Re-entrant spin glass (RSG) transitions in Ni-Mn and Au-Fe have been reassessed by acoustic measurements of the magneto-mechanical damping by domain walls. Stress-induced non-thermally activated domain wall dynamics is progressively replaced by an intense thermally activated relaxational response when the temperature approaches the RSG freezing point. A “frozen” state with negligible motion of domain walls on atomic and mesoscopic scales occurs in the RSG. We propose that RSG freezing has its origin in intrinsic properties of domain walls. The real part of the susceptibility, χ′(T), which shows a cusp during the SG transition is replaced at Tf by a decay of the ferromagnetic moment3 while the maximum in the imaginary part, χ″(T), is maintained. The very nature of the RSG transition is controversial. One model claims that RSG is simply a mixture of ferromagnetic and SG states with spin glass-like clusters embedded in a network of ferromagnetic domains12. Freezing of the clusters may generate random fields, provoking the breakdown of the ferromagnetic long-range order13. These magnetic clusters have not been observed experimentally, however. The co-existence of ferromagnetism and glass-like states may relate to atomic scale chemical disorder.Re-entrant spin glasses, RSGs, unlike ordinary spin glasses, SGTf15. This model involves a strong decrease of the domain wall mobility15 and explains the near vanishing of χ′. Similarly, the RSG-like behavior of high quality stoichiometric single crystals of magnetite has been explained by pinning of domain walls by unknown centers able to migrate and pin domain walls at temperatures around 25 K16. The RSG freezing around 40–50 K in Fe-Ni invar alloys20 has also been attributed to pinning by static defects such as grain boundaries or compositional fluctuations at the scale of the domain wall width. The wide scope of possible candidates for “pinners” during RSG freezing exemplifies the difficulties in identifying their possible origin. In addition to uncertainties about the pinning mechanism, the domain wall pinning concept does not explain the existence of “frustrated spins” in disordered RSGs. Finally, a similar line of thought, recently introduced in analogy with ferroelectrics22, involves a generic concept of “domain glasses”. It proposes that glassy behaviour does not necessarily require atomic-scale disorder, but can originate from extended defects in ferroic microstructure, such as domain walls and their mutual interaction, and internal substructures such as Bloch lines and Bloch points. We will argue in this paper that intrinsic freezing of domain wall is the origin of RSG transition.Another line of thinking, not invoking atomic-scale structural disorder, considers the role of magnetic domain walls in RSG materials, which were observed above and below δ. Damping (the imaginary part of the modulus) is particularly sensitive to the DW dynamics: the ideal material has a finite modulus and no losses. Thus a small change in the dynamics will yield only a small change in the modulus relative to the ideal modulus while a small change in the imaginary part of the modulus may be large compared to zero. MMD is defined as any damping that is suppressed by an external magnetic field. In the present context, linear damping δi is defined as being independent of the vibrational strain amplitude ε0. The components of MMD are26:δμ, measured at low strain amplitudes,a linear micro eddy current damping, δM, anda linear macro eddy current damping component, δh(ε0), emerging at higher strain amplitudes.a non-linear hysteretic damping, To clarify the involvement of domains walls in RSGs, we conducted new acoustic experiments studying the magneto-mechanical damping (MMD), which will be expressed as logarithmic decrement of oscillations δμ is commonly ascribed to short-range reversible domain wall displacements (much) less than the average dimensions of magnetic domains, δh is associated with larger-scale irreversible domain wall motions, more comparable with domain sizes. δM is measured at a scale comparable with the penetration depth of the macroscopic electromagnetic wave, averaging over many domains. All three MMD components depend on H and vanish at saturation. δi and δh(ε0) display their maximum values either for zero magnetization M = 0, H = 0, or in its vicinity, then decline monotonously with applied field H28. δM has its maximum value at H roughly equal half the saturating field; for M = 0, δM = 029. Thus, detailed studies of MMD components versus temperature, field and strain amplitude show aspects of the domain wall dynamics during RSG freezing at various length scales. Furthermore, δμ shows a Debye type maximum at frequencies f at ca.105–106 Hz30, and is usually negligibly small below 103 Hz and above 107 Hz. Here, in contrast to previous acoustic studies of SG and RSG freezing36, we use the frequency (~105 Hz) best suited for studying intrinsic properties of domain walls through δμ. We report results for three typical RSGs, namely Ni80Mn20, Ni77Mn23 and Au86Fe14.These components are associated with different length scales: δ with strain amplitude ε0 for Ni80Mn20 measured at 251 K > T > 16 K. The damping remains reasonably linear for strain amplitudes ε0 < 10−6 over the full temperature range. A strong non-linearity emerges above ε0 ≈ 10−6 between 251 and 212 K. The non-linear damping component is δh(ε0) = δ(ε0) − δi. It reaches a maximum at ca. 70 K  for the two Ni-Mn alloys  with the temperature axis  the discontinuity in dδi/dT  from the maximum slopes of the temperature dependences of Im(Z) and δh (maximum freezing rate at Tf); (ii) the intercepts of the steepest slopes of Im(Z) and δh versus T with the temperature axis. The latter temperatures, Tf*, correspond to a “frozen” state of the system. A comparison of these values, obtained from Im(Z) (AC permeability) and damping data are given in Table We determine ors Figs  and 3: . The E(T) dependences for H = 0 and large H coincide and Tf. δ - the out-of-phase component. The nonelastic strain vanishes when both δ vanish  and two near Tf (53 K and 48 K). At each temperature δi(H) and δ(H) data were measured for low and high strain amplitudes and a linear field ramp with extrema Hm = ±12 kA/m. The damping at low strain amplitudes and at high amplitudes (including the sum of linear and non-linear components) approach zero as they saturate near Hm = 10 kA/m. The linear damping term, δi, includes the micro- and macro-eddy terms and the relaxational damping δrel:The results of measurements of δ(H) registered at low, Fig. δh. We now describe the effect of H on the various MMD components.(i)δh(H)Hysteretic damping The difference between δh(H) δμ and δMMicro- and Macro- eddy current damping δi near H = 5 kA/m at 70 and 100 K and shoulders at 48 and 53 K in Fig. H below saturation. We identify them as due to the macro-eddy current component of damping δM. Since δM = 0 for M = 0 H = 0, the points for H = 0 represent the microeddy MMD and a new relaxational damping, δi|H = 0 = δμ + δrel, Fig. δi(H) do not permit further distinctions between micro- and macro-eddy MMD contributions.The side maxima of (iii)δrel(H)Relaxational damping (H) Fig.  is deter(H) Fig. . Each hy(H) Fig. .(ii)Micrδi(H) curves at 100 and 70 K do not include relaxational MMD components  ≈ δi. Accordingly, we use the entire δi curve as a measure of the background for δrel. The relaxational damping δrel and δrel is then determined as the difference δrel = δi − δi  and δrel(H) over a large range of the δh and δrel suppression by H.The nts Fig. . They al,H) Fig. , using d,H) Fig. . CompariTC and Tf, such as (i) an increased level of low amplitude linear damping δi, related to micro-eddy current damping δμ; (ii) the appearance of δh - the non-linear amplitude dependent hysteretic damping, (iii) existence of a peak in damping versus H that is independent of amplitude, which is the macro-eddy current peak, predicted by classical electromagnetic theory; macro-eddy damping disappears at H = 0 and H = Hsat; (iv) emergence of the ΔE – effect, and (v) suppression of all three types of damping by magnetic field. This classic picture changes dramatically near RSG freezing, however. The key observation is the proportionality between δrel(H) and δh(H). Since δh(H) is exclusively related to domain walls, the proportionality between δrel(H) and δh(H) suggests that δrel is also related to domain walls and, hence, the RSG transition is related to domain walls.Typical MMD features occur at temperatures between Z) in Fig. δi(T) in Fig. χ′(T) and χ″(T) in Ni7757Fe1Mn229. In addition, the peak of χ″ is a linear function of the magnetic field9, just as δrel is linear in strain amplitude. Finally, all non-linear effects in χ freeze out during RSG freezing9, just as δh in the damping pattern. The only difference between our damping measurements and the susceptibility measurements in9 is that the χ″ peak during RSG freezing was found to be independent of H9 while δi is completely suppressed by H in our work. A possible source of this disagreement is that the magnetic field employed in9 was below 400 A/m, which we find too low to show the field dependence of the relaxation, Fig. The behavior of Im (χ′and χ″) and that of the anelasticity (ΔE-effect and damping) indicate the same physical origin, namely the freezing of domain wall motion. This idea implies that domain walls are the key structural elements responsible for RSG freezing instead of SG-like clusters15. This proposal is consistent with the results of detailed studies by Sato et al.39 on neutron depolarization and small angle scattering in Ni77Mn23. They identified the SG-like clusters, coexisting in the RSG state with FM order, as vortex-like structures. All the properties of these vortices are perfectly consistent with those usually attributable to DWs: (i) the size and the total volume of the vortices decrease with applied field; (ii) the vortex-like structures exist also in the FM state at T > Tf, and are retained up to TC; (iii) the domain wall mobility decreases with lowering temperatures mainly due to freezing of spins in the vortices. The vortex-like structures always occur in complex arrays of domain walls as key ingredients of the RSG transition41.We propose, therefore, that the parallels between RSG-freezing of the susceptibility , which is not applicable in our case.Among microscopic mechanisms for atomic-scale domain wall relaxations, is escape from a Peierls potential. The corresponding relaxation process implies thermally activated creation and motion of atomic-scale kinks46. These linear structural entities can be susceptible to freezing similar to dislocations47 and to a transition from the non-thermally activated string-like behaviour to the thermally activated motion through creation and propagation of thermal kinks. In conclusion, an important consequence of the “domain glass” concept is that the glassy state is the consequence of intrinsic domain wall freezing and, therefore, may not necessarily require atomic structural disorder.The concept of a Peierls potential can be expanded to domain wall substructure, such as Bloch lines, able to control the DW mobilityfreezing is marked by a domain-wall related, relaxational linear magneto-mechanical damping peak, which is a new category of magnetomechanical damping;cooling through the freezing temperature is seen as the transition from the non thermally activated mode of domain wall motion to the thermally activated mode followed by a complete immobilization of domain walls on both atomic and mesoscopic scales;domain wall freezing implies variation of their structure on an atomic scale, resulting in complex spin arrangement and their immobilization. Specific mechanisms of domain wall freezing might include partitioning of domain walls into many low-symmetry variants or freezing of such elements of their internal substructure as the Bloch line in a Peierls-lattice potential.Detailed acoustic studies of domain wall dynamics show that the re-entrant spin glass transition is related to freezing of domain walls. This conclusion implies a qualitatively new interpretation of re-entrant spin glass transitions. The main features of the freezing process are:Ingots of Ni-Mn alloys were produced by induction casting from 99.99 wt% purity components and homogenized for 24 hrs at 1170 K, followed by water quenching. Samples with dimensions approximately 1.5 × 1.5 × 24 mm were cut by spark erosion.86Fe14 alloy was produced from 99.99 wt% purity ingredients. Fe pellets of appropriate mass were wrapped in Au foil and the cigar thus formed was sealed in an evacuated quartz tube, melted and water quenched. The ingot was re-melted 3 times, also in evacuated quartz tubes, and finally annealed at 1170 K for 24 hrs. The tube with the annealed ingot was taken rapidly from the furnace and the ingot was quenched in water immediately after breaking the quartz tube. Samples for ultrasonic experiments measured 1.5 × 1.5 × 12 mm. Compositions of all alloys were checked by EDS.A 10 g ingot of the Au48 at a frequency near 90 kHz. The experimental arrangement is described elsewhere49. It allows the determination of the logarithmic decrement, δ, and the resonant frequency, f, of the fundamental mode of the longitudinal oscillations of the sample. The effective Young’s modulus, E, was calculated from the resonant frequency, the density ρ and the length l of the sample, as E = 4ρl2f 2.Acoustic measurements were performed by three component piezoelectric composite oscillator techniqueδi and strain amplitude dependent internal friction δh between 15 and 300 K were registered simultaneously in the same temperature scan. Two values of the oscillatory strain amplitude ε0  × 10−5) were alternately stabilized for each temperature50. The strain amplitudes correspond to amplitude-independent and amplitude-dependent ranges of damping, respectively. This way, the linear damping versus temperature, δi(T), was directly registered for ε0 = 10−6. The damping measured at high strain amplitude contained both linear δi and non-linear δh components: δ(T) = δi(T) + δh(T). The non-linear damping temperature spectra were obtained as the difference between the spectra at high and low strain amplitudes: δh(T) = δ(T) − δi(T). Damping and Young’s modulus temperature spectra were recorded under different fixed polarizing magnetic fields H, including H = 0.In the first type of acoustic experiments, temperature spectra of the Young’s modulus, strain amplitude-independent ε0 were measured at different fixed temperatures. Finally, δ and E were measured for fixed temperatures under cyclic polarizing field at constant ε0 in the ranges of linear and non-linear an-elastic behavior.In a second experimental protocol, the dependences of ultrasonic absorption on δ(ε0) dependences at different temperatures were done consecutively in one heating scan, that is, without demagnetization between each measurement. A polarizing magnetic field was created by a solenoid 40 cm long, 5 cm in diameter, providing homogeneity of the field in the working space of a sample of better than 1%. A triangularly shaped wave was used to create a periodic magnetic field, parallel to the sample’s long axis, up to ±12 kA/m. The frequency of the applied field, 5 × 10−4 Hz, was sufficiently low to record 200 internal friction and frequency data points versus field in each cycle.Temperature, strain amplitude, and field spectra were measured with an Oxford closed-loop cryostat equipped with a heater free of magnetic fields and modified for acoustic studies with a temperature rate of 2 K/min. The oscillator was placed inside a closed copper chamber and the temperature of the sample was determined by means of an additional Lakeshore Cernox sensor placed in close vicinity (within 1–2 mm) to the sample. The samples were demagnetized thermally by cooling below the Curie temperature under zero field. The measurements of Z = Re(Z) + i Im(Z), for the same samples as used for ultrasonic studies, served to determine characteristic temperatures of magnetic transitions from the behavior of the imaginary part of the impedance, which, in such experiments and as a first approximation, can be considered proportional to the magnetic permeability µ: Im(Z) ∝ μ51. A standard four-terminal method with an excitation frequency 217 Hz and 686 Hz was used.Measurements of electric impedance,


Post-translational modification (PTM) of proteins by ubiquitination is an essential cellular regulatory process. Such regulation drives the cell cycle and cell division, signalling and secretory pathways, DNA replication and repair processes and protein quality control and degradation pathways. A huge range of ubiquitin signals can be generated depending on the specificity and catalytic activity of the enzymes required for attachment of ubiquitin to a given target. As a consequence of its importance to eukaryotic life, dysfunction in the ubiquitin system leads to many disease states, including cancers and neurodegeneration. This review takes a retrospective look at our progress in understanding the molecular mechanisms that govern the specificity of ubiquitin conjugation. A fubiquitin ,56 and Ebiquitin .44 patch) and RING–ubiquitin (Ile36 path) contacts, thus priming the complex for ubiquitin conjugation. Additional non-RING based E2 interaction elements are found in some RING ligases. The RING E3s gp78 and Cue1p both contain distal helical domains, Ube2g2-binding region (G2BR) and Ubc7p-binding region (UB7R) respectively, which bind the E2s ‘backside’ surface. The backside binding of the helical domains have different allosteric effects at their respective E2 active sites [Finally, there are three classes of E3 ubiquitin ligases; homologous to the E6–AP C-terminus (HECTs) , really ve sites ,84 neverve sites .In addition, RING domains also appear in multi-subunit protein complexes such as the Cullin RING ligase (CRL) family. The CRLs are a modular complex comprising an elongated cullin scaffold protein (six types) interacting with monomeric RINGs (Rbx1/Roc1/Hrt1) at the C-terminus and a wide range of substrate recognition modules (cullin adaptor proteins and substrate receptors) at the N-terminus . CRLs adThe HECT domain ligase family (28 members) have a C-terminal catalytic HECT domain that forms a catalytic intermediate with ubiquitin prior to substrate modification ,94. The Finally, the RBR family (13 members) features a RING domain and also bears a catalytic cysteine that forms a thioester–ubiquitin intermediate C, right Hundreds of E3 ligases confer specificity of ubiquitination and play crucial roles in almost every cellular process. Unsurprisingly, deregulation of these enzymes is linked to several human diseases. Mutations in Mdm2 , Von HipThe multi-step process of DNA damage repair relies on distinct ubiquitin signals to co-ordinate the damage response. Several RING E3 ligases play crucial roles in these pathways . DNA intEighteen proteins  along with several FA-associated proteins (FAAPs) participate in the FA pathway. Mutations in any of the FANC genes are linked to a failure in ICL repair –127. A cin vitro auto-ubiquitination. However, PHD zinc-fingers are not associated with ubiquitin E3 ligase activity [in vitro [FANCL was initially predicted to contain three WD (tryptophan-aspartate dipeptide) 40 repeats and a C-terminal plant homoeodomain (PHD) zinc-finger ,136. A Factivity  suggestiactivity ,139. Theactivity , where sactivity –143. Thiin vitro . The samin vitro  in the pDrosophila melanogaster (Dm) FANCL, which shares ∼20% sequence identities with human FANCL. This proved crucial to our success as we could purify DmFANCL and obtain diffraction quality crystals. The resulting structure, refined to 3.2 Å , revealed a remarkably different molecular architecture of FANCL to that predicted. FANCL comprises three domains  and notproteins . Mutatioproteins  and induproteins   are integral building blocks of multi-protein assemblies. Subsequent to our discovery of the DRWD domain, the same double-RWD architecture has been observed in multi-subunit protein complexes at both the inner and the outer kinetochore –153. Wit309 and Trp341 on FANCL RING domain and Phe63 on Ube2T) [63) results in a significant binding defect (>10 fold) in RING–Ube2T interaction [The inherent substrate specificity exhibited by FANCL helps explain how the E2–E3 pair of Ube2T–FANCL mediates FANCD2 monoubiquitination in absence of the FA core complex . Interesn Ube2T) . Mutatioeraction . Indeed,eraction .DmFANCL (PDB 3K1L) and Ube2T (PDB 1YH2) structures respectively. Thus, no major conformational changes occur during RING–E2 complex formation. The buried interface area in our structure (∼700 Å2) however, is markedly greater than other RING–E2 complexes . Accordingly, our structure reveals the RING–E2 interactions are extended beyond the generic E3–E2 interface to include an extended hydrophobic interface and an extensive network of polar and electrostatic contacts that stabilize the complex. In particular, Tyr311 of FANCL docks within an Ube2T pocket embraced by Arg6, Arg9 and Asn103 side chains. This residue is highly variable in other RINGs and is absent from the canonical RING–E2 interface. Furthermore, a critical basic residue on Ube2T (Arg60), predominantly acidic in other E2s, forms a salt bridge with Glu340 of FANCL serving as the positive selector for the FANCL RING–Ube2T pairing. Mutating residues at the canonical RING–E2 interface, as well as those unique to our structure  crystals. Despite numerous efforts we could not improve the crystals. As an alternative strategy, we expressed and purified a RING–Ube2T fusion protein bearing a short and flexible linker sequence between the E3 and E2 domains. The E3–E2 chimera successfully crystallized and diffracted to 2.4 Å (PDB 4CCG) . Incidentructure C disruptOur FANCL–Ube2T structure also offers insights into the site-specific substrate monoubiquitination events. A shared E2 interaction surface is required for both ubiquitin loading via the E1 and offloading via the E3 . FollowiE3 interactions with E2 govern the type of the ubiquitin signal that is conjugated on substrates and this is critical for the downstream outcomes of the signal. Our work on structure-function characterization of FANCL reveals how this E3 encodes inherent specificity for its substrates FANCD2 and FANCI. Further, we uncover the selective features on FANCL RING that control interactions with its physiological E2 partner Ube2T. This exquisite selectivity determines how the correct ubiquitin signal is generated for the ICL repair pathway to progress.A second model for understanding specificity in ubiquitination is Parkin, an E3 ligase mutated in heritable forms of PD. As an E3 ligase, Parkin is reported to have hundreds of putative targets including itself, can function with multiple E2 enzymes and is apparently capable of effecting multiple types of ubiquitin signals  Figure A.in vitro and in vivo. Thus, wild-type Parkin appears to be a dormant E3 ligase that is inhibited by a native Ubl domain. A series of experiments further uncovered an intramolecular interaction between an Ile44-centred surface on the Ubl domain and the rest of molecule. Structurally similar to ubiquitin, the Ubl domain also boasts an Ile44 hydrophobic surface. Interestingly, we find this ubiquitin surface to be required for auto-ubiquitination and Parkin variants with compromised intramolecular states enhance offloading of the E2–ubiquitin intermediate. The auto-regulation exerted by the Ubl domain thus involves the obscuring of catalytic interactions between Parkin and the ubiquitin loaded E2 [PD is the second most prevalent neurological disorder. PD affects approximately 1% of the population above the age of 50, with 5% of the cases being rare familial forms with an earlier onset (<45 years). Symptoms include bradykinesia, resting tremor and muscular rigidity, associated with the progressive loss of dopaminergic neurons in the substantia nigra. Research in the last two decades has uncovered multiple genetic causes underlying what was previously considered to be sporadic disease –161. Linoaded E2 .65 in the Ubl domain, leading to Parkin activation [27, Lys48 and Lys63-linked chains) [431–ubiquitin thioester intermediate [Our finding that Parkin is an auto-inhibited protein and hence constitutively inactive was not initially met with universal enthusiasm, despite several cell-based observations that hinted at a ‘latency’ in Parkin that required activation –197. A gtivation ,201. In  chains) –205. An rmediate –208. Thuin vitro and cell-based experiments. What we found is that not only are Parkin mutants rapidly degraded in cells, but that Parkin can only ubiquitinate Parkin in cis, not in trans. In other words, an active pathogenic mutant of Parkin only ubiquitinates itself, but does not modify another molecule of Parkin. In the heterozygous context, the mutant Parkin  would regulate its own status without influencing another copy, thus clarifying the recessive phenotype linked with the majority of pathogenic Parkin mutants [At this point, we understood that Parkin was auto-inhibited and could be activated by phosphorylation of the Ubl domain by PINK1. We also knew that pathogenic mutations in the Ubl domain lead to constitutively active Parkin and that the number of putative Parkin substrates was increasing rapidly. The inheritance of Parkin-related Parkinsonism is currently accepted to be autosomal recessive . There a mutants .431) and a small helical element, termed repressor element of Parkin (REP) that packs against the predicted E2-binding site. Thus, even in the absence of the first 140 amino acids, including the Ubl domain, Parkin adopts an apparently auto-inhibited conformation. Biochemical and structural studies of several multi-domain RBRs reveal auto-inhibition to be a characteristic feature of this enzyme family [65 phosphorylation, PINK1 directly phosphorylates ubiquitin at an equivalent Ser65 residue, thus uncovering an unprecedented functional link between phosphorylation and ubiquitination pathways [6, Lys11, Lys48 and Lys63-linked chains) on damaged mitochondria. In fact, activated Parkin is catalytically productive with nearly two-dozen E2s in vitro and ubiquitinates numerous outer mitochondrial membrane proteins in cells (>30 high confidence targets) [In 2013, several groups reported the structure of the RING0–RING1–BRcat–Rcat (R0RBR) domains of Parkin (residues 141–465) –172. Thee family ,210. Altpathways –214. Thepathways ,214. Quatargets) ,214,215.44-centred surface is central to the interface, including many of the residues we previously observed to be activating when mutated [2 surface area, between the RING0–RING1 domains. In the structures of Parkin lacking the Ubl domain, this distant site is remodelled when compared with intact Parkin. In particular, a trio of residues His227-glu300-His302, have side chains pointing north towards the interior of the protein when the Ubl is present, which diametrically flip to point south towards the surface of Parkin when the Ubl is absent. Using isothermal titration calorimetry, we had previously observed stable interactions in trans between the Ubl domain and the rest of Parkin (residues 77–465) whereas activating Ubl mutants weaken this connection [65 variants of the Ubl domain and ubiquitin. The binding of Ubl to R0RBR, regardless of Ser65 status, is an exothermic process driven by negative enthalpic and small entropic changes. However, altering the Ubl Ser65 side chain diminishes the R0RBR binding event (∼2-fold with Ser65Asp/Glu and ∼10-fold with phospho-Ser65). Interestingly, interactions between ubiquitin (wild-type and Ser65 variants) and R0RBR have endothermic signatures. In particular, phospho-ubiquitin binding with R0RBR binding is driven by large positive changes in enthalpy (+32 kJ/mol) and entropy (+261 J/mol°K), suggesting an increase in disorder of the system. Changes in ubiquitin Ser65 also dramatically improve its affinity with R0RBR (∼10 fold with Ser65Asp/Glu and ∼4000-fold with phospho-Ser65). Taken together, the affinity profiles suggest that although wild-type Ubl stabilises the tertiary conformation, Ser65 variants counter this effect. Furthermore, contrasting thermodynamic profiles of phospho-ubiquitin binding suggest it interacts with a distinct Parkin surface and alters its structural integrity [In order to understand the mechanisms of Parkin inhibition and activation, we needed to understand the molecular details of how the Ubl domain maintains the auto-regulated conformation and how the dual phosphorylation events offset this confirmation. Numerous crystallization trials with human full-length Parkin failed, however both the Ubl domain and the R0RBR region can be independently crystallized –172,216. mutated . Howevernnection . Using antegrity .65 lead to Parkin activation, we attempted to crystallize Ser65 variants of UblR0RBR (Ser65Asp/Glu or phospho-Ser65). Whereas the phospho-Ser65 UblR0RBR crystallization proved fruitless, we successfully obtained Ser65Asp UblR0RBR crystals and refined the structure to 2.4 Å (PDB 5C23). Superposition of Ser65Asp UblR0RBR and UblR0RBR (RMSD 0.58 Å) reveals no global conformational changes. Remarkably, however, inclusion of a negative charge at position 65 of the Ubl domain causes the His227-Glu300-His302 side chains in the RING0/RING1 interface to adopt the south facing orientation observed in the absence of the entire Ubl domain. This subtle remodelling serves to create a continuous basic patch at the RING0/RING1 interface , which is presented on the surface of Parkin. Our study, as well other independent studies, shows the phospho-ubiquitin binding patch to be at the RING0/RING1 interface of Parkin [65 phosphorylation of the Ubl domain weakens its packing with RING1 domains thus optimizing the RING0/RING1 interface for phospho–ubiquitin interactions.In order to understand how changes at Ubl Serf Parkin –220. A rf Parkin . These oIn an elegant series of competitive binding spectroscopy experiments carried out by Gary Shaw's laboratory, the consequence of phospho-ubiquitin binding was shown to be the displacement of the Ubl domain. First, the titration of R0RBR Parkin triggers a spectral peak transition of a labelled Ser65Glu Ubl species from an unbound to a bound state. Remarkably, these signals revert back to the unbound state when Ser65Glu ubiquitin is subsequently added into the system. In a reverse setup, titration of R0RBR Parkin reveals the unbound to bound transition of the labelled Ser65Glu ubiquitin species. However these signals are unaffected upon addition of Ser65Glu Ubl domain. These data show that R0RBR Parkin is unable to simultaneously bind both phospho-ubiquitin and the Ubl domain. Whereas phosphorylation of the Ubl domain optimizes the binding of phospho-ubiquitin, interaction with phospho-ubiquitin induces the complete displacement of the weakened phospho/Ubl interface from the RING1 surface C. Furthe2). The complete release of phospho-Ubl is predicted to expose a large interaction area including a RING1 surface that was once bound to the Ile44-hydrophobic patch on the Ubl domain. We had previously shown that a similar Ile44-hydrophobic patch on ubiquitin was required for Parkin auto-ubiquitination [65, giving rise to the allosteric displacement of the Ubl domain. This displacement, along with the presumed displacement of the REP creates the E2-binding surface, presents a ubiquitin-binding site that simultaneously recruits the E2–Ub intermediate. This model provides a molecular explanation for the apparent lack of E2 specificity displayed by Parkin, since association with the common denominator, ubiquitin, contributes to most of the binding energy. The ability to scaffold different E2s also explains how Parkin can support catalysis of diverse ubiquitin signals.In our UblR0RBR structure, the largest buried interface is formed between the Ubl domain and the rest of Parkin (∼2150 Åtination . Thus, ttination . These iOur studies show that achieving specificity within a given pathway can be established by specific interactions between the enzymatic components of the conjugation machinery, as seen in the exclusive FANCL–Ube2T interaction. By contrast, where a broad spectrum of modifications is required, this can be achieved through association of the conjugation machinery with the common denominator, ubiquitin, as seen in the case of Parkin. There are many outstanding questions to understanding the mechanisms governing substrate selection and lysine targeting. Importantly, we do not yet understand what makes a particular lysine and/or a particular substrate a good target for ubiquitination. Subunits and co-activators of the APC/C multi-subunit E3 ligase complex recognize short, conserved motifs (D  and KEN


BiP is the only Hsp70 chaperone in the endoplasmic reticulum (ER) and similar to other Hsp70s, its activity relies on nucleotide- and substrate-controllable docking and undocking of its nucleotide-binding domain (NBD) and substrate-binding domain (SBD). However, little is known of specific features of the BiP conformational landscape that tune BiP to its unique tasks and the ER environment. We present methyl NMR analysis of the BiP chaperone cycle that reveals surprising conformational heterogeneity of ATP-bound BiP that distinguishes BiP from its bacterial homologue DnaK. This unusual poise enables gradual post-translational regulation of the BiP chaperone cycle and its chaperone activity by subtle local perturbations at SBD allosteric ‘hotspots’. In particular, BiP inactivation by AMPylation of its SBD does not disturb Hsp70 inter-domain allostery and preserves BiP structure. Instead it relies on a redistribution of the BiP conformational ensemble and stabilization the domain-docked conformation in presence of ADP and ATP. The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells responsible for folding and maturation of the majority of secreted and membrane proteins. An immunoglobulin heavy-chain binding protein  is the only Hsp70 chaperone in the ER. BiP binds to the majority of unfolded and misfolded proteins in this organelle to promote their folding and prevent aggregation .E. coli Hsp70 (DnaK) and human cytosolic Hsp70s  conformation, the two Hsp70 domains behave independently and are structurally identical to the isolated NBD and SBD  Figure , left. RGrowing evidence has suggested that Hsp70 chaperones are extremely flexible in solution and crystallographic data have provided only ‘end-point’ snapshots of the chaperone landscape . In turnHowever, until now our ability to assess this hypothesis has been hampered by limited experimental information on the chaperone cycle of eukaryotic Hsp70s . In thisE. coli BiP homolog DnaK  binding to FL BiP* was entropically driven with the contribution from the entropy (-T∆S) to the binding energy of about −19–20 kcal/mol . This faTo obtain site-specific information about large changes in protein structure such as domain docking/undocking, we used methyl NMR  and intrATP binding to FL BiP* drastically affected the methyl peak pattern and the resulting NMR spectrum possessed two sets of peaks with near identical peak intensities , right, 1H (13C) chemical shifts and no significant line broadening was observed in the methyl NMR spectrum, we estimated that interconversion between the domain-undocked and -docked conformation is significantly slower that ~50 milliseconds . Such slow motions (and thus a large activation energy of the domain docking process) is not unexpected, given that domain docking is a multistep process that requires very large structural rearrangements  for Similar to DnaK,  binding Taking together, our NMR analysis suggests that despite structural similarity to DnaK, BiP has unique thermodynamic and kinetic features of its chaperone conformational landscape, apparently implied by a cumulative effect of amino acid sequence variations between these two Hsp70s .Next, we examined whether the BiP conformational equilibrium can be controlled allosterically. We introduced several ‘soft’ amino acid substitutions along the Hsp70 inter-domain allosteric network  connectiWould allosteric ‘tunability’ of the BiP conformation equilibrium provide a plausible explanation of how BiP is post-translationally regulated in cells? To test this hypothesis, we examined how the post-translational modification of BiP by AMPylation onto Thr518  affects 7,8 of the βSBD that connects allosterically important strands β7 and β8 , but also significantly stabilizes the domain docking in the absence of ATP  for the BiP*  and its Intriguingly, while the ADP-bound and ATP-bound domain-docked conformations are structurally almost identical, these two domain-docked conformations are thermodynamically different. First, domain docking dramatically favors only ATP binding, while ADP affinity is significantly less affected . MoreoveThe molecular mechanisms that underlie functional differences between different Hsp70s without significant structural variations between different family members have remained largely unknown. Whilst recent crystallographic descriptions of the ATP-bound states gave an impression that different Hsp70s adopt the same domain-docked and domain-undocked states, growing experimental evidence suggests that in solution each chaperone has a complex conformational landscape and co-exists as a heterogeneous ensemble of several conformations . In thisWe obtained the in-depth characterization of the BiP conformational cycle and revealed several unique features of the BiP conformational landscape . As discThe ‘divide-and-conquer’ analysis of methyl NMR spectra of the ATPase deficient variant of FL BiP and its NBD demonstrated that, despite the fact that BiP populates conformations structurally identical to DnaK, the distribution of the domain-docked and -undocked conformations for different ligand-bound states is drastically different , middle.We hypothesize that the enhanced conformational heterogeneity of BiP enables post-translational regulation of BiP activity and function. Our findings illustrate that local perturbations of the BiP SBD via ‘soft’ single-site amino acid substitutions enable allosteric controlling of the BiP conformational ensemble , right. Our results demonstrate that local perturbations in the BiP SBD fine-tune the pre-existing conformation ensemble populated upon binding to the certain ligands: For example, in the presence of ATP the I437V and I538V substitutions shift the thermodynamic equilibrium in the ensemble of the equally populated domain-docked and -undocked conformation toward the domain-undocked conformation  as well in vivo by covalent post-translational modifications and interactions with co-chaperones and other members of the ER protein quality control system.Intriguingly, local perturbations in the BiP SBD may also overpower conformational effects of nucleotide binding without decoupling of inter-domain and intra-domain allosteric transitions. For example, the V461F substitution and AMPylation of Thr518 . AstonisWhile mounting evidence suggests that BiP activity is linked to neurodegenerative diseases, diabetes, cardiovascular diseases, cancer progression and anticancer drug resistance,  these tuThe hamster BiP with a non-cleavable N-terminal 6x-His-tag was cloned into the pET28a vector. The N-terminal His-tagged BiP construct has been extensively characterized in the literature and the His-tag does not perturb BiP function . We desiE. coli BL21(DE3) competent cells (Novagen). A single colony was resuspended in 2 ml LB broth (Fisher Scientific) supplemented with kanamycin and grown overnight at 37°C. The small amount of the overnight culture were transferred into 500 ml LB broth media to reach starting optical density at a wavelength of 600 nm (OD600) ~0.1. The culture was incubated at 37°C with shacking until OD600 reached ~0.9. Then protein expression was induced with IPTG  and the culture was grown for another 6 hr. Cells were harvested by centrifugation, resuspended in the binding buffer  and frozen at 80°C until purification step.The labeled and unlabeled protein was expressed in 2H, 15N, Ileδ1-[ 13CH3]-labeled samples were performed according to published method (600 ~0.1) containing deuterated D-glucose , 15N-labeled ammonium chloride (15NH4Cl), 10% of deuterated Celtone Complete Medium  and D2O, and grew overnight at 30°C. The next day the pre-culture was transferred to 500 ml labeled M9 media (starting OD600 ~0.2) and incubated at 37°C. When the OD600 was reached ~0.7, 5 ml of methyl-13C-labeled alpha-ketobutyric acid (CLM-6820) solution  was added. After ~1 hr of incubation at 37°C, IPTG was added to the final concentration of 1 mM. The protein was expressed for 7 hr before harvesting as described above.Expression of d method  with min2, pH 7.6), flash frozen in liquid nitrogen and stored at −80°C. Protein concentrations were measured using UV absorbance at 280 nm. The purity of the samples were characterized using the SDS-PAGE gel and by measuring the A260/A280 ratio. The theoretical molar extinction coefficients of  were used for all calculations. Additionally, protein concentrations were measured using the Bradford protein assay (Bio-Rad) with bovine serum albumin as a standard.Thawed cells were incubated for 30 min on ice with 0.5 ml lysozyme , 0.1 ml DNaseI  and Protease Inhibitor Cocktail (Roche). After sonication, lysates were spun down at 20000 rpm for 45 min and filtered to remove cell debris. Supernatants were loaded onto a HisTrap HP nickel column  pre-equilibrated with the binding buffer . Following a wash step with the binding buffer containing 40 mM imidazole, bound protein was eluted by 500 mM imidazole. Collected fractions were extensively dialysed against the final buffer  and concentrated to 20 μM.The BiP SBD was obtained from the E. coli Origami 2 (DE3) cell (Novagen). A single colony was resuspended in 2 ml LB broth (Fisher Scientific) supplemented with ampicillin and grown overnight at 37°C. The small amount of the overnight culture were transferred into 500 ml LB broth media to reach starting optical density at a wavelength of 600 nm (OD600) ~0.1. The culture was incubated at 37°C with shacking until OD600 reached ~0.9. Them protein expression was induced with IPTG  and the culture was grown for another 3 hr. Cells were harvested by centrifugation, resuspended in the binding buffer  and frozen at 80°C until purification step.SubA was expressed and purified using a standard protocol from Prof David Ron’s laboratory (University of Cambridge). The protein was expressed in Thawed cells were incubated for 30 min on ice with 0.5 ml lysozyme , 0.1 ml DNaseI  and Protease Inhibitor Cocktail (Roche). After sonication, lysates were spun down at 20000 rpm for 45 min and filtered to remove cell debris. Supernatants were loaded onto a HisTrap HP nickel column  pre-equilibrated with the binding buffer . A gradual refolding of the protein was performed on-column using a linear urea gradient from 2M to 0M. Then, the protein was eluted using a linear imidazole gradient from 10 to 500 mM. Finally, the protein sample was dialysed against 50 mM HEPES, 300 mM NaCl, and 5% glycerol (pH 7.4).2, pH 7.6) in the presence 5 or 40 mM of ADP or ATP (no significant differences were observed in NMR spectra for different nucleotide and MgCl2 concentrations) A BEST version (1H-13C SOFAST-HMQC) was used for methyl NMR  and FL BiP* constructs were expressed in isotopically labeled media as described above. NMR acquisitions were carried out in the NMR buffer  and SBD. If we observed almost no changes in methyl NMR peak positions between the FL construct and the isolated NBD* and SBD x100%. Errors were calculated as standard deviations (SDs) from the means for three peak doublets.To calculate the populations of the domain-docked and -undocked conformations from spectra of FL BiP*, we used methyl peak intensities of three non-overlapping peak doublets shown in , grey. I2, pH 7.6). The final protein concentrations were between 20–80 μM and the stock nucleotide concentrations varied between 0.1–0.4 mM. All measurements were carried out at 25°C. For each protein construct measurements were repeated at least three times; ΔH and Kd values were obtained for each repeat using the non-linear least square curve-fitting algorithm implemented in MicroCal Origin . The ΔH and Kd values were averaged out over all repeats and the corresponding TΔS and ΔG values were calculated using the following equitations:All ITC experiments were performed on BiP* and its variants using MicroCal ITC200 system. The protein solution was dialyzed overnight against the ITC buffer .d is an ADP or ATP binding constant, R is the ideal gas constant, and T is the temperature in K. Populations of the domain-undocked and -docked conformations obtained from NMR spectra, were used to calculate the free energy of domain docking using the following equation: D and 1-pD are populations of the domain-docked and -undocked conformations, respectively  binding was obtained from the ITC data using the following equation: ectively .To obtain theoretical plot of free energy of nucleotide (ADP or ATP) binding, D and pU = 1 pD are the populations of the domain-docked and -undocked conformations, respectively. The free energy of docking were calculated:where D and pU = 1–pD are the populations of the domain-docked and –undocked conformations, respectively.where pet al  Triton X-100) using NAP-5 columns  and AMPyltaed for 8 hr at 30°C with bacterially expressed GST-FICD-E234G at ratio 1:100 in the presence of 3 mM ATP. Next, AMPylated protein was subjected to the standard purification protocol described above. AMPylation, was validated by mass spectrometry.The AMPylation protocol was adopted from Preissler et al .500 μL o−1. After valve switching, the bound protein was eluted by a wash of 95% solvent B in A for 3 min before re-equilibration at 10% solvent B in A ready for the next injection. The column eluent was directed in to the mass spectrometer via a Z-spray electrospray source. The MS was operated in positive TOF mode using a capillary voltage of 3kV, sample cone of 40V and source offset of 80V. Backing pressure was 7.9 mbar and trap bias 2V. The source temperature was 80°C and desolvation was 150°C. Argon was used as the buffer gas at a pressure of 9.1 × 10−3 mbar in the trap and transfer regions of the TriWave device. Mass calibration was performed by a separate injection of [Glu]-fibrinopeptide b at a concentration of 250 fmol µl−1 in MS/MS mode and a CID voltage (trap region) of 28V. Data processing was performed using the MassLynx v4.1 suite of software  supplied with the mass spectrometer.The mass analysis was performed by LC-MS using an M-class ACQUITY UPLC  interfaced to a Synapt G2S Q-IMT-TOF mass spectrometer . 1 µL of 5 µM sample was loaded onto a MassPREP protein desalting column  washed with 10% solvent B (0.1% formic acid in acetonitrile) in solvent A (0.1% formic acid in water) for 5 min at 25 µL min In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Randy Schekman as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Erik RP Zuiderweg (Reviewer #1).Thank you for submitting your article "Allosteric fine-tuning of the conformational equilibrium poises the chaperone BiP for post-translational regulation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The conformational cycle of BiP, the Hsp70 chaperone of the endoplasmic reticulum (ER), is less well understood than that of the bacterial Hsp70, DnaK. Hsp70s consist of an N-terminal nucleotide binding domain and a C-terminal substrate binding domain and their activity relies on ATP- and protein substrate-controllable docking and undocking of these domains. Based on a methyl NMR analysis of BiP, this study reveals a surprising conformational heterogeneity of ATP-bound BiP that distinguishes BiP from DnaK. This property allows regulation of the BiP chaperone cycle by post-translational modifications such as AMPylation. Interestingly, BiP inactivation by AMPylation of its substrate binding domain stabilizes the domain-docked conformation independent of the specific nucleotide bound.Major point to be addressed in the revised manuscript:1) The following important conclusion is not represented in the manuscript, although it is supported by the data: The authors show, for the first time, that the NMR-observed conformations (docked vs. not-docked) are thermodynamically relevant in a quantitative way. This is validating earlier work from the Gierasch and other NMR labs, as well as establishing the base for future work by the authors. This conclusion should be stressed in the revised manuscript. This point refers to the following results:"Next, to examine how AMPylation affects thermodynamics of nucleotide binding, we performed ITC measurements for the AMPylated and non-AMPylated V461F BiP* variant. ATP affinity increased from Kd of 800 nM for BiP* (that populated 53% of the domain-docked conformation) to Kd of 210 nM for the V461F variant (66% of the domain-docked conformation), and finally to Kd of 70 nM for the AMPylated V461F BiP (89% of the domain docked conformation) ."Plotting the free energy of ATP binding for these 3 results versus the free energy of the Docking/Undocking percentages results in a very good correlation with a slope of 1.1. This suggests that the NMR-observed states are thermodynamically relevant in a quantitative way. It seems important to make this correlation clear in the Discussion, with the caveat that these chemical changes are of course not equilibrium thermodynamics. Thus, please include a Table and plot.Other questions raised by the reviewers:2) Too many figures of chemical shift differences are shown. Showing it once is sufficient.3) Re. Materials and methods: Is the His tag on the C or N terminus? Was it cleaved off?4) Re. Discussion: With respect to 5) Why is the conversion between the D and U states in the ATP-bound conformation slow? Any idea?6) Do the authors understand why the population of the U state is so much higher in BiP compared to DnaK?7) Why is there a perfect resonance overlap in the isolated NBD and FL-BiP spectra in the D state? Wouldn't someone expect that in the docked state some NBD residues would be affected giving rise to different chemical shifts?8) How were the D and U states assigned? Major point to be addressed in the revised manuscript:1) The following important conclusion is not represented in the manuscript, although it is supported by the data: The authors show, for the first time, that the NMR-observed conformations (docked vs. not-docked) are thermodynamically relevant in a quantitative way. This is validating earlier work from the Gierasch and other NMR labs, as well as establishing the base for future work by the authors. This conclusion should be stressed in the revised manuscript. This point refers to the following results:"Next, to examine how AMPylation affects thermodynamics of nucleotide binding, we performed ITC measurements for the AMPylated and non-AMPylated V461F BiP* variant. ATP affinity increased from Kd of 800 nM for BiP* (that populated 53% of the domain-docked conformation) to Kd of 210 nM for the V461F variant (66% of the domain-docked conformation), and finally to Kd of 70 nM for the AMPylated V461F BiP (89% of the domain docked conformation) ."Plotting the free energy of ATP binding for these 3 results versus the free energy of the Docking/Undocking percentages results in a very good correlation with a slope of 1.1. This suggests that the NMR-observed states are thermodynamically relevant in a quantitative way. It seems important to make this correlation clear in the Discussion, with the caveat that these chemical changes are of course not equilibrium thermodynamics. Thus, please include a Table and plot.” as well as a new Materials and methods section: “Analysis of the thermodynamic linkage between domain docking and nucleotide binding”. As reviewers suggested, we also highlighted the fact that the observed correlation between thermodynamics of nucleotide binding and domain docking validates that BiP conformations observed by NMR indeed represent thermodynamically relevant conformational distributions in the BiP functional ensemble .We thank the reviewers for this constructive suggestion. As the reviewers suggested, we added the plot and table that show the correlation between the free energy of ATP binding and the free energy of domain docking for BiP* and its non-AMPylated and AMPylated V461F variant  in the rOther questions raised by the reviewers:2) Too many figures of chemical shift differences are shown. Showing it once is sufficient.As suggested, we reduced the amount of figures with NMR methyl spectra. Particularly, we removed 3) Re. Materials and methods: Is the His tag on the C or N terminus? Was it cleaved off?We thank the reviewers for pointing this out. In this study all FL BiP and NBD constructs have a noncleavable 6x-His-tag as it has been shown previously that the N-terminal His-tag does not perturb BiP function : 26670-26676. We added these details on Materials and methods .4) Re. Discussion: With respect to The reviewers raised a very interesting and important question! We don’t think that the current study provides enough experimental information to unambiguously answer this intriguing question. However, we speculate that weaker communication between NBD subdomains may cause the conformational heterogeneity of the ATP-bound NBD .5) Why is the conversion between the D and U states in the ATP-bound conformation slow? Any idea?To address this question, we estimated the time-scale of domain docking in BiP using slow exchange limit with the respect to the chemical shift differences between the two conformations. We found that the transition should be significantly slower than 50 ms, which is not completely unexpected, given that domain docking is a multistep process that requires very large structural rearrangements. We discussed this in the revised version of the manuscript: subsection “Conformational features of the BiP allosteric cycle”, third paragraph.6) Do the authors understand why the population of the U state is so much higher in BiP compared to DnaK?It has been previously demonstrated that even subtle (one amino-acid) perturbations in the DnaK sequence can cause very significant redistributions in the chaperone conformational ensemble ). To this end, one should expect very different populations of the U and D conformations for BiP, which share only ~60% sequence identity with DnaK. Consequently, the differences between DnaK and BiP are likely to rely on a cumulative effect from aminoacid differences between these two Hsp70s. Interestingly, our data revealed that the BiP conformational landscape has not only unique thermodynamic but also kinetic features. Particularly, the transition between the docked and undocked conformations is significantly slower in BiP as compared with DnaK. We described these observations in the revised manuscript, Subsection “Conformational features of the BiP allosteric cycle”, third and last paragraphs.7) Why is there a perfect resonance overlap in the isolated NBD and FL-BiP spectra in the D state? Wouldn't someone expect that in the docked state some NBD residues would be affected giving rise to different chemical shifts?8) How were the D and U states assigned?We apologize for the confusion. In the presence of ATP, two sets of peaks were observed in the NMR spectrum. For each peak doublet, one peak that overlaps perfectly with the corresponding peak from the spectrum of the isolated NBD, was assigned to the domain-undocked (U) conformation. The almost perfect overlapping between peaks for FL BiP and its isolated NBD suggests that the chemical environment in the FL protein and its isolated domain is very similar. On the contrary, the second peak from the FL doublet had distinct chemical shifts from the corresponding isolated NBD peak. This suggests that the second conformation of the ATP-bound FL BiP is significantly different from the conformation of the isolated NBD. We assigned these peaks to the domain-docked (D) conformation. We modified the Materials and methods  and the legend for


ADGRA2 genetic lesions found in previously generated zebrafish and mouse models, the zebrafish ouchless allele encodes an aberrantly-spliced and inactive receptor lacking a single leucine-rich repeat (LRR) unit within its N-terminus. By characterizing this allele we uncover that, in contrast to all other extracellular domains, the precise composition of the LRR domain determines proper receptor trafficking to the plasma membrane. Using CRISPR/Cas9 engineered cells, we further show that Adgra2 trafficking occurs in a Reck-independent manner and that, similarly, Reck reaches the plasma membrane irrespective of Adgra2 expression or localization, suggesting that the partners meet at the plasma membrane after independent intracellular trafficking events.Adgra2, formerly known as Gpr124, is a key regulator of cerebrovascular development in vertebrates. Together with the GPI-anchored glycoprotein Reck, this adhesion GPCR (aGPCR) stimulates Wnt7-dependent Wnt/β-catenin signaling to promote brain vascular invasion in an endothelial cell-autonomous manner. Adgra2 and Reck have been proposed to assemble a receptor complex at the plasma membrane, but the molecular modalities of their functional synergy remain to be investigated. In particular, as typically found in aGPCRs, the ectodomain of Adgra2 is rich in protein-protein interaction motifs whose contributions to receptor function are unknown. In opposition to the severe  Summary: This work uncovers molecular determinants of Adgra2/Gpr124 and Reck trafficking to the plasma membrane where the partners meet to act as potent Wnt7-specific Wnt/β-catenin signaling co-activators required for brain vascularization. Adhesion G protein-coupled receptors (aGPCRs) constitute the second largest group of GPCRs in humans. Most aGPCRs are orphan receptors with no identified ligands that function through remarkably diverse mechanisms . They diRECK in the mouse leads to CNS-specific vascular defects, thereby demonstrating the evolutionary conserved role of RECK in cerebrovascular development  lacking the third LRR motif of the LRR/CT domain. The zebrafish ouchless mutant thereby constitutes the first in vivo model of adgra2 N-terminal domain-specific variation.Building a proper understanding of Adgra2 function will benefit from delineating the contribution of each N-terminal domain to receptor function. An Adgra2 variant exhibiting an altered N-terminal domain architecture was recently identified in the zebrafish  mutants . As the ouchless variant mislocalizes to the endoplasmic reticulum (ER), we undertook a comparative analysis of the contribution of the different Adgra2 N-terminal domains to Adgra2 and Reck intracellular trafficking and function. Detailed mutagenesis and chimeragenesis reveals that the LRR/CT domain controls Adgra2 trafficking. Investigations in genetically-engineered cultured cells further suggest that Adgra2 and Reck proceed independently through the secretory pathway and hence tentatively assign their synergistic effect on Wnt7-stimulated Wnt/β-catenin signaling to subsequent events occurring at the level of the plasma membrane.In this work, starting from the observation that the Adgra2adgra2 variant found in ouchless mutants differs from adgra2 reference sequences by four non-synonymous SNPs as well as a 72 bp deletion corresponding to exon 4 (ouchless inactivation (ouchless (Adgra2ouchless) and ΔLRR3 (Adgra2ΔLRR3) variants. This latter variant reproduces the exon 4 deletion found in ouchless in a WT allele of adgra2, and hence lacks the ouchless-associated SNPs  zebrafish blastula, WT Adgra2-EGFP labeled the plasma membrane where it colocalized with a membrane-tethered lyn-RFP marker D. By conThe mislocalization of the ΔLRR3 variant prompted us to perform a more detailed molecular dissection of the impact of the LRR/CT domain on Adgra2 progression through the secretory pathway. In-frame deletion of any of the four LRR repeats individually (ΔLRR1-4) or together (ΔLRR) resulted in ER retention  and 2A-CMechanistically, the LRR domain could be directly involved in trafficking through its recognition by an ER-resident binding partner that would assist Adgra2 progression. Alternatively, the absence or alteration of the LRR domain could act indirectly, for example by affecting Adgra2 folding. In the first scenario, the LRR domain should be strictly necessary for trafficking and hence any receptor deletion variant encompassing the LRR domain is predicted to accumulate in the ER. This was tested by analyzing the intracellular distribution of increasingly larger deletion variants with deletions ranging from the first LRR motif to the LRR C-terminal domain (ΔLRR/CT), the Ig-like domain (ΔLRR/CT/Ig-like) or the HRM domain (ΔLRR/CT/Ig-like/HRM). While the ΔLRR/CT variant exhibited an intermediate phenotype, with the most protein within the ER and a minor pool at the plasma membrane A-C, the LRR domains are composed of tandemly-arranged units that organize in arched solenoid assemblies contributing to the overall three-dimensional arrangement of proteins. Deleting one unit is thus anticipated to impact on the spatial arrangement of adjacent protein domains. The essential trafficking role revealed by the LRR/CT deletion variants could thus reflect a mere structural role of this domain that would fulfill its function sequence-independently. In agreement with this hypothesis, it has been previously demonstrated that the LRR/CT domain of Adgra2 can be substituted with the equivalent domain of Adgra3, a closely related but distinct aGPCR . We exteADGRA2 and RECK . As in WT cells, both proteins accumulated at the plasma membrane when expressed individually, indicating that each partner can reach its final destination independently  maintain functionality while chimeras with the more distantly related Adgra2 LRR1, LRR2 (54% similarity) and CPN2 LRR7 (42% similarity) do not. No other N-terminal domain of Adgra2 appears to be required for Adgra2 localization at the plasma membrane, underlying the specific requirement for the LRR/CT domain in this process.The ER retention of the in vivo  and in vitro (HEK293T cells).The LRR/CT-dependent Adgra2 localization results are in apparent conflict with previous findings. Indeed, Posokhova et al. analyzed the subcellular distribution of a set of N-terminal deletion variants of ADGRA2 and reported a robust trafficking mechanism to the plasma membrane independent of any of the extracellular domains, including the LRR/CT domain . We noteMechanistically, the ER retention of Adgra2 molecules exhibiting LRR variations or deletions is most easily explained by improper folding. As (i) receptor variants lacking the entire LRR/CT domain accumulate in the ER and (ii) the deletion of the adjacent Ig-like domain is sufficient to suppress the defective trafficking of LRR/CT variants, we propose that improper folding of the Ig-like domain and not the LRR/CT domain itself is causing ER retention of the LRR/CT variants. The LRR/CT domain might help instruct folding of the adjacent Ig-like domain through intramolecular contacts involving specific residues within the LRR domain.While this study describes the role of the LRR/CT domain in promoting Adgra2 progression through the ER, it does not exclude additional roles for this domain in the Adgra2/Reck signaling pathway. The LRR/CT domain might, for instance, be additionally implicated in the interactions with Reck, Wnt7 or Fzd/Lrp5/6 occurring at the plasma membrane. By extension, the contribution of the other extracellular domains of Adgra2 and Reck to Adgra2/Reck signaling will require further investigation. These future studies will be important not only for their insights into the molecular mechanisms governing essential developmental processes, but also because they hold the key to understanding the thus-far elusive mechanisms of Wnt ligand-specific signaling pathways.Danio rerio) were raised and maintained under standard conditions. The following lines were used: s843Tg(kdrl:GFP) (s896Tg(kdrl:ras-mCherry)  (w61Tg(-17.0neurog1:EGFP) (s984adgra2 (Zebrafish (GFP)s843 , Tg(kdrlrry)s896 , Tg(flie4FF)ubs4 , Tg(-17.EGFP)w61  and adgrgra2s984 . All aniadgra2 deletion mutants and hybrids were generated by In-Fusion cloning . Deletions correspond to the following amino acids: Adgra2ΔLRR1: 77-100; Adgra2ΔLRR2: 101-124; Adgra2ΔLRR3: 125-148; Adgra2ΔLRR4: 149-169; Adgra2ΔLRR: 70-166; Adgra2ΔLRR/CT: 70-228; Adgra2ΔIg-like: 230-344; Adgra2ΔHRM: 353-406; Adgra2ΔGAIN: 416-727; Adgra2ΔLRR/CT/Ig-like: 70-344; Adgra2ΔLRR/CT/Ig-like/HRM: 70-406. Chimeras were generated by substituting the Adgra2 LRR3 (125-148) with Adgra3 LRR3 (122-145), CPN2 LRR7 (247-270), Adgra2 LRR1 (77-100) or Adgra2 LRR2 (101-124). Capped messenger RNA was synthesized using the mMESSAGE mMACHINE kit . One-cell stage embryos were injected either with 100 pg of the indicated mRNA or 4 ng of a previously validated adgra2 splice-blocking morpholino (adgra2 in pCS2 by In-Fusion cloning (Takara). The fusion products were then subcloned in a pTol2-5xUAS expression construct.The rpholino . The EGF5xUAS:adgra2-EGFP, pTol2-ouchless-EGFP5xUAS:adgra2 and pTol2-ΔLRR3-EGFP5xUAS:adgra2 constructs into double transgenic s896Tg(kdrl:ras-mCherry); ubs4Tg embryos at the one-cell stage.Transgenic endothelial mosaic overexpression was achieved by co-injecting 25 pg of Tol2 transposase mRNA and 25 pg of the pTol2-−1 and 10 µg ml−1, respectively. HEK293T cells were transfected with Lipofectamine 2000  and grown in glass-coated imaging chambers  for 48 h before being fixed for 15 min in 4% paraformaldehyde at room temperature. Where indicated, cells were permeabilized with 0.5% saponin in PBS for 10 min at room temperature. PLA assays were performed following the manufacturer's instructions .The following antibodies were used: mouse monoclonal anti-FLAG M2, 1:10,000 for PLA , purified polyclonal rabbit anti-HA, 1:400 for PLA and 1:250 for immunofluorescence assays  and anti-rabbit Alexa594-conjugated secondary antibody, 1:5000 . DAPI and Hoechst counterstaining were performed for 2 min at 5 µg ml+ HEK293T cells were isolated 48 h after transfection by FACS  and clonally expanded before homozygous mutant selection by high resolution melt analysis and lesion identification through Sanger sequencing.CRISPR/Cas9 guide sequences were cloned in pSpCas9(BB)-2A-GFP . GFP+ HEImages were acquired on a Zeiss LSM710 confocal microscope. Colocalization was assessed using the Pearson correlation coefficient (PCC) calculated with coloc2 in ImageJ (NIH). Applied to colocalization analysis, PCC is a measure of the linear correlation between the intensities observed in the two channels of interest. A value of 1 indicates that the two signals are positively and perfectly linearly related; a value of −1 indicates a perfectly linear, but negative correlation; and a value close to zero indicates that the distributions of the two signals are uncorrelated.P-values were calculated by the Kruskal–Wallis test (post hoc Dunn's test).Statistical analyses were performed using the GraphPad Prism software. Sample size was determined with the G*power 3.1.5 software to reach adequate statistical power. Each dot plot value represents an independent cell or embryo and every experiment was conducted three times independently.


The influence of misfit strain on the ECE of PbTiO3 nanoparticle with the double-vortex domain structure is investigated, and results show that the compress misfit strain can enhance the ECE, but the tensile misfit strain reduces the ECE. This work reveals a way to obtain giant ECE of ferroelectric materials by domain engineering and strain engineering in a wide temperature range.Electrocaloric effect (ECE) has the potential applications in solid-state refrigeration with the features of high efficiency and environmentally friendly. Large adiabatic temperature change in a wide temperature range is needed for electrocaloric effect to meet the requirement of commercially application. In this work, giant electrocaloric effect is found in PbTiO Large ECEs near ferroelectric-paraelectirc (F-P) phase transition have been reported in the low dimensional ferroelectric materials8, which proved that low dimensional ferroelectric materials have great potential for practical applications and can be commercially exploited for electronic devices. However, the working temperature of many devices near the room temperature, which much lower than the temperature of F-P phase transition11. Therefore, it is important to find an effective way to design and develop ferroelectric materials which can generate giant adiabatic temperature change near the room temperature.Electrocaloric effect (ECE), a temperature change of a given material in response to external electric field, has attracted considerable attention because it can be used as solid-state refrigeration to replace Freon-based cooling systems with the feature of high efficiency15. For example, Peng et al. reported that the field-induced polar nanodomain formation and alignment could induced a twin-peak ECEs12. Wang et al. found that the transition from multidomain to monodomain driven by temperature can largely enhance the ECE of PbTiO313. Especially, our previous works have found a large temperature change related to the toroidal moment change in the ferroelectric nanoparticle with vortex domain structure17. These works indicate that the ferroelectric nanostructure with the vortex domain structure can provide an effective way to achieving a large ECE through domain engineering.At room temperature, domains are formed in ferroelectric materials when polarization has the same direction. The domain engineering, which control and adjust the properties of ferroelectric materials through changing the domain structure and number, has been proved an effective way to control and adjust the ECE of ferroelectric materials20. For example, Haeni et al. has observed a Curie temperature TC shift of hundreds of degrees in strained ferroelectric thin films owing to the strong coupling between strain and ferroelectricity19. The epitaxial strain from the substrate could be applied to increase of TC and raise the produce room-temperature ferroelectricity. Cruz et al. find that a film-substrate misfit strain may significantly affect the thermodynamic stability of domain walls in epitaxial BiFeO3 film20. So, it is an effective way to improve the ECE of ferroelectric nanostructure with vortex domain structure by combining the domain engineering and strain engineering.In addition, in order to better satisfy the requirement of commercially application, it has necessary to further improve the ECE of ferroelectric nanostructure with vortex domain structure near the room temperature. It is noted that, controlling and adjusting the properties of ferroelectric materials through the strain, what is the so called strain engineering, is another effective way to control and adjust the properties of ferroelectric materials3 ferroelectric nanoparticle with a double-vortex domain structure is investigated by using phase field method. A large adiabatic temperature change is found in the PbTiO3 nanoparticle. And the mechanism of this large adiabatic temperature change is analyzed in detail.In this letter, the influence of misfit strain on the ECE of PbTiOx* = Δy* = Δz* = 1 with a cell size of Δx = Δy = Δz = 1 nm. In order to obtain double-vortex domain structure, the polarization boundary condition of PTO nanoparticle is set as zero boundary condition and open-circuit electrical boundary condition is employed to solve Eq.  generated after the circular electric field is applied. However, most of the area generate positive ECE(ΔT > 0) on x-y surface. Apart from the variability of adiabatic temperature change along different axis, the adiabatic temperature change are also different between planes along the same axis. Figures x, y, z axis, respectively. It is apparently that the distribution of adiabatic temperature change is different between the y-z middle plane  because of the inhomogeneity of adiabatic temperature change in 3D.Figure ane Fig. and y-z sT) increases from 1.8 K to 6.5 K when the vorticity vector of circular electric field (Q) increases from 0.1 mV/Å2 to 0.5 mV/Å2, which reveals that the ECE of PTO nanoparticle with double-vortex domain can be improved by the strength of circular electric field. A further study shows that the large adiabatic temperature change associates with the domain structure change closely under the circular electric field. The simulation result shows that double-vortex domain turns to a single-vortex domain when external circular electric filed is applied, which is shown in Fig. Q = 0.5 mV/Å2 is exhibited in Fig. T > 0) and negative ECE (ΔT < 0) are generated in local area, and the final adiabatic temperature change is 6.5 K under the vorticity vector of circular electric field Q = 0.5 mV/Å2 after thermal conduction.The adiabatic temperature change under different value of the vorticity vector of circular electric field is exhibited in Fig. The generation of positive and negative ECE can be explained through the domain structure change. As it has mentioned in previous paragraph, the double-vortex domain could turn into a single-vortex domain when the circular electric field is applied, the polarization of the new generated vortex center decreases largely  is employed to investigate the influence of misfit strain on the ECE of PTO nanoparticle with the double-vortex domain structure, at shown in Fig. ε11 = ε22 > 0) and compress misfit strain (ε11 = ε22 < 0) on ECE is different. The adiabatic temperature change increases as the absolute value of the compress misfit strain increases, which means compress can improve the ECE of PTO nanoparticle with the double-vortex domain structure. Tensile misfit strain has a opposite influence, which it reduces the ECE as the absolute value of the tensile misfit strain increases.The misfit strain induced by the lattice constant mismatch between the ferroelectrics and substrate has an important influence on the properties of ferroelectric materialsQ = 0.5 mV/Å2 and equiaxial misfit strain ε11 = ε22 = −0.009. Giant ECE is obtained at the temperature range 0 ~ 675 °C, and the lowest and highest adiabatic temperature change is 7.2 K and 16.5 K, respectively. The PTO nanoparticle shows different electrocaloric property in different temperature range. In the region I where the temperature range is 0 ~ 150 °C, the ΔT of PTO nanoparticle increases linearly as the temperature increases. When the temperature is higher than 165 °C . The difference of electrocaloric property of PTO particle in region I and II  is chosen as the order parameter to describe the domain structure of PbTiO3 nanoparticle, which can be obtained by solving the time-dependent Ginzburg-Landau equation,F is the total free energy of PbTiO3 nanoparticle, r is spatial vector. The total free energy F of PbTiO3 nanoparticle can be written as25,fLD is Landau-Devonshire energy density, fG is gradient energy density, felas is elastic energy density, felec is electric energy density, and V denotes volume. The energy density fLD, fG, felas, felec can be expressed as following equations, respectively27,α1, α11, α12, α111, α112, α123 are the dielectric stiffness in equation (α1 = (T−T0)/2ε0C0. ε0 is the dielectric constant of vacuum, C0 is the Curie constant, T and T0 denote the temperature and the Curie-Weiss temperature, respectively. G11, G12, G44 and cijkl are the elastic energy constants in equation  can be calculated by using the following equation28,ρ is the density of material, CE is the heat capacity. Ea and Eb denote initial and final electric field, respectively, T is the temperature which the unit is K, S is entropy and σ is stress.The adiabatic temperature change (Δ


Numerical modeling of microstructure evolution in various regions during uniaxial compression and canning compression of NiTi shape memory alloy (SMA) are studied through combined macroscopic and microscopic finite element simulation in order to investigate plastic deformation of NiTi SMA at 400 °C. In this approach, the macroscale material behavior is modeled with a relatively coarse finite element mesh, and then the corresponding deformation history in some selected regions in this mesh is extracted by the sub-model technique of finite element code ABAQUS and subsequently used as boundary conditions for the microscale simulation by means of crystal plasticity finite element method (CPFEM). Simulation results show that NiTi SMA exhibits an inhomogeneous plastic deformation at the microscale. Moreover, regions that suffered canning compression sustain more homogeneous plastic deformation by comparison with the corresponding regions subjected to uniaxial compression. The mitigation of inhomogeneous plastic deformation contributes to reducing the statistically stored dislocation (SSD) density in polycrystalline aggregation and also to reducing the difference of stress level in various regions of deformed NiTi SMA sample, and therefore sustaining large plastic deformation in the canning compression process. Owing to its functional properties, including shape memory effect, superelasticity, and biological compatibility, NiTi shape memory alloy (SMA) has attracted increasing attention in the field of materials science and engineering. It is generally accepted that hot plastic deformation, rather than cold plastic deformation, plays a significant role in transforming as-cast NiTi SMA ingots into NiTi SMA products, including NiTi wire, bar, tube, strip, and sheet ,2,3. On Historically, the finite element method (FEM), as a numerical method, is the main approach applied in the industry to simulate large scale forming processes. This method is a perfect candidate for clarifying the plastic deformation law of a metallic alloy by describing material behavior as a continuum ,15. HoweIn the present study, numerical modeling of microstructure evolution in various regions during uniaxial compression and canning compression of NiTi SMA are conducted through combined macroscopic and microscopic finite element simulation in order to investigate plastic deformation of NiTi SMA at 400 °C. The corresponding deformation history in some selected regions of the macroscopic finite element model is directly extracted by means of the sub-model technique of finite element code ABAQUS and subsequently used as the boundary conditions for the microscale simulation by means of CPFEM.50.9Ti49.1 (at.%) by means of electro-discharge machining (EDM). In addition, the as-received NiTi SMA bar was prepared by virtue of vacuum induction melting method and subsequent rolling at 800 °C. In the case of canning compression, the NiTi SMA sample was canned in the low carbon steel cans with the inner diameter of 4 mm and the outer diameter of 10 mm. The elastic and plastic data used in macroscale finite element simulation is directly obtained from the mechanical response of NiTi SMA subjected to uniaxial compression at 400 °C at the constant strain rate of 0.001 s−1 by using an INSTRON-5500R universal testing machine  equipped with a heating device. In this way, this model provides general distribution of displacement, strain and stress at the macroscale level.In the present study, interest is mainly focus on understanding the heterogeneous deformation and microstructure evolution in the selected regions with the corresponding boundary conditions extracted from the macroscale finite element simulation. Therefore, a polycrystalline model with refined mesh is constructed and used, since the accurate representation of grain shape and especially grain boundary is important in capturing the heterogeneous deformation in polycrystal. To acquire the initial microstructure and texture of NiTi SMA, a scanning electron microscope (SEM) experiment and an electron back-scattered diffraction (EBSD) experiment were conducted on the as-received specimen using a Zeiss Supra 55 scanning electron microscope  coupled with OXFORD EBSD instrument . By applying the intercept method on individual grains, it has been confirmed that the as-received NiTi SMA are composed of equiaxed grains with an equivalent grain diameter of about 25 μm. Based on this experimental result, the polycrystalline model on the basis of Voronoi tessellation is constructed by means of a free open source software package NEPER , as showIn the present study, the framework of crystal plasticity theory is based on dislocation slip, and three slip systems, including {110}<100>, {010}<100>, and {110}<111>, are introduced into the proposed crystal plasticity finite element model . As it hIn the framework of rate-dependent single crystal plasticity, the elastic constitutive equation is specified by the following equation ,24.(1)σn stands for the rate dependency. If the material is rate-independent, a large value can be chosen up to 50, whereas if the material is highly rate-dependent, a typical value of 10 can be used [The crystal was assumed to behave as an elasto-viscoplastic solid, so the slipping shear rate  be used . In the  be used . γ˙0 is q = 1.4 is used in the present study [A simple hardening model is given as follows :(5)hαα=hnt study .The aforementioned crystallographic formulations are implemented numerically into ABAQUS standard solver through a user-defined material subroutine (UMAT), where the implicit (Euler backward) integration algorithm is adopted . MoreoveOn the one hand, dislocation is the vehicle to sustain plastic deformation. On the other hand, region 1 possesses the maximal plastic strain. Therefore, polycrystalline models corresponding to region 1 have the highest dislocation density during uniaxial compression and canning compression. In order to investigate the distribution of statistically stored dislocation (SSD) density during uniaxial compression and canning compression of NiTi SMA, the methodology described in  is adopt15 m−2 in the case of uniaxial compression and the corresponding value in the case of canning compression is 3.15 × 1015 m−2. This can be served as an evidence that at the same deformation degree, canning compression contributes to relieving the SSD density within the deformed sample than uniaxial compression. As a result, canning compression is favorable of sustaining large plastic deformation.Due to the aforementioned analysis, the results obtained from the macroscale numerical modeling by FEM and the microscale numerical modeling on the basis of CPFEM contribute to presenting a clear understanding of microstructure evolution in various regions during uniaxial compression and canning compression.(1)At the macroscale, it can be concluded that canning compression contributes to relieving the inhomogeneous plastic strain as the compression of the low carbon steel can results in a three-dimensional compressive loading state of NiTi SMA sample.(2)The sub-model technique in the finite element code ABAQUS is used to extracted the accurate deformation history of selected regions at the macroscale simulation. Then, this deformation history, which reflects the complex interaction between the polycrystalline aggregation and neighboring grains, can serve as boundary conditions used at the microsacle simulation.(3)At the microscale, it can be concluded that the mitigation of inhomogeneous plastic deformation in canning compression contributes to various microstructure evolution in selected regions. Moreover, the ease of inhomogeneity in plastic strain contributes to reducing the difference of the stress level in various regions and reducing statistically stored dislocation (SSD) density within the deformed NiTi SMA sample. Therefore, canning compression is favorable of sustaining large plastic deformation.


DYRK1A is a cause of a neurodevelopmental syndrome termed mental retardation autosomal dominant 7 (MRD7). Several truncation mutations, microdeletions and missense variants have been identified and result in a recognizable phenotypic profile, including microcephaly, intellectual disability, epileptic seizures, autism spectrum disorder and language delay. DYRK1A is an evolutionary conserved protein kinase which achieves full catalytic activity through tyrosine autophosphorylation. We used a heterologous mammalian expression system to explore the functional characteristics of pathogenic missense variants that affect the catalytic domain of DYRK1A. Four of the substitutions eliminated tyrosine autophosphorylation , indicating that these variants lacked kinase activity. Tyrosine phosphorylation of DYRK1A-L295F in mammalian cells was comparable to wild type, although the mutant showed lower catalytic activity and reduced thermodynamic stability in cellular thermal shift assays. In addition, we observed that one variant (DYRK1A-T588N) with a mutation outside the catalytic domain did not differ from wild-type DYRK1A in tyrosine autophosphorylation, catalytic activity or subcellular localization. These results suggest that the pathogenic missense variants in the catalytic domain of DYRK1A impair enzymatic function by affecting catalytic residues or by compromising the structural integrity of the kinase domain.Haploinsufficiency of This article has an associated First Person interview with the first author of the paper. Summary: We have analyzed the functional consequences of amino acid substitutions in the protein kinase DYRK1A that have been identified as pathogenic in patients with microcephaly, intellectual disability and autism. DYRK1A is a member of the DYRK [dual-specificity tyrosine (Y) phosphorylation-regulated kinase] family of protein kinases which play important roles in the regulation of cell differentiation, proliferation, and survival . DYRKs aDYRK1A is highly dependent on its gene dosage  (OMIM 614104). Patients with heterozygous truncation mutations or deletion of DYRK1A share a phenotype with microcephaly, intellectual disability, language delay, epileptic seizures, neonatal feeding problems, and facial dysmorphism  Study and account for around 0.5% of syndromic intellectual disability in this cohort  (DYRK1A haploinsufficiency syndrome (The function of e dosage . Human Dosome 21 . Overexposome 21 . Decreasmorphism . Pathoges cohort . DYRK1A er (ASD) . Autistisyndrome .Drosophila gene (mnb) result in a ‘minibrain phenotype’ of adult flies with reduced optic lobes and central brain hemispheres  in the core of the kinase domain . This seF308 is a key component of the hydrophobic ‘R-spine’ (regulatory) that is a hallmark signature of every active kinase conformation . This stIn contrast to the DFG motif, the neighboring amino acids (S310-S311-C312) at the beginning of the activation loop are distinctive for the DYRK family of protein kinases. C312 can form an intramolecular disulfide bridge with a cysteine in the catalytic loop (C286) that is conserved in all members of the DYRK family . The repS346 is located in the middle of the F-helix, which acts as a central scaffold of the C-lobe . As alreThe C-terminal part of the kinase domain  is rather far from the substrate and ATP-binding sites and is rarely affected by disease-causing SNPs . HoweverFor comparison, we included one variant that results in the substitution of a residue in the non-catalytic C-terminal domain and was also classified as likely pathogenic T588N, . T588 isTo examine the consequences of the pathogenic missense mutations on the function of DYRK1A, we introduced the respective amino acid substitutions into a GFP-DYRK1A fusion protein. Wild-type and mutant constructs were immunoprecipitated from transiently transfected HEK293 cells and subjected to immunoblot analysis to determine the phosphorylation of Y321. Autophosphorylation of the tyrosine in the activation loop is a key step in the maturation of DYRKs, and we used the phosphorylation status of Y321 as an indicator of correct folding and conformational integrity of DYRK1A. For comparison, we included two variants that do not affect the catalytic domain, the T588N missense and the Q576* nonsense variant. In addition, GFP-DYRK1A-K188R and D287N served as catalytically inactive controls, and DYRK1A-Y321F was used as a control for antibody specificity.Next we determined the enzymatic activity of DYRK1A-L295F and T588N towards an exogenous substrate. GFP-DYRK1A constructs were immunoprecipitated from HEK293 cells and subjected to kinase assays using the peptide substrate, DYRKtide. DYRK1A-Y321F was included as a control with strongly reduced but detectable catalytic activity . ConsistOur results show that the substitution of L295 by phenylalanine still allows the kinase domain to obtain an active conformation. To assess whether the amino acid exchange compromised the conformational stability of the kinase domain, we used a modified version of the cellular thermal shift assay (CETSA) . To estain vitro translation system to assess whether the amino acid exchange affected tyrosine autophosphorylation in a cell free system. Under these conditions, tyrosine autophosphorylation of DYRK1A-L295F was significantly reduced as compared to the wild-type enzyme . Most cases of MRD7 are caused by deletion of the function . TherefoThe complete lack of phosphotyrosine in most of the DYRK1A mutants shows that the catalytic domain cannot attain a conformation capable of ATP binding and/or phosphoryl transfer. F308, S346 and R467 have conserved structural functions in the kinase domain fold (see above) and are hotspots of disease-causing mutations in the protein kinase superfamily . Intriguin vitro. Furthermore, DYRK1A-L295F showed decreased tyrosine autophosphorylation in a bacterial in vitro translation system. It seems plausible that chaperones can partially compensate for the thermodynamic instability of the kinase domain when DYRK1A-L295F is expressed in mammalian cells. It is also possible that a fraction of DYRK1A-L295F molecules fails to autophosphorylate in HEK293 cells but escapes detection because it is eliminated by the ubiquitin proteasome system.In contrast to the other amino acid exchanges in the catalytic domain, L295F did not impair the autophosphorylation of Y321 in mammalian cells. Interestingly, a patient carrying this allele seemed to display a relatively mild phenotype, and was not affected by feeding difficulties, seizures or intellectual disability at the age of 21 months . Neverthhttp://exac.broadinstitute.org), which excludes individuals affected by severe pediatric disease  was detectably mislocalized when the GFP-DYRK1A fusion proteins were ectopically expressed in COS-7 cells. This experimental model system was previously used to characterize the nuclear localization signal in the N-terminal domain and the speckle-targeting function of the polyhistidine track in the C-terminus of DYRK1A . NeverthDYRK1A is one of the genes that was subject to the strongest selective pressure after introgression of Neanderthal alleles in the gene pools of modern humans  or by compromising the structural integrity of the kinase domain. As previously noted by n humans .Fig. 7.DYRK1A missense variants were described since we initiated our study. Several further in vitro translation experiments, partial DYRK1A cDNA encoding for amino acids 28-499 was inserted into the expression plasmid pET-ST2 to create an N-terminal fusion with the Strep-tag 2 sequence . Rabbit polyclonal antibody against phospho-HIPK2 (pTyr361) was purchased from Thermo Fisher Scientific  and used at 1:1000 dilution for western blot detection. We have previously shown that this antibody is suitable for detection of pY321 in DYRK1A, due to the sequence similarity of the activation loop in HIPK2 and DYRK1A (Research Resource Identifiers (RRID) of antibodies are indicated if available . Dishes were shaken for 30 min on ice. Lysates were vortexed, sonicated and cleared by centrifugation . Supernatants were incubated for 1 h at 4°C with 10 µl GFP Trap_M, which contains a recombinant fragment of alpaca anti-GFP-antibody covalently coupled to the surface of paramagnetic beads . The beads were washed three times with washing buffer  and split in two aliquots. One aliquot was used for radiometric assay to determine kinases activities with the peptide substrate DYRKtide as described . Two days after transfection, cells in a 10-cm dish were washed with cold PBS and lysed using 1 ml non-denaturing buffer . Membranes were blocked using 3% bovine albumin in TBST  and incubated with primary antibodies overnight at 4°C. Incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies was done at room temperature for 1 h, followed by washing with TBST. Chemiluminescence signals were detected using LAS-300 Imaging system  and densitometrically evaluated with the help of Aida Image Analyzer 3.52 software .E. coli-based in vitro transcription-translation system  was used to express a DYRK1A(28-499) construct fused to an N-terminal Strep-tag 2 sequence. Reactions were incubated in a volume of 10 μl with 10 ng/μl plasmid DNA at 37°C for 90 min. Autophosphorylation of Y321 in DYRK1A was assessed by immunoblot blot with rabbit anti phospho-HIPK2 (Tyr361) antibody. Band intensities were normalized to total protein levels as detected by Strep-Tactin conjugated to HRP .A reconstituted g at 4°C). The abundance of DYRK1A in the soluble fraction was quantified by western blotting.The assay was developed based on the protocol of cellular thermal shift assay CETSA,  with mod4Cl in PBS for 5 min. All incubations were done at room temperature. After washing with PBS, the coverslips were mounted onto slides using Vectashields Mounting Medium  containing 1.5 µg/ml 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining. Images were obtained with a Zeiss LSM 710 confocal microscope  using the C-apochromat 63×/1.20 water objective of Zeiss. GFP was excited with the 488 nm line of an argon laser and DAPI was excited with 405 nm laser diode. Images were analyzed with the Zen 2012 software .COS7 cells cultured on coverslips in 12-well plates were transiently transfected with different expression constructs and incubated for 48 h. The cells were washed with PBS, fixed in 4% paraformaldehyde for 20 min and quenched with 50 mM NHMolecular visualization and analysis of the kinase domain structure of DYRK1A  was applied to test the hypothesis that readout parameters were different between wild-type DYRK1A and a particular missense variant . Samples sizes (n) reflect independent experiments.The one-sample