A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death.

BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.

CDKL Family Kinases Have Evolved Distinct Structural Features and Ciliary Function.

Various kinases, including a cyclin-dependent kinase (CDK) family member, regulate the growth and functions of primary cilia, which perform essential roles in signaling and development. Neurological disorders linked to CDK-Like (CDKL) proteins suggest that these underexplored kinases may have similar functions. Here, we present the crystal structures of human CDKL1, CDKL2, CDKL3, and CDKL5, revealing their evolutionary divergence from CDK and mitogen-activated protein kinases (MAPKs), including an unusual ?J helix important for CDKL2 and CDKL3 activity. C. elegans CDKL-1, most closely related to CDKL1-4 and localized to neuronal cilia transition zones, modulates cilium length; this depends on its kinase activity and ?J helix-containing C terminus. Human CDKL5, linked to Rett syndrome, also localizes to cilia, and it impairs ciliogenesis when overexpressed. CDKL5 patient mutations modeled in CDKL-1 cause localization and/or cilium length defects. Together, our studies establish a disease model system suggesting cilium length defects as a pathomechanism for neurological disorders, including epilepsy.

Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16.

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts.

Sensitive, high throughput detection of proteins in individual, surfactant-stabilized picoliter droplets using nanoelectrospray ionization mass spectrometry.

Droplet-based fluidics is emerging as a powerful platform for single cell analysis, directed evolution of enzymes, and high throughput screening studies. Due to the small amounts of compound compartmentalized in each droplet, detection has been primarily by fluorescence. To extend the range of experiments that can be carried out in droplets, we have developed the use of electrospray ionization mass spectrometry (ESI-MS) to measure femtomole quantities of proteins in individual pico- to nanoliter droplets. Surfactant-stabilized droplets containing analyte were produced in a flow-focusing droplet generation microfluidic device using fluorocarbon oil as the continuous phase. The droplets were collected off-chip for storage and reinjected into microfluidic devices prior to spraying the emulsion into an ESI mass spectrometer. Crucially, high quality mass spectra of individual droplets were obtained from emulsions containing a mixture of droplets at >150 per minute, opening up new routes to high throughput screening studies.

The p53 cofactor Strap exhibits an unexpected TPR motif and oligonucleotide-binding (OB)-fold structure.

Activation of p53 target genes for tumor suppression depends on the stress-specific regulation of transcriptional coactivator complexes. Strap (stress-responsive activator of p300) is activated upon DNA damage by ataxia telangiectasia mutated (ATM) and Chk2 kinases and is a key regulator of the p53 response. In addition to antagonizing Mdm2, Strap facilitates the recruitment of p53 coactivators, including JMY and p300. Strap is a predicted TPR-repeat protein, but shows only limited sequence identity with any protein of known structure. To address this and to elucidate the molecular mechanism of Strap activity we determined the crystal structure of the full-length protein at 2.05 Å resolution. The structure of Strap reveals an atypical six tetratricopeptide repeat (TPR) protein that also contains an unexpected oligonucleotide/oligosaccharide-binding (OB)-fold domain. This previously unseen domain organization provides an extended superhelical scaffold allowing for protein-protein as well as protein-DNA interaction. We show that both of the TPR and OB-fold domains localize to the chromatin of p53 target genes and exhibit intrinsic regulatory activity necessary for the Strap-dependent p53 response.

Carboxyl pK(a) values and acid denaturation of BBL.

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK(a) values of all the carboxylic residues in this pH range. We employed (13)C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK(a) values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK(a) values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK(a), and, using thermodynamic cycles, we could calculate their pK(a)s in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured (13)C(α) chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.

The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces.

Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Hückel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution.

Structural basis for Par-4 recognition by the SPRY domain- and SOCS box-containing proteins SPSB1, SPSB2, and SPSB4.

The mammalian SPRY domain- and SOCS box-containing proteins, SPSB1 to SPSB4, belong to the SOCS box family of E3 ubiquitin ligases. Substrate recognition sites for the SPRY domain are identified only for human Par-4 (ELNNNL) and for the Drosophila orthologue GUSTAVUS binding to the DEAD-box RNA helicase VASA (DINNNN). To further investigate this consensus motif, we determined the crystal structures of SPSB1, SPSB2, and SPSB4, as well as their binding modes and affinities for both Par-4 and VASA. Mutation of each of the three Asn residues in Par-4 abrogated binding to all three SPSB proteins, while changing EL to DI enhanced binding. By comparison to SPSB1 and SPSB4, the more divergent protein SPSB2 showed only weak binding to Par-4 and was hypersensitive to DI substitution. Par-4((59-77)) binding perturbed NMR resonances from a number of SPSB2 residues flanking the ELNNN binding site, including loop D, which binds the EL/DI sequence. Although interactions with the consensus peptide motif were conserved in all structures, flanking sites in SPSB2 were identified as sites of structural change. These structural changes limit high-affinity interactions for SPSB2 to aspartate-containing sequences, whereas SPSB1 and SPSB4 bind strongly to both Par-4 and VASA peptides.

The folding mechanism of BBL: Plasticity of transition-state structure observed within an ultrafast folding protein family.

Studies on members of protein families with similar structures but divergent sequences provide insights into the effects of sequence composition on the mechanism of folding. Members of the peripheral subunit-binding domain (PSBD) family fold ultrafast and approach the smallest size for cooperatively folding proteins. Phi-Value analysis of the PSBDs E3BD and POB reveals folding via nucleation-condensation through structurally very similar, polarized transition states. Here, we present a Phi-value analysis of the family member BBL and found that it also folds by a nucleation-condensation mechanism. The mean Phi values of BBL, E3BD, and POB were near identical, indicating similar fractions of non-covalent interactions being formed in the transition state. Despite the overall conservation of folding mechanism in this protein family, however, the pattern of Phi values determined for BBL revealed a larger dispersion of the folding nucleus across the entire structure, and the transition state was less polarized. The observed plasticity of transition-state structure can be rationalized by the different helix-forming propensities of PSBD sequences. The very strong helix propensity in the first helix of BBL, relative to E3BD and POB, appears to recruit more structure formation in that helix in the transition state at the expense of weaker interactions in the second helix. Differences in sequence composition can modulate transition-state structure of even the smallest natural protein domains.

Downhill versus barrier-limited folding of BBL 1: energetic and structural perturbation effects upon protonation of a histidine of unusually low pKa.

A dispersion of melting temperatures at pH5.3 for individual residues of the BBL protein domain has been adduced as evidence for barrier-free downhill folding. Other members of the peripheral subunit domain family fold cooperatively at pH7. To search for possible causes of anomalies in BBL's denaturation behavior, we measured the pH titration of individual residues by heteronuclear NMR. At 298 K, the pK(a) of His142 was close to that of free histidine at 6.47+/-0.04, while that of the more buried His166 was highly perturbed at 5.39+/-0.02. Protonation of His166 is thus energetically unfavorable and destabilizes the protein by approximately 1.5 kcal/mol. Changes in C(alpha) secondary shifts at pH5.3 showed a decrease in helicity of the C-terminus of helix 2, where His166 is located, which was accompanied by a measured decrease of 1.1+/-0.2 kcal/mol in stability from pH7 to 5.3. Protonation of His166 perturbs, therefore, the structure of BBL. Only approximately 1% of the structurally perturbed state will be present at the biologically relevant pH7.6. Experiments at pH5.3 report on a near-equal mixture of the two different native states. Further, at this pH, small changes of pH and pK(a) induced by changes in temperature will have near-maximal effects on pH-dependent conformational equilibria and on propagation of experimental error. Accordingly, conventional barrier-limited folding predicts some dispersion of measured thermal unfolding curves of individual residues at pH5.3.

Downhill versus barrier-limited folding of BBL 2: mechanistic insights from kinetics of folding monitored by independent tryptophan probes.

Barrier-free downhill folding has been proposed for the peripheral subunit-binding domain BBL. To date, ultrafast kinetic experiments on BBL, which are crucial for a mechanistic understanding of folding, have been hampered by the lack of good intrinsic spectroscopic probes. Here, we present a detailed kinetic characterization of three single-point tryptophan mutants of BBL that have suitable fluorescence properties for following microsecond and nanosecond folding kinetics using temperature jump fluorescence spectroscopy. Experiments were performed at pH 7, which is optimal for stability and minimizes complications that arise from the presence of an alternative native-state conformation of BBL at lower pH. We examined the dependence of rate and equilibrium constants on concentration of denaturant and found that they follow well-established laws allowing kinetic transients to be related to events in folding and compared with equilibrium data. Logarithms of rate constants versus denaturant concentration yielded plots (chevrons) that are characteristic of barrier-limited folding for all mutants investigated, including a truncated sequence that was previously used in the proposal of downhill folding. The thermodynamic quantities calculated from the rate constants were in excellent agreement with those directly determined from equilibrium denaturation based on empirical two-state equations. We found that sequence truncation of BBL as used in studies proposing downhill folding leads to a large loss in helical content and protein stability, which were exacerbated at the low pH used in those studies. The kinetics and equilibria of folding of BBL fit to conventional barrier-limited kinetics.

Conservation of transition state structure in fast folding peripheral subunit-binding domains.

Phi-value analysis was used to characterise the structure of the transition state (TS) for folding of POB L146A Y166W, a peripheral subunit-binding domain that folds in microseconds. Helix 2 was structured in the TS with consolidating interactions from the structured loop that connects the two alpha-helices. This distribution of Phi-values was very similar to that determined for E3BD F166W, a homologue with high sequence and structural similarity. The extrapolated folding rate constants in water at 298 K were 210,000 s(-1) for POB and 27,500 s(-1) for E3BD. A contribution to the faster folding of POB came from its having significantly greater helical propensity in helix 2, the folding nucleus. The folding rate also appeared to be influenced by differences in the sequence and structural properties of the loop connecting the two helices. Unimodal downhill folding has been proposed as a conserved, biologically important property of peripheral subunit-binding domains. POB folds five times faster and E3BD folds slower than a proposed limit of 40,000 s(-1) for barrier-limited folding. However, experimental evidence strongly suggests that both POB L146A Y166W and E3BD F166W fold in a barrier-limited process through a very similar TS ensemble.

Structural biology: analysis of 'downhill' protein folding.

There is controversy as to whether homologues from the peripheral subunit binding domain family of small proteins fold 'downhill' (that is, non-cooperatively, in the absence of free-energy barriers between conformations) and whether they modulate their size for biological function. Sadqi et al. claim that Naf-BBL--a naphthylalanine-labelled, truncated version of this domain--folds in this way, on the grounds that they recorded a wide spread of melting temperatures of individual atoms measured by proton nuclear magnetic resonance (NMR) during their thermal denaturation. But their data are not of adequate quality to distinguish, within experimental error, between downhill folding and folding with a cooperative transition. Accordingly, their results offer no compelling evidence that Naf-BBL folds downhill, particularly as non-truncated, unmodified peripheral subunit binding domains seem to fold cooperatively.

Distinguishing between cooperative and unimodal downhill protein folding.

Conventional cooperative protein folding invokes discrete ensembles of native and denatured state structures in separate free-energy wells. Unimodal noncooperative ("downhill") folding, however, proposes an ensemble of states occupying a single free-energy well for proteins folding at >/=4 x 10(4) s(-1) at 298 K. It is difficult to falsify unimodal mechanisms for such fast folding proteins by standard equilibrium experiments because both cooperative and unimodal mechanisms can present the same time-averaged structural, spectroscopic, and thermodynamic properties when the time scale used for observation is longer than for equilibration. However, kinetics can provide the necessary evidence. Chevron plots with strongly sloping linear refolding arms are very difficult to explain by downhill folding and are a signature for cooperative folding via a transition state ensemble. The folding kinetics of the peripheral subunit binding domain POB and its mutants fit to strongly sloping chevrons at observed rate constants of >6 x 10(4) s(-1) in denaturant solution, extrapolating to 2 x 10(5) s(-1) in water. Protein A, which folds at 10(5) s(-1) at 298 K, also has a well-defined chevron. Single-molecule fluorescence energy transfer experiments on labeled Protein A in the presence of denaturant demonstrated directly bimodal distributions of native and denatured states.

General structural motifs of amyloid protofilaments.

Human CA150, a transcriptional activator, binds to and is co-deposited with huntingtin during Huntington's disease. The second WW domain of CA150 is a three-stranded beta-sheet that folds in vitro in microseconds and forms amyloid fibers under physiological conditions. We found from exhaustive alanine scanning studies that fibrillation of this WW domain begins from its denatured conformations, and we identified a subset of residues critical for fibril formation. We used high-resolution magic-angle-spinning NMR studies on site-specific isotopically labeled fibrils to identify abundant long-range interactions between side chains. The distribution of critical residues identified by the alanine scanning and NMR spectroscopy, along with the electron microscopy data, revealed the protofilament repeat unit: a 26-residue non-native beta-hairpin. The structure we report has similarities to the hairpin formed by the A(beta)((1-40)) protofilament, yet also contains closely packed side-chains in a "steric zipper" arrangement found in the cross-beta spine formed from small peptides from the Sup35 prion protein. Fibrillation of unrelated amyloidogenic sequences shows the common feature of zippered repeat units that act as templates for fiber elongation.

The transition state for folding of a peripheral subunit-binding domain contains robust and ionic-strength dependent characteristics.

The denaturant dependencies of the folding and unfolding kinetics were used to characterize the structure of the transition state for folding of E3BD, a peripheral subunit-binding domain. For the majority of E3BD mutants, the Phi-values calculated at 298 K from the analysis of chevron plots were in good agreement with those previously determined at 325 K using Arrhenius analysis. This agreement further demonstrates the general robustness of Phi-value analyses, since different experiments, methods of denaturation and thermodynamic assumptions were used to determine each set of Phi(F) values. The structure of the transition state for folding was grossly conserved at 298 K and 325 K, with residues in Helix I playing a lesser role in folding than those located in the 3(10) helix, disordered loop and Helix II. However, the energetic contributions of a cluster of basic residues close to the N-terminus and Helix I, which are an integral part of the ligand-binding site, were susceptible to ionic strength effects because of electrostatic strain in native and transition states of E3BD at low ionic strength. We found no evidence of the downhill folding previously proposed for E3BD, even though the conditions employed in this study significantly increased the energetic bias towards the native state.

Ultra-fast barrier-limited folding in the peripheral subunit-binding domain family.

We have determined the solution structures, equilibrium properties and ultra-fast folding kinetics for three bacterial homologues of the peripheral subunit-binding domain (PSBD) family. The mesophilic homologue, BBL, was less stable than the thermophilic and hyper-thermophilic variants (E3BD and POB, respectively). The broad unfolding transitions of each PSBD, when probed by different techniques, were essentially superimposable, consistent with co-operative denaturation. Temperature-jump and continuous-flow fluorescence methods were used to measure the folding kinetics for E3BD, POB and BBL. E3BD folded fairly rapidly at 298K (folding half-time approximately 25 micros) and BBL and POB folded even faster (folding half-times approximately 3-5 micros). The variations in equilibrium and kinetic behaviour observed for the PSBD family resembles that of the homeodomain family, where the folding pattern changes from apparent two-state transitions to multi-state kinetics as the denatured state becomes more structured. The faster folding of POB may be a consequence of its higher propensity to form helical structure in the region corresponding to the folding nucleus of E3BD. The ultra-fast folding of BBL appears to be a consequence of residual structure in the denatured ensemble, as with engrailed homeodomain. We discuss issues concerning "one-state", downhill folding, and find no evidence for, and strong evidence against, it occurring in these PSBDs. The shorter construct used previously for BBL was destabilized significantly and the stability further perturbed by the introduction of fluorescent probes. Thermal titrations for 11 side-chains scattered around the protein, when probed by (13)C-NMR experiments, could be fit globally to a common co-operative transition.

One-state downhill versus conventional protein folding.

Classical protein folding invokes a cooperative transition between distinct thermodynamic states that are individually populated at equilibrium and separated by an energy barrier. It has been proposed, however, that the small protein, BBL, undergoes one-step downhill folding whereby it folds non-cooperatively to its native state without encountering an appreciable energy barrier. Only a single conformational ensemble is populated under given conditions, and so the denatured state ensemble progressively changes into the native structure. A wide dispersion of thermal denaturation midpoints that was observed for an extrinsically labelled fragment of BBL is proposed to be evidence for its one-state, downhill folding, a phenomenon that is also suggested to be functionally important for BBL and its homologues. We found, however, that thermal denaturation of unlabelled wild-type BBL was highly cooperative, with very similar transition midpoints for the melting of secondary and tertiary interactions, as well as for individual residues when monitored by NMR. Similar results were also observed for two other homologues, E3BD and POB. Further, the extrinsic fluorophores perturbed the unfolding energetics of labelled BBL, and complicated its equilibrium behaviour. One-step downhill folding may well occur for some proteins that do not have distinct folded states but not for BBL and its well-folded homologues.

Rapid amyloid fiber formation from the fast-folding WW domain FBP28.

The WW domains are small proteins that contain a three-stranded, antiparallel beta-sheet. The 40-residue murine FBP28 WW domain rapidly formed twirling ribbon-like fibrils at physiological temperature and pH, with morphology typical of amyloid fibrils. These ribbons were unusually wide and well ordered, making them highly suitable for structural studies. Their x-ray and electron-diffraction patterns displayed the characteristic amyloid fiber 0.47-nm reflection of the cross-beta diffraction signature. Both conventional and electron cryomicroscopy showed clearly that the ribbons were composed of many 2.5-nm-wide subfilaments that ran parallel to the long axis of the fiber. There was a region of lower density along the center of each filament. Lateral association of these filaments generated twisted, often interlinked, sheets up to 40 nm wide and many microns in length. The pitch of the helix varied from 60 to 320 nm, depending on the width of the ribbon. The wild-type FBP28 fibers were formed under conditions in which multiexponential folding kinetics is observed in other studies and which was attributed to a change in the mechanism of folding. It is more likely that those phases result from initial events in the off-pathway aggregation observed here.