-kinase. Point mutations in the PH domain might abolish membrane binding, preventing signalling via Btk. RESULTS We have determined the crystal structures of the wild-type PH domain and a gain-of-function mutant E41K in complex with D-myo-inositol 1,3,4,5-tetra-kisphosphate (Ins (1,3,4,5)P4). The inositol Ins (1,3,4,5)P4 binds to a site that is similar to the inositol 1,4,5-trisphosphate binding site in the PH domain of phospholipase C-delta. A second Ins (1,3,4,5)P4 molecule is associated with the domain of the E41K mutant, suggesting a mechanism for its constitutive interaction with membrane. The affinities of Ins (1,3,4,5)P4 to the wild type (Kd = 40 nM), and several XLA-causing mutants have been measured using isothermal titration calorimetry. CONCLUSIONS Our data provide an explanation for the specificity and high affinity of the interaction with phosphatidylinositol 3,4,5-trisphosphate and lead to a classification of the XLA mutations that reside in the Btk PH domain. Mis-sense mutations that do not simply destabilize the PH fold either directly affect the interaction with the phosphates of the lipid head group or change electrostatic properties of the lipid-binding site. One point mutation (Q127H) cannot be explained by these facts, suggesting that the PH domain of Btk carries an additional function such as interaction with a Galpha protein.

Structural basis for the inhibition of activin signalling by follistatin

The secreted, multidomain protein follistatin binds activins with high affinity, inhibiting their receptor interaction. We have dissected follistatins domain structure and shown that the minimal activin‐inhibiting fragment of follistatin is comprised of the first and second Fs domains (Fs12). This protein can bind to activin dimer and form a stable complex containing two Fs12 molecules and one activin dimer. We have solved crystal structures of activin A alone and its complex with Fs12 fragment to 2 Å resolution. The complex structure shows how Fs12 molecules wrap around the back of the ‘wings’ of activin, blocking the type II receptor‐binding site on activin A. Arginine 192 in Fs2 is a key residue in this interaction, inserting itself in between activins fingers. Complex formation imposes a novel orientation for the EGF‐ and Kazal‐like subdomains in the Fs2 domain and activin A shows further variation from the canonical TGF‐β family fold. The structure provides a detailed description of the inhibitory mechanism and gives insights into interactions of follistatin with other TGF‐β family proteins.

Using a Fragment‐Based Approach To Target Protein–Protein Interactions

The ability to identify inhibitors of protein–protein interactions represents a major challenge in modern drug discovery and in the development of tools for chemical biology. In recent years, fragment‐based approaches have emerged as a new methodology in drug discovery; however, few examples of small molecules that are active against chemotherapeutic targets have been published. Herein, we describe the fragment‐based approach of targeting the interaction between the tumour suppressor BRCA2 and the recombination enzyme RAD51; it makes use of a screening pipeline of biophysical techniques that we expect to be more generally applicable to similar targets. Disruption of this interaction in vivo is hypothesised to give rise to cellular hypersensitivity to radiation and genotoxic drugs. We have used protein engineering to create a monomeric form of RAD51 by humanising a thermostable archaeal orthologue, RadA, and used this protein for fragment screening. The initial fragment hits were thoroughly validated biophysically by isothermal titration calorimetry (ITC) and NMR techniques and observed by X‐ray crystallography to bind in a shallow surface pocket that is occupied in the native complex by the side chain of a phenylalanine from the conserved FxxA interaction motif found in BRCA2. This represents the first report of fragments or any small molecule binding at this protein–protein interaction site.

Pleckstrin homology domains: a fact file.

Structures of three different pleckstrin homology domains have been determined within the past year. They have a common core consisting of a seven-stranded and strongly bent beta-sheet and a C-terminal alpha-helix that packs against the beta-sheet. Phosphatidylinositol 4,5-bisphosphate and related compounds specifically bind to pleckstrin homology domains, suggesting that the domain may be involved in reversible anchorage to membranes or in recognition of a second messenger, such as inositol 1,4,5-trisphosphate. Pleckstrin homology domains have also been suggested to bind to the G beta gamma complex, but direct evidence for this is missing.

An efficient, multiply promiscuous hydrolase in the alkaline phosphatase superfamily

We report a catalytically promiscuous enzyme able to efficiently promote the hydrolysis of six different substrate classes. Originally assigned as a phosphonate monoester hydrolase (PMH) this enzyme exhibits substantial second-order rate accelerations ((kcat/KM)/kw), ranging from 107 to as high as 1019, for the hydrolyses of phosphate mono-, di-, and triesters, phosphonate monoesters, sulfate monoesters, and sulfonate monoesters. This substrate collection encompasses a range of substrate charges between 0 and -2, transition states of a different nature, and involves attack at two different reaction centers (P and S). Intrinsic reactivities (half-lives) range from 200 days to 105 years under near neutrality. The substantial rate accelerations for a set of relatively difficult reactions suggest that efficient catalysis is not necessarily limited to efficient stabilization of just one transition state. The crystal structure of PMH identifies it as a member of the alkaline phosphatase superfamily. PMH encompasses four of the native activities previously observed in this superfamily and extends its repertoire by two further activities, one of which, sulfonate monoesterase, has not been observed previously for a natural enzyme. PMH is thus one of the most promiscuous hydrolases described to date. The functional links between superfamily activities can be presumed to have played a role in functional evolution by gene duplication.

Targeting Protein–Protein Interactions and Fragment-Based Drug Discovery

Protein-protein interactions (PPI) are integral to the majority of biological functions. Targeting these interactions with small molecule inhibitors is of increased interest both in academia as well as in the pharmaceutical industry, both for therapeutic purposes and in the search for chemical tools for basic science. Although the number of well-characterised examples is still relatively modest, it is becoming apparent that many different kinds of interactions can be inhibited using drug-like small molecules. Compared to active site targeting, PPI inhibition suffers from the particular problem of more exposed and less defined binding sites, and this imposes significant experimental challenges to the development of PPI inhibitors. PPI interfaces are large, up to thousands of square angstroms, and there is still debate as to what part of the interface one should target. We will review recent developments in the field of PPI inhibition, with emphasis on fragment-based methods, and discuss various factors one should take into account when developing small molecule inhibitors targeted at PPI interfaces.

Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics.

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.

A new member of the alkaline phosphatase superfamily with a formylglycine nucleophile: structural and kinetic characterisation of a phosphonate monoester hydrolase/phosphodiesterase from Rhizobium leguminosarum.

The alkaline phosphatase superfamily comprises a large number of hydrolytic metalloenzymes such as phosphatases and sulfatases. We have characterised a new member of this superfamily, a phosphonate monoester hydrolase/phosphodiesterase from Rhizobium leguminosarum (R/PMH) both structurally and kinetically. The 1.42 A crystal structure shows structural homology to arylsulfatases with conservation of the core alpha/beta-fold, the mononuclear active site and most of the active-site residues. Sulfatases use a unique formylglycine nucleophile, formed by posttranslational modification of a cysteine/serine embedded in a signature sequence (C/S)XPXR. We provide mass spectrometric and mutational evidence that R/PMH is the first non-sulfatase enzyme shown to use a formylglycine as the catalytic nucleophile. R/PMH hydrolyses phosphonate monoesters and phosphate diesters with similar efficiency. Burst kinetics suggest that substrate hydrolysis proceeds via a double-displacement mechanism. Kinetic characterisation of active-site mutations establishes the catalytic contributions of individual residues. A mechanism for substrate hydrolysis is proposed on the basis of the kinetic data and structural comparisons with E. coli alkaline phosphatase and Pseudomonas aeruginosa arylsulfatase. R/PMH represents a further example of conservation of the overall structure and mechanism within the alkaline phosphatase superfamily.

From crystal packing to molecular recognition: prediction and discovery of a binding site on the surface of polo-like kinase 1

Protein–protein interactions are notoriously difficult to target with small molecules as large, discontinuous surfaces are often involved1, 2 that can adopt different conformations to interact with diverse binding partners.3–5 In addition, protein surfaces are inherently flexible, exemplified by reports of hits from high-throughput screens that were found to bind in previously unidentified pockets resulting from surface flexibility.6 These factors significantly complicate structure-based drug discovery in the context of protein–protein interfaces.7 The ability to understand the flexibility of the protein surface and predict its adaptive changes conditioned by molecular recognition of a ligand would open up new avenues for targeting protein–protein interactions. However, despite significant interest there are few systematic methods to accomplish this.8, 9