Directed evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome.

LC8 dynein light chain (DYNLL) is a eukaryotic hub protein that is thought to function as a dimerization engine. Its interacting partners are involved in a wide range of cellular functions. In its dozens of hitherto identified binding partners DYNLL binds to a linear peptide segment. The known segments define a loosely characterized binding motif: [D/S](-4)K(-3)X(-2)[T/V/I](-1)Q(0)[T/V](1)[D/E](2). The motifs are localized in disordered segments of the DYNLL-binding proteins and are often flanked by coiled coil or other potential dimerization domains. Based on a directed evolution approach, here we provide the first quantitative characterization of the binding preference of the DYNLL binding site. We displayed on M13 phage a naïve peptide library with seven fully randomized positions around a fixed, naturally conserved glutamine. The peptides were presented in a bivalent manner fused to a leucine zipper mimicking the natural dimer to dimer binding stoichiometry of DYNLL-partner complexes. The phage-selected consensus sequence V(-5)S(-4)R(-3)G(-2)T(-1)Q(0)T(1)E(2) resembles the natural one, but is extended by an additional N-terminal valine, which increases the affinity of the monomeric peptide twentyfold. Leu-zipper dimerization increases the affinity into the subnanomolar range. By comparing crystal structures of an SRGTQTE-DYNLL and a dimeric VSRGTQTE-DYNLL complex we find that the affinity enhancing valine is accommodated in a binding pocket on DYNLL. Based on the in vitro evolved sequence pattern we predict a large number of novel DYNLL binding partners in the human proteome. Among these EML3, a microtubule-binding protein involved in mitosis contains an exact match of the phage-evolved consensus and binds to DYNLL with nanomolar affinity. These results significantly widen the scope of the human interactome around DYNLL and will certainly shed more light on the biological functions and organizing role of DYNLL in the human and other eukaryotic interactomes.

GAP43 shows partial co-localisation but no strong physical interaction with prolyl oligopeptidase.

It has recently been proposed that prolyl oligopeptidase (POP), the cytosolic serine peptidase with neurological implications, binds GAP43 (Growth-Associated Protein 43) and is implicated in neuronal growth cone formation, axon guidance and synaptic plasticity. We investigated the interaction between GAP43 and POP with various biophysical and biochemical methods in vitro and studied the co-localisation of the two proteins in differentiated HeLa cells. GAP43 and POP showed partial co-localisation in the cell body as well as in the potential growth cone structures. We could not detect significant binding between the recombinantly expressed POP and GAP43 using gel filtration, CD, ITC and BIACORE studies, pull-down experiments, glutaraldehyde cross-linking and limited proteolysis. However, glutaraldehyde cross-linking suggested a weak and transient interaction between the proteins. Both POP and GAP43 interacted with artificial lipids in our in vitro model system, but the presence of lipids did not evoke binding between them. In native polyacrylamide gel electrophoresis, GAP43 interacted with one of the three forms of a polyhistidine-tagged prolyl oligopeptidase. The interaction of the two proteins was also evident in ELISA and we have observed co-precipitation of the two proteins during co-incubation at higher concentrations. Our results indicate that there is no strong and direct interaction between POP and GAP43 at physiological conditions.

Methylene substitution at the alpha-beta bridging position within the phosphate chain of dUDP profoundly perturbs ligand accommodation into the dUTPase active site.

dUTP pyrophosphatase, a preventive DNA repair enzyme, contributes to maintain the appropriate cellular dUTP/dTTP ratio by catalyzing dUTP hydrolysis. dUTPase is essential for viability in bacteria and eukaryotes alike. Identification of species-specific antagonists of bacterial dUTPases is expected to contribute to the development of novel antimicrobial agents. As a first general step, design of dUTPase inhibitors should be based on modifications of the substrate dUTP phosphate chain, as modifications in either base or sugar moieties strongly impair ligand binding. Based on structural differences between bacterial and human dUTPases, derivatization of dUTP-analogous compounds will be required as a second step to invoke species-specific character. Studies performed with dUTP analogues also offer insights into substrate binding characteristics of this important and structurally peculiar enzyme. In this study, alpha,beta-methylene-dUDP was synthesized and its complex with dUTPase was characterized. Enzymatic phosphorylation of this substrate analogue by pyruvate kinase was not possible in contrast to the successful enzymatic phosphorylation of alpha,beta-imino-dUDP. One explanation for this finding is that the different bond angles and the presence of the methylene group may preclude formation of a catalytically competent complex with the kinase. Crystal structure of E. coli dUTPase:alpha,beta-methylene-dUDP and E. coli dUTPase:dUDP:Mn complexes were determined and analyzed in comparison with previous data. Results show that the "trans" alpha-phosphate conformation of alpha,beta-methylene-dUDP differs from the catalytically competent "gauche" alpha-phosphate conformation of the imino analogue and the oxo substrate, manifested in the shifted position of the alpha-phosphorus by more than 3 A. The three-dimensional structures determined in this work show that the binding of the methylene analogue with the alpha-phosphorus in the "gauche" conformation would result in steric clash of the methylene group with the protein atoms. In addition, the metal ion cofactor was not bound in the crystal of the complex with the methylene analogue while it was clearly visible as coordinated to dUDP, arguing that the altered phosphate chain conformation also perturbs metal ion complexation. Isothermal calorimetry titrations indicate that the binding affinity of alpha,beta-methylene-dUDP toward dUTPase is drastically decreased when compared with that of dUDP. In conclusion, the present data suggest that while alpha,beta-methylene-dUDP seems to be practically nonhydrolyzable, it is not a strong binding inhibitor of dUTPase probably due to the altered binding mode of the phosphate chain. Results indicate that in some cases methylene analogues may not faithfully reflect the competent substrate ligand properties, especially if the methylene hydrogens are in steric conflict with the protein.

Active site closure facilitates juxtaposition of reactant atoms for initiation of catalysis by human dUTPase.

Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:alpha,beta-imino-dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C-terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker water oxygen and the alpha-phosphorus of the substrate, decreasing their distance below the van der Waals limit. Complex interactions of the C-terminus with both substrate and product were observed via a specifically designed tryptophan sensor, suitable for further detailed kinetic and ligand binding studies. Results explain the key functional role of the C-terminus.

Temperature effects on alpha-crystallin structure probed by 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive two-wavelength fluorescent dye covalently attached to the single Cys residue.

The single Cys residue in the C-terminal domain of bovine eye lens alpha-crystallin was covalently labelled with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone. This novel SH-reactive two-band ratiometric fluorescent dye is characterized by excited state intramolecular proton transfer reaction yielding two highly emissive N* and T* bands separated by more than 100 nm. Their relative intensities are known to be highly sensitive to the H-bonding ability of the environment. Properties of the environment of the dye attached to the protein were studied under native-like conditions and at a range of elevated temperatures that are known to facilitate alpha-crystallin chaperone-like activity. We observe that on heating, the environment of the dye becomes more flexible and the H-bonding of the dye with the protein vicinity decreases. The spectroscopic properties observed on heating were partially restored after cooling, but the initial values were not reached on the time scale of our experiments (up to 3 h). This suggests that the changes of the dye microenvironment are connected with the rearrangements of alpha-crystallin quaternary structure. Since there is only one Cys residue in alphaA subunit of alpha-crystallin (whereas alphaB subunit contains no Cys), we attributed the observed temperature-induced changes of the dye's microenvironment to the particular site within alpha-crystallin molecule.

Chaperone-like activity of alpha-crystallin is enhanced by high-pressure treatment.

alpha-Crystallin, an oligomeric protein in vertebrate eye lens, is a member of the small heat-shock protein family. Several papers pointed out that its chaperone-like activity could be enhanced by increasing the temperature. We demonstrate in the present study that structural perturbations by high hydrostatic pressures up to 300 MPa also enhance this activity. In contrast with temperature-induced changes, the pressure-induced enhancement is reversible. After pressure release, the extra activity is lost with a relaxation time of 2.0+/-0.5 h. Structural alterations contributing to the higher activity were studied with IR and fluorescence spectroscopy, and light-scattering measurements. The results suggest that while the secondary structure barely changes under pressure, the interactions between the subunits weaken, the oligomers dissociate, the area of accessible hydrophobic surfaces significantly increases and the environment of tryptophan residues becomes slightly more polar. It seems that structural flexibility and the total surface area of the oligomers are the key factors in the chaperone capacity, and that the increase in the chaperone activity does not require the increase in the oligomer size as was assumed previously [Burgio, Kim, Dow and Koretz (2000) Biochem. Biophys. Res. Commun. 268, 426-432]. After pressure release, the structure of subunits are reorganized relatively quickly, whereas the oligomer size reaches its original value slowly with a relaxation time of 33+/-4 h. In our interpretation, both the fast and slow structural rearrangements have an impact on the functional relaxation.