Structural basis for feedforward control in the PINK1/parkin pathway

PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson's disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observe that phospho-ubiquitin can bind to two distinct sites on parkin, a high affinity site on RING1 that controls parkin localization, and a low affinity site on RING0 that releases parkin autoinhibition. Surprisingly, NMR titrations and ubiquitin vinyl sulfone assays show that the RING0 site has higher affinity for phospho-ubiquitin than the phosphorylated Ubl. Parkin could be activated by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic a mitochondrion bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development.

Calculating metalation in cells reveals CobW acquires CoII for vitamin B12 biosynthesis while related proteins prefer ZnII

Protein metal-occupancy (metalation) in vivo has been elusive. To address this challenge, the available free energies of metals have recently been determined from the responses of metal sensors. Here, we use these free energy values to develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. We use the calculator to understand the function and mechanism of GTPase CobW, a predicted CoII-chaperone for vitamin B12. Upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator also reveals that related GTPases with comparable ZnII affinities to CobW, preferentially acquire ZnII due to their relatively weaker CoII affinities. The calculator is made available here for use with other proteins.

Calculating metalation in cells reveals CobW acquires CoII for vitamin B12 biosynthesis upon binding nucleotide

Protein metal-occupancy (metalation) in vivo has been elusive. Here we develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. The calculations are based on available free-energies of metals determined from the responses of metal sensors. We use the calculator to understand the function and mechanism of CobW, a predicted CoII-chaperone for vitamin B12. CobW is calculated to acquire negligible metal alone: But, upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator reveals how CobW acquires its metal and is made available for use with other proteins.

In vivo PET Imaging of [11C]CIMBI-5, a 5-HT2AR Agonist Radiotracer in Nonhuman Primates

Purpose: 5-HT2AR exists in high and low affinity states. Agonist PET tracers measure binding to the active high affinity site and thus provide a functionally relevant measure of the receptor. Limited in vivo data have been reported so far for a comparison of agonist versus antagonist tracers for 5-HT2AR used as a proof of principle for measurement of high and low affinity states of this receptor. We compared the in vivo binding of [11C]CIMBI-5, a 5-HT2AR agonist, and of the antagonist [11C]M100907, in monkeys and baboons. Methods: [11C]CIMBI-5 and [11C]M100907 baseline PET scans were performed in anesthetized male baboons (n=2) and male vervet monkeys (n=2) with an ECAT EXACT HR+ and GE 64-slice PET/CT Discovery VCT scanners. Blocking studies were performed in vervet monkeys by pretreatment with MDL100907 (0.5 mg/kg, i.v.) 60 minutes prior to the scan. Regional distribution volumes and binding potentials were calculated for each ROI using the likelihood estimation in graphical analysis and Logan plot, with either plasma input function or reference region as input, and simplified reference tissue model approaches. Results: PET imaging of [11C]CIMBI-5 in baboons and monkeys showed the highest binding in 5-HT2AR-rich cortical regions, while the lowest binding was observed in cerebellum, consistent with the expected distribution of 5-HT2AR. Very low free fractions and rapid metabolism were observed for [11C]CIMBI-5 in baboon plasma. Binding potential values for [11C]CIMBI-5 were 25-33% lower than those for [11C]MDL100907 in the considered brain regions. Conclusion: The lower binding potential of [11C]CIMBI-5 in comparison to [11C]MDL100907 is likely due to the preferential binding of the former to the high affinity site in vivo in contrast to the antagonist, [11C]MDL100907, which binds to both high and low affinity sites.

A gonadotropin-releasing hormone type neuropeptide with a high affinity binding site for copper(ii) and nickel(ii)

Gonadotropin releasing hormone from Asterias rubens binds Cu(ii) in a nitrogen-rich, high-affinity site. Cu(ii)-binding is an evolutionarily conserved feature of GnRH-type neuropeptides.

Hooking She3p onto She2p for myosin-mediated cytoplasmic mRNA transport

The segregation of approximately two dozen distinct mRNAs from yeast mother to daughter cell cytoplasm is a classical paradigm for eukaryotic mRNA transport. The information for transport resides in an mRNA element 40–100 nt in length, known as “zipcode.” Targeted transport requires properly positioned actin filaments and cooperative loading of mRNA cargo to myosin. Cargo loading to myosin uses myosin 4 protein (Myo4p), swi5p-dependent HO expression 2 protein (She2p) and 3 protein (She3p), and zipcode. We previously determined a crystal structure of Myo4p and She3p, their 1:2 stoichiometry and interactome; we furthermore showed that the motor complex assembly requires two Myo4p⋅She3p heterotrimers, one She2p tetramer, and at least a single zipcode to yield a stable complex of [Myo4p⋅She3p⋅She2p⋅zipcode] in 2:4:4:1 stoichiometry in vitro. Here, we report a structure at 2.8-Å resolution of a cocrystal of a She2p tetramer bound to a segment of She3p. In this crystal structure, the She3p segment forms a striking hook that binds to a shallow hydrophobic pocket on the surface of each She2p subunit of the tetramer. Both She3p hook and cognate She2p binding pocket are composed of highly conserved residues. We also discovered a highly conserved region of She3p upstream of its hook region. Because this region consists of basic and aromatic residues, it likely represents part of She3p’s binding activity for zipcode. Because She2p also exhibits zipcode-binding activity, we suggest that “hooking” She3p onto She2p aligns each of their zipcode-binding activities into a high-affinity site, thereby linking motor assembly to zipcode.

Effect Of Lipids On The Sequence-Specific Interactions Of Kindlin-3 and Talin-1 With The Cytosolic Tail Of The Integrin β3 Subunit

Activating the platelet integrin αIIbβ3 is an essential step for primary hemostasis. Physiologic αIIbβ3 activation occurs when platelet agonist-generated inside-out signals induce binding of the FERM domains of the cytosolic proteins talin-1 and kindlin-3 to the cytosolic tail (CT) of the β3 subunit of αIIbβ3. While talin-1 binding is thought to activate αIIbβ3 by physically causing separation of the αIIb and β3 cytosolic and transmembrane domains, αIIbβ3 activation in platelets does not occur in the absence of kindlin-3 binding to the β3 CT. Nonetheless, it is unclear whether it is necessary for talin-1 and kindlin-3 to be concurrently bound to the β3 CT in order to activate αIIbβ3, and if that is the case, whether there is a preferred order of binding and whether binding is cooperative. It is noteworthy in this regard that the sequences of the core binding motifs on the β3 CT for the talin-1 and kindlin-3 FERM domains, N744PLY747 and N756ITY759, respectively, are quite similar. To begin to address these questions, we have expressed and purified recombinant forms of the integrin-binding talin-1 head domain (THD) and full-length kindlin-3 and measured their interaction with a peptide corresponding to the β3 CT by surface plasmon resonance (SPR). For these experiments, the β3 CT was anchored to the dextran matrix of a CM5 SPR sensor chip and the equilibrium kinetics of THD and kindlin-3 binding was measured. Analysis of the THD binding data was compatible with two classes of binding sites, a high affinity site with a Kd of 155 nM and a low affinity site with a Kd of 3.5 µM. Similar analysis of kindlin-3 binding was also consistent with two classes of binding sites, a high affinity site with a Kd of 5 nM and a lower affinity site with a Kd of 2.2 µM. Next, we tested the effect of mutating the core binding motifs for the THD and kindlin-3 on the β3 CT. We found that replacing Y759 in the core kindlin-3 binding motif with Ala eliminated high affinity kindlin-3 binding, whereas replacing Y747 in the core THD binding motif with Ala eliminated low affinity kindlin-3 binding. Conversely, the Y747A replacement eliminated high affinity THD binding, while the Y759A replacement eliminated low affinity THD binding. Thus, these experiments demonstrate that the talin-1 and kindlin-3 FERM domains each recognize the general NXXY motif, but their high affinity interactions with this motif are highly sequence-specific. Previously, we found that appending the β3 CT to acidic phospholipids increased its affinity for the THD by three orders of magnitude, likely through interactions involving an extended positively-charged surface on the THD F2 and F3 subdomains. Further, kindlins contain a pleckstrin homology domain with a conserved lipid-binding loop that has been found to be essential for αIIbβ3 activation. Accordingly, we investigated the effect on THD and kindlin-3 binding of tethering the β3 CT to DOPC-coated L1 SPR chips. Unexpectedly, we found that when the β3 CT was tethered to lipid, the Kd for THD binding increased to 430 nM, comparable to the Kd we previously found using isothermal titration calorimetry for THD binding to the β3 CT appended to liposomes. We also found that kindlin-3 binding to the β3 CT tethered to lipids was unexpectedly weaker than binding in the absence of lipid, but it remained approximately 3-fold stronger than THD binding under the same conditions. Previous NMR and hydrogen-deuterium exchange studies of the β3 CT appended to liposomes have revealed that the regions of the β3 CT containing the THD and kindlin-3 binding sites consist of two dynamic amphiphilic helices that are stabilized by interacting with lipid bilayers. Thus, the results presented here suggest that the folding of the β3 CT and the interaction of the folded structure with lipids are important determinants of the strength of the interaction of the THD and kindlin-3 with the β3 CT and consequently are important factors in the regulation of αIIbβ3 activation. Disclosures: No relevant conflicts of interest to declare.

Two Binding Sites for [3H]PBR28 in Human Brain: Implications for TSPO PET Imaging of Neuroinflammation

[11C]PBR28, a radioligand targeting the translocator protein (TSPO), does not produce a specific binding signal in approximately 14% of healthy volunteers. This phenomenon has not been reported for [11C]PK11195, another TSPO radioligand. We measured the specific binding signals with [3H]PK11195 and [3H]PBR28 in brain tissue from 22 donors. Overall, 23% of the samples did not generate a visually detectable specific autoradiographic signal with [3H]PBR28, although all samples showed [3H]PK11195 binding. There was a marked reduction in the affinity of [3H]PBR28 for TSPO in samples with no visible [3H]PBR28 autoradiographic signal ( K i=188±15.6 nmol/L), relative to those showing normal signal ( K i=3.4±0.5 nmol/L, P<0.001). Of this latter group, [3H]PBR28 bound with a two-site fit in 40% of cases, with affinities ( K i) of 4.0±2.4 nmol/L (high-affinity site) and 313±77 nmol/L (low-affinity site). There was no difference in Kd or Bmax for [3H]PK11195 in samples showing no [3H]PBR28 autoradiographic signal relative to those showing normal [3H]PBR28 autoradiographic signal. [3H]PK11195 bound with a single site for all samples. The existence of three different binding patterns with PBR28 (high-affinity binding (46%), low-affinity binding (23%), and two-site binding (31%)) suggests that a reduction in [11C]PBR28 binding may not be interpreted simply as a reduction in TSPO density. The functional significance of differences in binding characteristics warrants further investigation.