Tousled-like kinase 2 targets ASF1 histone chaperones through client mimicry.

Tousled-like kinases (TLKs) are nuclear serine-threonine kinases essential for genome maintenance and proper cell division in animals and plants. A major function of TLKs is to phosphorylate the histone chaperone proteins ASF1a and ASF1b to facilitate DNA replication-coupled nucleosome assembly, but how TLKs selectively target these critical substrates is unknown. Here, we show that TLK2 selectivity towards ASF1 substrates is achieved in two ways. First, the TLK2 catalytic domain recognizes consensus phosphorylation site motifs in the ASF1 C-terminal tail. Second, a short sequence at the TLK2 N-terminus docks onto the ASF1a globular N-terminal domain in a manner that mimics its histone H3 client. Disrupting either catalytic or non-catalytic interactions through mutagenesis hampers ASF1 phosphorylation by TLK2 and cell growth. Our results suggest that the stringent selectivity of TLKs for ASF1 is enforced by an unusual interaction mode involving mutual recognition of a short sequence motifs by both kinase and substrate.

Structure and activation mechanism of the yeast RNA Pol II CTD kinase CTDK-1 complex.

The C-terminal domain (CTD) kinase I (CTDK-1) complex is the primary RNA Polymerase II (Pol II) CTD Ser2 kinase in budding yeast. CTDK-1 consists of a cyclin-dependent kinase (CDK) Ctk1, a cyclin Ctk2, and a unique subunit Ctk3 required for CTDK-1 activity. Here, we present a crystal structure of CTDK-1 at 1.85-Å resolution. The structure reveals that, compared to the canonical two-component CDK-cyclin system, the third component Ctk3 of CTDK-1 plays a critical role in Ctk1 activation by stabilizing a key element of CDK regulation, the T-loop, in an active conformation. In addition, Ctk3 contributes to the assembly of CTDK-1 through extensive interactions with both Ctk1 and Ctk2. We also demonstrate that CTDK-1 physically and genetically interacts with the serine/arginine-like protein Gbp2. Together, the data in our work reveal a regulatory mechanism of CDK complexes.

Mechanism of self/nonself-discrimination in Brassica self-incompatibility.

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.

Transient Deprotonation of the Chromophore Affects Protein Dynamics Proximal and Distal to the Linear Tetrapyrrole Chromophore in Phytochrome Cph1.

Phytochromes are biological red/far-red light sensors found in many organisms. Prototypical phytochromes, including Cph1 from the cyanobacterium Synechocystis 6803, act as photochemical switches that interconvert between stable red (Pr)- and metastable far-red (Pfr)-absorbing states induced by photoisomerization of the bilin chromophore. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY) and the C-terminal transmitter (output) module, usually a histidine kinase, as in the case of Cph1. The chromophore deprotonates transiently during the Pr → Pfr photoconversion in association with extensive global structural changes required for signal transmission. Here, we performed equilibrium studies in the Pr state, involving pH titration of the linear tetrapyrrole chromophore in different Cph1 constructs, and measurement of pH-dependent structural changes at various positions in the protein using picosecond time-resolved fluorescence anisotropy. The fluorescent reporter group was attached at positions 371 (PHY domain), 305 (GAF domain), and 120 (PAS domain), as well as at sites in the PAS-GAF bidomain. We show direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains. Our results suggest that chromophore deprotonation is closely associated with a higher protein mobility (conformational space) both in proximal and in distal protein sites, implying a causal relationship that might be important for the global large protein arrangements and thus intramolecular signal transduction.

InsP6 binding to PIKK kinases revealed by the cryo-EM structure of an SMG1-SMG8-SMG9 complex.

We report the 3.45-Å resolution cryo-EM structure of human SMG1-SMG8-SMG9, a phosphatidylinositol-3-kinase (PI(3)K)-related protein kinase (PIKK) complex central to messenger RNA surveillance. Structural and MS analyses reveal the presence of inositol hexaphosphate (InsP6) in the SMG1 kinase. We show that the InsP6-binding site is conserved in mammalian target of rapamycin (mTOR) and potentially other PIKK members, and that it is required for optimal in vitro phosphorylation of both SMG1 and mTOR substrates.

Insights into the EGFR SAR of N-phenylquinazolin-4-amine-derivatives using quantum mechanical pairwise-interaction energies.

Protein kinases are an important class of enzymes that play an essential role in virtually all major disease areas. In addition, they account for approximately 50% of the current targets pursued in drug discovery research. In this work, we explore the generation of structure-based quantum mechanical (QM) quantitative structure-activity relationship models (QSAR) as a means to facilitate structure-guided optimization of protein kinase inhibitors. We explore whether more accurate, interpretable QSAR models can be generated for a series of 76 N-phenylquinazolin-4-amine inhibitors of epidermal growth factor receptor (EGFR) kinase by comparing and contrasting them to other standard QSAR methodologies. The QM-based method involved molecular docking of inhibitors followed by their QM optimization within a ~ 300 atom cluster model of the EGFR active site at the M062X/6-31G(d,p) level. Pairwise computations of the interaction energies with each active site residue were performed. QSAR models were generated by splitting the datasets 75:25 into a training and test set followed by modelling using partial least squares (PLS). Additional QSAR models were generated using alignment dependent CoMFA and CoMSIA methods as well as alignment independent physicochemical, e-state indices and fingerprint descriptors. The structure-based QM-QSAR model displayed good performance on the training and test sets (r2 ~ 0.7) and was demonstrably more predictive than the QSAR models built using other methods. The descriptor coefficients from the QM-QSAR models allowed for a detailed rationalization of the active site SAR, which has implications for subsequent design iterations.

Structure and assembly mechanism of plant C2S2M2-type PSII-LHCII supercomplex.

In plants, the photosynthetic machinery photosystem II (PSII) consists of a core complex associated with variable numbers of light-harvesting complexes II (LHCIIs). The supercomplex, comprising a dimeric core and two strongly bound and two moderately bound LHCIIs (C2S2M2), is the dominant form in plants acclimated to limited light. Here we report cryo-electron microscopy structures of two forms of C2S2M2 (termed stacked and unstacked) from Pisum sativum at 2.7- and 3.2-angstrom resolution, respectively. In each C2S2M2, the moderately bound LHCII assembles specifically with a peripheral antenna complex CP24-CP29 heterodimer and the strongly bound LHCII, to establish a pigment network that facilitates light harvesting at the periphery and energy transfer into the core. The high mobility of peripheral antennae, including the moderately bound LHCII and CP24, provides insights into functional regulation of plant PSII.

Structural basis of the day-night transition in a bacterial circadian clock.

Circadian clocks are ubiquitous timing systems that induce rhythms of biological activities in synchrony with night and day. In cyanobacteria, timing is generated by a posttranslational clock consisting of KaiA, KaiB, and KaiC proteins and a set of output signaling proteins, SasA and CikA, which transduce this rhythm to control gene expression. Here, we describe crystal and nuclear magnetic resonance structures of KaiB-KaiC,KaiA-KaiB-KaiC, and CikA-KaiB complexes. They reveal how the metamorphic properties of KaiB, a protein that adopts two distinct folds, and the post-adenosine triphosphate hydrolysis state of KaiC create a hub around which nighttime signaling events revolve, including inactivation of KaiA and reciprocal regulation of the mutually antagonistic signaling proteins, SasA and CikA.

NMR elucidation of reduced B-Z transition activity of PKZ protein kinase at high NaCl concentration.

A Z-DNA binding protein (ZBP)-containing protein kinase (PKZ) in fish species has an important role in the innate immune response. Previous structural studies of the Zα domain of the PKZ from Carassius auratus (caZαPKZ) showed that the protein initially binds to B-DNA and induces B-Z transition of double stranded DNA in a salt concentration-dependent manner. However, the significantly reduced B-Z transition activity of caZαPKZ at high salt concentration was not fully understood. In this study, we present the binding affinity of the protein for B-DNA and Z-DNA and characterize its extremely low B-Z transition activity at 250 mM NaCl. Our results emphasize that the B-DNA-bound form of caZαPKZ can be used as molecular ruler to measure the degree of B-Z transition.

Non-degradative Ubiquitination of Protein Kinases.

Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

Structure of Ctk3, a subunit of the RNA polymerase II CTD kinase complex, reveals a noncanonical CTD-interacting domain fold.

CTDK-I is a yeast kinase complex that phosphorylates the C-terminal repeat domain (CTD) of RNA polymerase II (Pol II) to promote transcription elongation. CTDK-I contains the cyclin-dependent kinase Ctk1 (homologous to human CDK9/CDK12), the cyclin Ctk2 (human cyclin K), and the yeast-specific subunit Ctk3, which is required for CTDK-I stability and activity. Here we predict that Ctk3 consists of a N-terminal CTD-interacting domain (CID) and a C-terminal three-helix bundle domain. We determine the X-ray crystal structure of the N-terminal domain of the Ctk3 homologue Lsg1 from the fission yeast Schizosaccharomyces pombe at 2.0 Å resolution. The structure reveals eight helices arranged into a right-handed superhelical fold that resembles the CID domain present in transcription termination factors Pcf11, Nrd1, and Rtt103. Ctk3 however shows different surface properties and no binding to CTD peptides. Together with the known structure of Ctk1 and Ctk2 homologues, our results lead to a molecular framework for analyzing the structure and function of the CTDK-I complex.

Snapshots of Conformational Changes Shed Light into the NtrX Receiver Domain Signal Transduction Mechanism.

Brucella abortus is an important pathogenic bacterium that has to overcome oxygen deficiency in order to achieve a successful infection. Previously, we proved that a two-component system formed by the histidine kinase NtrY and the response regulator NtrX is essential to achieve an adaptive response to low oxygen tension conditions. Even though the relevance of this signaling pathway has already been demonstrated in other microorganisms, its molecular activation mechanism has not yet been described in detail. In this article, we report the first crystal structures from different conformations of the NtrX receiver domain from B. abortus, and we propose a sequence of events to explain the structural rearrangements along the activation process. The analysis of the structures obtained in the presence of the phosphoryl group analog beryllofluoride led us to postulate that changes in the interface formed by the α4 helix and the ß5 strand are important for the activation, producing a reorientation of the α5 helix. Also, a biochemical characterization of the NtrX receiver domain enzymatic activities was performed, describing its autophosphorylation and autodephosphorylation kinetics. Finally, the role of H85, an important residue, was addressed by site-directed mutagenesis. Overall, these results provide significant structural basis for understanding the response regulator activation in this bacterial two-component system.

AFM characterization of biomolecules in physiological environment by an advanced nanofabricated probe.

Many relevant questions in biology and medicine require both topography and chemical information with high spatial resolution. Several biological events that occur at the nanometer scale level need to be investigated in physiological conditions. In this regard Atomic Force Microscopy (AFM) is one of the most powerful tools for label-free nanoscale characterization of biological samples in liquid environment. Recently, the coupling of Raman spectroscopy to scanning probe microscopies has opened new perspectives on this subject; however, the coupling of quality AFM spectroscopy with Raman spectroscopy in the same probe is not trivial. In this work we report about the AFM capabilities of an advanced high-resolution probe that has been previously nanofabricated by our group for coupling with Raman spectroscopy applications. We investigate its use for liquid AFM measurements on biological model samples like lipid bilayers, amyloid fibrils, and titin proteins. We demonstrate topography resolution down to nanometer level, force measurement and stable imaging capability. We also discuss about its potential as nanoscale chemical probe in liquid phase.

Phosphorylation primes vinculin for activation.

Vinculin phosphorylation has been implicated as a potential mechanism for focal adhesion growth and maturation. Four vinculin residues-Y100, S1033, S1045, and Y1065-are phosphorylated by kinases during focal adhesion maturation. In this study, phosphorylation at each of these residues is simulated using molecular dynamics models. The simulations demonstrate that once each phosphorylated vinculin structure is at equilibrium, significant local conformational changes result that may impact either vinculin activation or vinculin binding to actin and PIP2. Simulation of vinculin activation after phosphorylation shows that the added phosphoryl groups can prime vinculin for activation. It remains to be seen if vinculin can be phosphorylated at S1033 in vivo, but these simulations highlight that in the event of a S1033 phophorylation vinculin will likely be primed for activation.

Are titin properties reflected in single myofibrils?

Titin is a structural protein in muscle that spans the half sarcomere from Z-band to M-line. Although there are selected studies on titin's mechanical properties from tests on isolated molecules or titin fragments, little is known about its behavior within the structural confines of a sarcomere. Here, we tested the hypothesis that titin properties might be reflected well in single myofibrils. Single myofibrils from rabbit psoas were prepared for measurement of passive stretch-shortening cycles at lengths where passive titin forces occur. Three repeat stretch-shortening cycles with magnitudes between 1.0 and 3.0µm/sarcomere were performed at a speed of 0.1µm/s·sarcomere and repeated after a ten minute rest at zero force. These tests were performed in a relaxation solution (passive) and an activation solution (active) where cross-bridge attachment was inhibited with 2,3 butanedionemonoxime. Myofibrils behaved viscoelastically producing an increased efficiency with repeat stretch-shortening cycles, but a decreased efficiency with increasing stretch magnitudes. Furthermore, we observed a first distinct inflection point in the force-elongation curve at an average sarcomere length of 3.5µm that was associated with an average force of 68±5nN/mm. This inflection point was thought to reflect the onset of Ig domain unfolding and was missing after a ten minute rest at zero force, suggesting a lack of spontaneous Ig domain refolding. These passive myofibrillar properties observed here are consistent with those observed in isolated titin molecules, suggesting that the mechanics of titin are well preserved in isolated myofibrils, and thus, can be studied readily in myofibrils, rather than in the extremely difficult and labile single titin preparations.

Assembly of a protein "brush" by end-grafting titin fragments to liposomes.

Titin is a giant filamentous protein of striated muscle composed of >300 immunoglobulin-like domains linked in tandem. Here, we demonstrate that a six-immunoglobulin fragment of titin carrying a poly-histidine tag forms a protein "brush" on liposomes containing metallochelating lipids. These specimens might allow the direct visualization by cryo-EM of frozen hydrated and unstained titin chains with preserved architectural features.

Molecular origin of the hierarchical elasticity of titin: simulation, experiment, and theory.

This review uses the giant muscle protein titin as an example to showcase the capability of molecular dynamics simulations. Titin is responsible for the passive elasticity in muscle and is a chain composed of immunoglobulin (Ig)-like and fibronectin III (FN-III)-like domains, as well as PEVK segments rich in proline (P), glutamate (E), valine (V), and lysine (K). The elasticity of titin is derived in stages of extension under increasing external force: Ig domain straightening occurs first (termed tertiary structure elasticity), followed by the extension of the disordered PEVK segments. At larger extension and force, Ig domains unfold one by one (termed secondary structure elasticity). With the availability of crystal structures of single and connected Ig domains, the tertiary and secondary structure elasticity of titin was investigated through molecular dynamics simulations, unveiling the molecular origin of titin's elasticity.

Roles of titin in the structure and elasticity of the sarcomere.

The giant protein titin is thought to play major roles in the assembly and function of muscle sarcomeres. Structural details, such as widths of Z- and M-lines and periodicities in the thick filaments, correlate with the substructure in the respective regions of the titin molecule. Sarcomere rest length, its operating range of lengths, and passive elastic properties are also directly controlled by the properties of titin. Here we review some recent titin data and discuss its implications for sarcomere architecture and elasticity.

Tertiary and secondary structure elasticity of a six-Ig titin chain.

The protein titin functions as a mechanical spring conferring passive elasticity to muscle. Force spectroscopy studies have shown that titin exhibits several regimes of elasticity. Disordered segments bring about a soft, entropic spring-type elasticity; secondary structures of titin's immunoglobulin-like (Ig-) and fibronectin type III-like (FN-III) domains provide a stiff elasticity. In this study, we demonstrate a third type of elasticity due to tertiary structure and involving domain-domain interaction and reorganization along the titin chain. Through 870 ns of molecular dynamics simulations involving 29,000-635,000 atom systems, the mechanical properties of a six-Ig domain segment of titin (I65-I70), for which a crystallographic structure is available, are probed. The results reveal a soft tertiary structure elasticity. A remarkably accurate statistical mechanical description for this elasticity is derived and applied. Simulations also studied the stiff, secondary structure elasticity of the I65-I70 chain due to the unraveling of its domains and revealed how force propagates along the chain during the secondary structure elasticity response.

Shape and flexibility in the titin 11-domain super-repeat.

Titin is a giant protein of striated muscle with important roles in the assembly, intracellular signalling and passive mechanical properties of sarcomeres. The molecule consists principally of approximately 300 immunoglobulin and fibronectin domains arranged in a chain more than 1 mum long. The isoform-dependent N-terminal part of the molecule forms an elastic connection between the end of the thick filament and the Z-line. The larger, constitutively expressed C-terminal part is bound to the thick filament. Through most of the thick filament part, the immunoglobulin and fibronectin domains are arranged in a repeating pattern of 11 domains termed the 'large super-repeat'. There are 11 contiguous copies of the large super-repeat making up a segment of the molecule nearly 0.5 mum long. We have studied a set of two-domain and three-domain recombinant fragments from the large super-repeat region by electron microscopy, synchrotron X-ray solution scattering and analytical ultracentrifugation, with the goal of reconstructing the overall structure of this part of titin. The data illustrate different average conformations in different domain pairs, which correlate with differences in interdomain linker lengths. They also illustrate interdomain bending and flexibility around average conformations. Overall, the data favour a helical conformation in the super-repeat. They also suggest that this region of titin is dimerized when bound to the thick filament.