S100 proteins interact with the N-terminal domain of MDM2

S100 proteins interact with the transactivation domain and the C-terminus of p53. Further, S100B has been shown to interact with MDM2, a central negative regulator of p53. Here, we show that S100B bound directly to the folded N-terminal domain of MDM2 (residues 2-125) by size exclusion chromatography and surface plasmon resonance experiments. This interaction with MDM2 (2-125) is a general feature of S100 proteins; S100A1, S100A2, S100A4 and S100A6 also interact with MDM2 (2-125). These interactions with S100 proteins do not result in a ternary complex with MDM2 (2-125) and p53. Instead, we observe the ability of a subset of S100 proteins to disrupt the extent of MDM2-mediated p53 ubiquitylation in vitro.

Structured summary

MINT-7905256: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A6 (uniprotkb:P06703) by surface plasmon resonance (MI:0107)MINT-7905063: MDM2 (uniprotkb:Q00987) and s100A1 (uniprotkb:P23297) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905376: s100A4 (uniprotkb:P26447) and MDM2 (uniprotkb:Q00987) physically interact (MI:0915) by competition binding (MI:0405)MINT-7905130: s100A6 (uniprotkb:P06703) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905207: s100A6 (uniprotkb:P06703) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905043: s100B (uniprotkb:P04271) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905196: p53 (uniprotkb:P04637) and s100A4 (uniprotkb:P26447) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905358: p53 (uniprotkb:P04637) and s100A4 (uniprotkb:P26447) physically interact (MI:0915) by fluorescence polarization spectroscopy (MI:0053)MINT-7905220: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100B (uniprotkb:P04271) by surface plasmon resonance (MI:0107)MINT-7905104: s100A4 (uniprotkb:P26447) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905229: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A1 (uniprotkb:P23297) by surface plasmon resonance (MI:0107)MINT-7905317, MINT-7905162: s100B (uniprotkb:P04271) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905238: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A2 (uniprotkb:P29034) by surface plasmon resonance (MI:0107)MINT-7905174, MINT-7905308: s100A1 (uniprotkb:P23297) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905247: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A4 (uniprotkb:P26447) by surface plasmon resonance (MI:0107)MINT-7905090: s100A2 (uniprotkb:P29034) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905142, MINT-7905326: MDM2 (uniprotkb:Q00987) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905185, MINT-7905347: s100A2 (uniprotkb:P29034) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)     

Stoichiometric protein complex formation and over-expression using the prokaryotic native operon structure

In prokaryotes, operon encoded proteins often form protein-protein complexes. Here, we show that the native structure of operons can be used to efficiently overexpress protein complexes. This study focuses on operons from mycobacteria and the use of Mycobacterium smegmatis as an expression host. We demonstrate robust and correct stoichiometric expression of dimers to higher oligomers. The expression efficacy was found to be largely independent of the intergenic distances. The strategy was successfully extended to express mycobacterial protein complexes in Escherichia coli, showing that the operon structure of gram-positive bacteria is also functional in gram-negative bacteria. The presented strategy could become a general tool for the expression of large quantities of pure prokaryotic protein complexes for biochemical and structural studies.

Structured summary

MINT-7542207: ESAT-6 (uniprotkb:Q50206) and CFP-10 (uniprotkb:O33084) bind (MI:0407) by blue native page (MI:0276)MINT-7542534: ESAT-6 (uniprotkb:P0A564) and CFP-10 (uniprotkb:P0A566) bind (MI:0407) by X-ray crystallography (MI:0114)MINT-7542187: CFP-10 (uniprotkb:P0A566) and ESAT-6 (uniprotkb:P0A564) bind (MI:0407) by blue native page (MI:0276)MINT-7542652: CFP-10 (uniprotkb:P0A566) and ESAT-6 (uniprotkb:P0A564) bind (MI:0407) by molecular sieving (MI:0071)MINT-7542474, MINT-7542303: CFP-10 (uniprotkb:P0A566) physically interacts (MI:0915) with ESAT-6 (uniprotkb:P0A564) by pull down (MI:0096)     

Defining the structural requirements for ribose 5-phosphate-binding and intersubunit cross-talk of the malarial pyridoxal 5-phosphate synthase

Most organisms synthesise the B₆ vitamer pyridoxal 5-phosphate (PLP) via the glutamine amidotransferase PLP synthase, a large enzyme complex of 12 Pdx1 synthase subunits with up to 12 Pdx2 glutaminase subunits attached. Deletion analysis revealed that the C-terminus has four distinct functionalities: assembly of the Pdx1 monomers, binding of the pentose substrate (ribose 5-phosphate), formation of the reaction intermediate I₃₂₀, and finally PLP synthesis. Deletions of distinct C-terminal regions distinguish between these individual functions. PLP formation is the only function that is conferred to the enzyme by the C-terminus acting in trans, explaining the cooperative nature of the complex.

Structured summary

MINT-7994448: PfPdx1 (uniprotkb:C6KT50) and PfPdx1 (uniprotkb:C6KT50) bind (MI:0407) by molecular sieving (MI:0071)MINT-7994425, MINT-7994413, MINT-7994435: PfPdx1 (uniprotkb:C6KT50) and PfPdx1 (uniprotkb:C6KT50) bind (MI:0407) by cosedimentation in solution (MI:0028).     

Phosphorylation of more than one site is required for tight interaction of human tau protein with 14-3-3ζ

Serine residues phosphorylated by protein kinase A (PKA) in the shortest isoform of human tau protein (τ3) were sequentially replaced by alanine and interaction of phosphorylated τ3 and its mutants with 14-3-3 was investigated. Mutation S156A slightly decreased interaction of phosphorylated τ3 with 14-3-3. Double mutations S156A/S267A and especially S156A/S235A, strongly inhibited interaction of phosphorylated τ3 with 14-3-3. Thus, two sites located in the Pro-rich region and in the pseudo repeats of τ3 are involved in phosphorylation-dependent interaction of τ3 with 14-3-3. The state of τ3 phosphorylation affects the mode of 14-3-3 binding and by this means might modify tau filament formation.

Structured summary

MINT-7233358, MINT-7233372, MINT-7233384: 14-3-3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636-3) bind (MI:0407) by molecular sieving (MI:0071)MINT-7233323, MINT-7233334, MINT-7233346: Tau 3 (uniprotkb:P10636-3) and 14-3-3 zeta (uniprotkb:P63104) bind (MI:0407) by crosslinking studies (MI:0030)MINT-7233285, MINT-7233297, MINT-7233310: 14-3-3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636-3) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)     

Characterisation of the interaction of colicin A with its co-receptor TolA

Colicin A enters Escherichia coli cells through interaction with endogenous TolA and TolB proteins. In vitro, binding of the colicin A translocation domain to TolA leads to unfolding of TolA. Through NMR studies of the colicin A translocation domain and polypeptides representing the individual TolA and TolB binding epitopes of colicin A we question if the unfolding of TolA induced by colicin A is likely to be physiologically relevant. The NMR data further reveals that the colicin A binding site on TolA is different from that for colicin N which explains why there is a difference in colicin toxicity for E. coli carrying a TolA-III homologue from Yersina enterocolitica in place of its own TolA-III.

Structured summary

MINT-7888512: TolA (uniprotkb:P19934) and Col-A (uniprotkb:P04480) bind (MI:0407) by nuclear magnetic resonance (MI:0077)MINT-7888526: TolA (uniprotkb:P19934) and TolB (uniprotkb:P0A857) bind (MI:0407) by nuclear magnetic resonance (MI:0077)MINT-7888999: TolA (uniprotkb:P19934), TolB (uniprotkb:P0A855) and Col-A (uniprotkb:P04480) physically interact (MI:0915) by molecular sieving (MI:0071)MINT-7888982: TolA (uniprotkb:P19934), TolB (uniprotkb:P0A855) and Col-A (uniprotkb:P04480) physically interact (MI:0915) by nuclear magnetic resonance (MI:0077)     

DnaK-mediated association of ClpB to protein aggregates. A bichaperone network at the aggregate surface

Intracellular protein aggregates formed under severe thermal stress can be reactivated by the concerted action of the Hsp70 system and Hsp100 chaperones. We analyzed here the interaction of DnaJ/DnaK and ClpB with protein aggregates. We show that aggregate properties modulate chaperone binding, which in turn determines aggregate reactivation efficiency. ClpB binding strictly depends on previous DnaK association with the aggregate. The affinity of ClpB for the aggregate-DnaK complex is low (K_d = 5-10 μM), indicating a weak interaction. Therefore, formation of the DnaK-ClpB bichaperone network is a three step process. After initial DnaJ binding, the cochaperone drives association of DnaK to aggregates, and in the third step, as shown here, DnaK mediates ClpB interaction with the aggregate surface.

Structured summary

MINT-7258957: G6PDH (uniprotkb:P0AC53) and G6PDH (uniprotkb:P0AC53) bind (MI:0407) by dynamic light scattering (MI:0038)MINT-7258951: alpha glucosidase (uniprotkb:P21517) and alpha glucosidase (uniprotkb:P21517) bind (MI:0407) by dynamic light scattering (MI:0038)MINT-7258903: AdhE (uniprotkb:P0A9Q7) and AdhE (uniprotkb:P0A9Q7) bind (MI:0407) by dynamic light scattering (MI:0038)MINT-7258900: G6PDH (uniprotkb:P0AC53) and G6PDH (uniprotkb:P0AC53) bind (MI:0407) by biophysical (MI:0013)MINT-7258974: DnaK (uniprotkb:P0A6Y8), ClpB (uniprotkb:P63284), DnaJ (uniprotkb:P08622) and G6PDH (uniprotkb:P0AC53) physically interact (MI:0914) by cosedimentation (MI:0027)     

A proposed reaction mechanism for rice NADPH thioredoxin reductase C, an enzyme with protein disulfide reductase activity

NADPH thioredoxin reductase C (NTRC) is an interesting NTR with a thioredoxin (Trx) domain at the C-terminus, able to conjugate both activities for 2-Cys peroxiredoxin (Prx) reduction. NTRC is dimeric in the presence of NADPH and interacted with dimeric 2-Cys Prx through the Trx module by a mixed disulfide between Cys377 of NTRC and Cys61 of the 2-Cys Prx. NTRC variants of both NTR and Trx active sites were inactive, but 1:1 mixtures of both variants allowed partial recovery of activity suggesting inter-subunit transfer of electrons during catalysis. Based on these results we propose a model for the reaction mechanism of NTRC.

Structured summary

MINT-7017333: 2cys Prx (uniprotkb:Q6ER94) and 2cys Prx (uniprotkb:Q6ER94) bind (MI:0407) by molecular sieving (MI:0071)MINT-7017101, MINT-7017183: NTRC (uniprotkb:Q70G58) and 2cys Prx (uniprotkb:Q6ER94) bind (MI:0407) by enzymatic studies (MI:0415)     

Direct visualization of the small hydrophobic protein of human respiratory syncytial virus reveals the structural basis for membrane permeability

Human respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract disease in infants. The HRSV small hydrophobic (SH) protein plays an important role in HRSV pathogenesis, although its mode of action is unclear. Analysis of the ability of SH protein to induce membrane permeability and form homo-oligomers suggests it acts as a viroporin. For the first time, we directly observed functional SH protein using electron microscopy, which revealed SH forms multimeric ring-like objects with a prominent central stained region. Based on current and existing functional data, we propose this region represents the channel that mediates membrane permeability.

Structured summary

MINT-7890792, MINT-7890805: SH (uniprotkb:P04852) and SH (uniprotkb:P04852) bind (MI:0407) by chromatography technology (MI:0091)MINT-7890784, MINT-7890776: SH (uniprotkb:P04852) and SH (uniprotkb:P04852) bind (MI:0407) by electron microscopy (MI:0040)     

The domain structure of talin: Residues 1815-1973 form a five-helix bundle containing a cryptic vinculin-binding site

Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. Its rod region consists of a series of helical bundles. Here we show that residues 1815-1973 form a 5-helix bundle, with a topology unique to talin which is optimally suited for formation of a long rod such as talin. This is much more stable than the 4-helix (1843-1973) domain described earlier and as a result its vinculin binding sequence is inaccessible to vinculin at room temperature, with implications for the overall mechanism of the talin-vinculin interaction.

Structured summary

MINT-7722300, MINT-7760951: Talin-1 (uniprotkb:P26039) and Vinculin (uniprotkb:P12003) bind (MI:0407) by molecular sieving (MI:0071)     

Characterization of GmCaMK1, a member of a soybean calmodulin-binding receptor-like kinase family

Calmodulin(CaM)-regulated protein phosphorylation forms an important component of Ca²⁺ signaling in animals but is less understood in plants. We have identified a CaM-binding receptor-like kinase from soybean nodules, GmCaMK1, a homolog of Arabidopsis CRLK1. We delineated the CaM-binding domain (CaMBD) of GmCaMK1 to a 24-residue region near the C-terminus, which overlaps with the kinase domain. We have demonstrated that GmCaMK1 binds CaM with high affinity in a Ca²⁺-dependent manner. We showed that GmCaMK1 is expressed broadly across tissues and is enriched in roots and developing nodules. Finally, we examined the CaMBDs of the five-member GmCaMK family in soybean, and orthologs present across taxa.

Structured summary

MINT-8051564: AtCRLK2 (uniprotkb:Q9LFV3) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051416: GmCaMK3 (uniprotkb:C6ZRS6) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051258: CaM (uniprotkb:P62199) and GmCaMK1 (genbank_protein_gi:223452504) bind (MI:0407) by isothermal titration calorimetry (MI:0065)MINT-8051400: GmCaMK2 (uniprotkb: C6ZRY5) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051242, MINT-8051295, MINT-8051313, MINT-8051327, MINT-8051341, MINT-8051355: GmCaMK1 (genbank_protein_gi:223452504) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051467: GmCaMK4 (uniprotkb: C6TIQ0) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051276: CaM (uniprotkb:P62199) and GmCaMK1 (genbank_protein_gi:223452504) bind (MI:0407) by comigration in non denaturing gel electrophoresis (MI:0404)MINT-8051374: CaM (uniprotkb:P62199) and GmCaMK1 (genbank_protein_gi:223452504) bind (MI:0407) by mass spectrometry studies of complexes (MI:0069)     

Heparin binding domain in vitronectin is required for oligomerization and thus enhances integrin mediated cell adhesion and spreading

Vitronectin is a multi-functional protein found predominantly as a monomer in blood and as an oligomer in the extracellular matrix. We have dissected the minimal regions of vitronectin protein needed for effective integrin dependent cell adhesion and spreading. A fragment of vitronectin containing the RGD integrin binding site showed similar binding affinity as that of full vitronectin protein to purified integrin αvβ3 but had diminished cell adhesion and spreading function in vivo. We demonstrate that the oligomeric state of the protein is responsible for this effect. We provide compelling evidence for the involvement of the heparin binding domain of vitronectin in the oligomerization process and show that such oligomerization reinforces the activity of vitronectin in cell adhesion and spreading.

Structured summary

MINT-7905703: Vn (uniprotkb:P04004) and Vn (uniprotkb:P04004) bind (MI:0407) by molecular sieving (MI:0071)     

HO1 and PcyA proteins involved in phycobilin biosynthesis form a 1:2 complex with ferredoxin-1 required for photosynthesis

The HO1 and PcyA genes, encoding heme oxygenase-1 (HO1) and phycocyanobilin (PCB):ferredoxin (Fd) oxidoreductase (PcyA), respectively, are required for chromophore synthesis in photosynthetic light-harvesting complexes, photoreceptors, and circadian clocks. In the PCB biosynthetic pathway, heme first undergoes cleavage to form biliverdin. I confirmed that Fd1 induced the formation of a stable and functional HO1 complex by the gel mobility shift assay. Furthermore, analysis by a chemical cross-linking technique designed to detect protein-protein interactions revealed that HO1 and PcyA directly interact with Fd in a 1:2 ratio. Thus, Fd1, a one-electron carrier protein in photosynthesis, drives the phycobilin biosynthetic pathway.

Structured summary

MINT-7014657: Fd1 (uniprotkb:P0A3C9) and HO1 (uniprotkb:Q8DLW1) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)MINT-7014666: HO1 (uniprotkb:Q8DLW1 and Fd1 (uniprotkb:P0A3C9) bind (MI:0407) by cross-linking studies (MI:0030)MINT-7014675: PcyA (uniprotkb:P59288) and Fd1 (uniprotkb:P0A3C9) bind (MI:0407) by cross-linking studies (MI:0030)     

Abi1/Hssh3bp1 pY213 links Abl kinase signaling to p85 regulatory subunit of PI-3 kinase in regulation of macropinocytosis in LNCaP cells

Macropinocytosis is regulated by Abl kinase via an unknown mechanism. We previously demonstrated that Abl kinase activity is, itself, regulated by Abi1 subsequent to Abl kinase phosphorylation of Abi1 tyrosine 213 (pY213) [1]. Here we show that blocking phosphorylation of Y213 abrogated the ability of Abl to regulate macropinocytosis, implicating Abi1 pY213 as a key regulator of macropinocytosis. Results from screening the human SH2 domain library and mapping the interaction site between Abi1 and the p85 regulatory domain of PI-3 kinase, coupled with data from cells transfected with loss-of-function p85 mutants, support the hypothesis that macropinocytosis is regulated by interactions between Abi1 pY213 and the C-terminal SH2 domain of p85—thereby linking Abl kinase signaling to p85-dependent regulation of macropinocytosis.

Structured summary

MINT-7908602: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to SHIP2 (uniprotkb:O15357) by array technology (MI:0008)MINT-7908362: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Emt (uniprotkb:Q08881) by array technology (MI:0008)MINT-7908235: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Lyn (uniprotkb:P07948) by array technology (MI:0008)MINT-7908075: Abi1 (uniprotkb:Q8IZP0)binds (MI:0407) to Fgr (uniprotkb:P09769) by array technology (MI:0008)MINT-7908330, MINT-7908522: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Vav1 (uniprotkb:P15498) by array technology (MI:0008)MINT-7907962: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Fyn (uniprotkb:P06241) by array technology (MI:0008)MINT-7908203: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Src (uniprotkb:P12931) by array technology (MI:0008)MINT-7908570: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to SHP-2 (uniprotkb:P35235) by array technology (MI:0008)MINT-7908187, MINT-7908586: Abi1(uniprotkb:Q8IZP0) binds (MI:0407) to Gap (uniprotkb:P20936) by array technology (MI:0008)MINT-7907981, MINT-7907995: Abi1 (uniprotkb:Q8IZP0) physically interacts (MI:0915) with p85a (uniprotkb:P26450) by anti tag coimmunoprecipitation (MI:0007)MINT-7908251: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to PLCG1 (uniprotkb:P19174) by array technology (MI:0008)MINT-7908346: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Grb2 (uniprotkb:P62993) by array technology (MI:0008)MINT-7907945: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Abl (uniprotkb:P00519) by array technology (MI:0008)MINT-7908474: Abi1 (uniprotkb:Q8IZP0)binds (MI:0407) to p85b (uniprotkb:O00459) by array technology (MI:0008)MINT-7908107: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Hck (uniprotkb:P08631) by array technology (MI:0008)MINT-7908011: p85a (uniprotkb:P26450) physically interacts (MI:0915) with Abi1 (uniprotkb:Q8IZP0) by pull down (MI:0096)MINT-7908155: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to FynT (uniprotkb:P06241-2) by array technology (MI:0008)MINT-7908283, MINT-7908490: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to p55g (uniprotkb:Q92569) by array technology (MI:0008)MINT-7907929, MINT-7907815, MINT-7907832, MINT-7907865, MINT-7907897, MINT-7907913, MINT-7907881, MINT-7907848: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to p85a (uniprotkb:P27986) by array technology (MI:0008)MINT-7908059: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Frk (uniprotkb:P42685) by array technology (MI:0008)MINT-7908378: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to CblC (uniprotkb:Q9ULV8) by array technology (MI:0008)MINT-7908618: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to CblA (uniprotkb:B5MC15) by array technology (MI:0008)MINT-7908139, MINT-7908538: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Nap4 (uniprotkb:O14512) by array technology (MI:0008)MINT-7908426: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to CblB (uniprotkb:Q13191) by array technology (MI:0008)MINT-7908506: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Crk (uniprotkb:P46108) by array technology (MI:0008)MINT-7908554: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to mAbl (uniprotkb:P00520) by array technology (MI:0008)MINT-7908043, MINT-7908394: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Vav2 (uniprotkb:P52735) by array technology (MI:0008)MINT-7908458: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to mSck/ShcB (uniprotkb:Q8BMC3) by array technology (MI:0008)MINT-7908091: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Yes (uniprotkb:P07947) by array technology (MI:0008)MINT-7908219: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Src (uniprotkb:P00523) by array technology (MI:0008)MINT-7908123: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Fer (uniprotkb:P16591) by array technology (MI:0008)MINT-7908410: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to CrkL (uniprotkb:P46109) by array technology (MI:0008)MINT-7908314, MINT-7908442: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Arg (uniprotkb:P42684) by array technology (MI:0008)MINT-7908299: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to PLCG1 (uniprotkb:P10686) by array technology (MI:0008)MINT-7908171: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Fes (uniprotkb:P07332) by array technology (MI:0008)MINT-7908027: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Lck (uniprotkb:P06239) by array technology (MI:0008)     

Inhibition of human initiator caspase 8 and effector caspase 3 by cross-class inhibitory bovSERPINA3-1 and A3-3

Serpins are a superfamily of structurally conserved proteins. Inhibitory serpins use a suicide substrate-like mechanism. Some are able to inhibit cysteine proteases in cross-class inhibition. Here, we demonstrate for the first time the strong inhibition of initiator and effector caspases 3 and 8 by two purified bovine SERPINA3s. SERPINA 3-1 (uniprotkb:Q9TTE1) binds tighly to human CASP3 (uniprotkb:P42574) and CASP8 (uniprotkb:Q14790) with k_ass of 4.2 × 10⁵ and 1.4 × 10⁶ M⁻¹ s⁻¹, respectively. A wholly similar inhibition of human CASP3 and CASP8 by SERPINA3-3 (uniprotkb:Q3ZEJ6) was also observed with k_ass of 1.5 × 10⁵ and 2.7 × 10⁶ M⁻¹ s⁻¹, respectively and form SDS-stable complexes with both caspases. By site-directed mutagenesis of bovSERPINA3-3, we identified Asp³⁷¹ as the potential P1 residue for caspases. The ability of other members of this family to inhibit trypsin and caspases was analysed and discussed.

Structured summary

MINT-7234656: CASP8 (uniprotkb:Q14790) and SERPINA3-1 (uniprotkb:Q9TTE1) bind (MI:0407) by biochemical (MI:0401)MINT-7234634: SERPINA3-3 (uniprotkb:Q3ZEJ6) and CASP3 (uniprotkb:P42574) bind (MI:0407) by biochemical (MI:0401)MINT-7234663: CASP8 (uniprotkb:Q14790) and SERPINA3-3 (uniprotkb:Q3ZEJ6) bind (MI:0407) by biochemical (MI:0401)MINT-7234625: SERPINA3-1 (uniprotkb:Q9TTE1) and CASP3 (uniprotkb:P42574) bind (MI:0407) by biochemical (MI:0401)     

AFM visualization of clathrin triskelia under fluid and in air

Atomic force microscopy (AFM) is used to characterize the structure and interactions of clathrin triskelia. Time sequence images of individual, wet triskelia resting on mica surfaces clearly demonstrate conformational fluctuations of the triskelia. AFM of dried samples yields images having nanometric resolution comparable to that obtainable by electron microscopy of shadowed samples. Increased numbers of triskelion dimers and assembly intermediates, as well as structures having dimensions similar to those of clathrin cages, are observed when the triskelia were immersed in a low salt, low pH buffer. These entities have been quantified by AFM protein volume computation.

Structured summary

MINT-7299119, MINT-7299136:Clathrin (uniprotkb:P49951) and Clathrin (uniprotkb:P49951) bind (MI:0407) by atomic force microscopy (MI:0872)     

Isolation of a point-mutated p47 lacking binding affinity to p97ATPase

p47, a p97-binding protein, functions in Golgi membrane fusion together with p97 and VCIP135, another p97-binding protein. We have succeeded in creating p47 with a point mutation, F253S, which lacks p97-binding affinity. p47 mapping experiments revealed that p47 had two p97-binding regions and the F253S mutation occurred in the first p97-binding site. p47(F253S) could not form a complex with p97 and did not caused any cisternal regrowth in an in vitro Golgi reassembly assay. In addition, mutation corresponding to the p47 F253S mutation in p37 and ufd1 also abolished their binding ability to p97.

Structured summary

MINT-7987189, MINT-7987207, MINT-7987303: p47 (uniprotkb:O35987) binds (MI:0407) to p97 (uniprotkb:Q01853) by pull down (MI:0096)MINT-7987226: p97 (uniprotkb:P46462) binds (MI:0407) to p47 (uniprotkb:O35987) by pull down (MI:0096)MINT-7987348: p97 (uniprotkb:P46462) physically interacts (MI:0915) with Ufd1 (uniprotkb:P70362) by pull down (MI:0096)MINT-7987264: p97 (uniprotkb:P46462) and p47 (uniprotkb:O35987) bind (MI:0407) by competition binding (MI:0405)MINT-7987326: p97 (uniprotkb:P46462) binds (MI:0407) to p37 (uniprotkb:Q0KL01) by pull down (MI:0096)     

An S-locus receptor-like kinase in plasma membrane interacts with calmodulin in Arabidopsis

Calmodulin-regulated protein phosphorylation plays a pivotal role in amplifying and diversifying the action of calcium ion. In this study, we identified a calmodulin-binding receptor-like protein kinase (CBRLK1) that was classified into an S-locus RLK family. The plasma membrane localization was determined by the localization of CBRLK1 tagged with a green fluorescence protein. Calmodulin bound specifically to a Ca²⁺-dependent calmodulin binding domain in the C-terminus of CBRLK1. The bacterially expressed CBRLK1 kinase domain could autophosphorylate and phosphorylates general kinase substrates, such as myelin basic proteins. The autophosphorylation sites of CBRLK1 were identified by mass spectrometric analysis of phosphopeptides.

Structured summary

MINT-6800947:CBRLK1 (uniprotkb:Q9ZT06) and AtCaM2 (uniprotkb:P25069) bind (MI:0407) by electrophoretic mobility shift assay (MI:0413)MINT-6800966:AtCaM2 (uniprotkb:P25069) and CBRLK1 (uniprotkb:Q9ZT06) bind (MI:0407) by competition binding (MI:0405)MINT-6800930:CBRLK1 (uniprotkb:Q9ZT06) binds (MI:0407) to AtCaM2 (uniprotkb:P25069) by far Western blotting (MI:0047)MINT-6800978:AtCaM2 (uniprotkb:P25069) physically interacts (MI:0218) with CBRLK1 (uniprotkb:Q9ZT06) by cytoplasmic complementation assay (MI:0228)     

The N-terminal domain of human holocarboxylase synthetase facilitates biotinylation via direct interaction with the substrate protein

Human holocarboxylase synthetase shows a high degree of sequence homology in the catalytic domain with bacterial biotin ligases such as Escherichia coli BirA, but differs in the length and sequence of the N-terminus. Despite several studies having been undertaken on the N-terminal region of hHCS, the role of this region remains unclear. We determined the structure of the N-terminal domain of hHCS by limited proteolysis and showed that this domain has a crucial effect on the enzymatic activity. The domain interacts not only with biotin acceptor protein, but also with the catalytic domain of hHCS, as shown by nuclear magnetic resonance (NMR) experiments. We propose that the N-terminal domain of hHCS recognizes the charged region of biotin acceptor protein, distinctly from the recognition by the catalytic domain.

Structured summary

MINT-7543113: hHCS (uniprotkb:P50747) and hHCS (uniprotkb:P50747) bind (MI:0407) by nuclear magnetic resonance (MI:0077)MINT-7543096, MINT-7543129: ACC75 (uniprotkb:O00763) and hHCS (uniprotkb:P50747) bind (MI:0407) by nuclear magnetic resonance (MI:0077)MINT-7543053: hHCS (uniprotkb:P50747) enzymaticly reacts (MI:0414) ACC75 (uniprotkb:O00763) by nuclear magnetic resonance (MI:0077)MINT-7543070: hHCS (uniprotkb:P50747) enzymaticly reacts (MI:0414) ACC75 (uniprotkb:O00763) by enzymatic study (MI:0415)     

A disulfide driven domain swap switches off the activity of Shigella IpaH9.8 E3 ligase

We show that the monomeric form of Shigella IpaH9.8 E3 ligase catalyses the ubiquitination of human U2AF35 in vitro, providing a molecular mechanism for the observed in vivo effect. We further discover that under non-reducing conditions IpaH9.8 undergoes a domain swap driven by the formation of a disulfide bridge involving the catalytic cysteine and that this dimer is unable to catalyse the ubiquitination of U2AF35. The crystal structure of the domain-swapped dimer is presented. The redox inactivation of IpaH9.8 could be a mechanism of regulating the activity of the IpaH9.8 E3 ligase in response to cell damage so that the host cell in which the bacteria resides is maintained in a benign state suitable for bacterial survival.

Structured summary

MINT-7993779: ipaH9.8 (uniprotkb:Q8VSC3) and ipaH9.8 (uniprotkb:Q8VSC3) bind (MI:0408) by X-ray crystallography (MI:0114) MINT-7993812: ipaH9.8 (uniprotkb:Q8VSC3) and ipaH9.8 (uniprotkb:Q8VSC3) bind (MI:0407) by affinity chromatography technology (MI:0004) MINT-7993790: ipaH9.8 (uniprotkb:Q8VSC3) and ipaH9.8 (uniprotkb:Q8VSC3) bind (MI:0407) by blue native page (MI:0276)