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Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography
利用合理化诱变和X射线晶体学分析在不同功能状态下的信号复合体DesK:DesR   

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Abstract

We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR complexes and further purification. The 3D structures of the complex trapped in the phosphotransferase and in the phosphatase reaction steps, were solved by X-ray crystallography using molecular replacement. The solution was not trivial, and we found that in silico-generated models used as search probes, were instrumental to succeeding in placing a large portion of the complex in the asymmetric unit. Electron density maps were then clear enough to allow for manual model building attaining complete atomic models. These methods contribute to tackling a major challenge in the bacterial signaling field, namely obtaining stable kinase:regulator complexes, in distinct conformational states, amenable for high-resolution crystallographic studies.

Keywords: Signaling proteins(信号蛋白), Protein phosphorylation(蛋白磷酸化), Trapping conformational rearrangements(捕获构象重排), Structure-based mutagenesis(基于结构的诱变), X-ray crystallography(X射线晶体学), Protein engineering(蛋白质工程)

Background

Structural information about bacterial signaling complexes, especially of two-component systems (TCSs), is still scarce (Casino et al., 2009; Gao and Stock, 2009). TCSs comprise a sensory histidine-kinase (HK) and a response regulator (RR) partner, present in almost all bacteria, they allow the cells to perceive the environment and to react accordingly through adaptive responses. Structural information is even more limited when it comes to TCS complexes adopting different functional states, despite the importance of such switching mechanism in signal transmission (Trajtenberg et al., 2016). We have studied the DesK-DesR pathway (de Mendoza, 2014), a TCS from Bacillus subtilis involved in regulating the cell membrane composition in adaptation to cues that reduce the bilayer’s fluidity, such as cold shock.

The protocols we have developed were aimed at overcoming major technical bottlenecks, encompassing complex purification, crystallization and X-ray structure determination. Most of these hurdles likely arise from the intrinsic flexibility and heterogeneity that characterize TCS proteins. With the purpose of trapping the DesK:DesR complex in defined signaling steps, it is useful to recall some details based on previous findings from our laboratory. The protocols have been developed to work with DesKC, a truncated DesK variant comprising the entire cytoplasmic region of DesK, without the trans-membrane sensory domain, which is catalytically competent to phosphotransfer to DesR, as well as to dephosphorylate P~DesR (Albanesi et al., 2004). As for the response regulator partner, DesR, we have chosen to use a truncated form, including the entire receiver domain (REC), competent for all DesK-mediated phosphotransfer reactions (Trajtenberg et al., 2014), but lacking the C-terminal DNA-binding domain, and thus minimizing potential inter-domain flexibility issues.

In order to trap the DesKC:DesR complex in the phosphotransfer step of the signaling pathway, we chose to use the phosphomimetic point mutant DesKC-His188Glu. This variant, when not bound to DesR, adopts a structural conformation very similar to the phosphorylated form of wild-type DesKC (Albanesi et al., 2009), hence an attractive template to mimic the phosphorylated HK just prior to the transfer reaction, also avoiding effective transfer to take place.

On the other hand, in order to trap the DesKC:DesR complex in the dephosphorylation step, previous work was instrumental by uncovering a switch mechanism of DesK, swapping between ‘active’ (kinase-on/phosphatase-off) and ‘inactive’ (phosphatase-on/kinase-off traits) states of the kinase (Albanesi et al., 2009). Briefly, the conformational transition of DesK from its kinase-active to the inhibited form, implicates the assembly of a coiled-coil structure within the central Dimerization and His-phosphotransfer (DHp) domain, a coiled-coil that is otherwise ‘broken’ when the kinase is active. The DHp, an all-helical domain, connects the trans-membrane sensor with the Catalytic ATP-binding (CA) domains, hence the identified DHp’s conformational switching plays a key role in signal transmission through long-range allosteric rearrangements. Such mechanistic insights later led to constructing a coiled-coil hyper-stabilized variant (DesKSTA) (Saita et al., 2015), harboring point-mutations at key positions (Ser150Ile, Ser153Leu and Arg157Ile) that stabilize a phosphatase-constitutive form (Saita et al., 2015). The corresponding soluble construct, with the trans-membrane domain truncated (DesKCSTAB), indeed displays a phosphatase-trapped 3D structure (Trajtenberg et al., 2016). DesKCSTAB was used to trap the DesKC:DesR complex in the dephosphorylation step, as described in this protocol.

Materials and Reagents

  1. P2, P200 and P1000 micropipette tips, autoclaved (Gilson, catalog numbers: F161630 , F161930 and F161670 )
  2. 1.5 ml Eppendorf tubes (Eppendorf, catalog number: 022364111 )
  3. 15 and 50 ml Falcon tubes (Corning, catalog numbers: 352097 and 352098 )
  4. Minisart 0.45 µm syringe filter (Sartorius, catalog number: 16555-K )
  5. 96-Well Clear V-Bottom 2 ml polypropylene deep well plate (Corning, catalog number: 3960 )
  6. Minisart 0.22 µm syringe filter (Sartorius, catalog number: 16532-K )
  7. 1 L polypropylene bottles (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3140-1002 )
  8. SnakeSkin Dialysis Tubing 3.5K MWCO, 35 mm Dry I.D., 35 feet (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88244 )
  9. Vivaspin 6 ml 10,000 MWCO centrifugal concentrator devices (Sartorius, catalog number VS0601 )
  10. Vivaspin 20 ml 10,000 MWCO centrifugal concentrator devices (Sartorius, catalog number VS2001 )
  11. Linbro 24-well plates (MP Biomedicals, catalog number: CPL-101 )
  12. Cryo-loops (HAMPTON RESEARCH, catalog number: HR4-955 )
  13. Cover slide
  14. Escherichia coli BL21 (DE3) and TOP10F’ strains from stocks stored at -80 °C
  15. pACYCDuet-1 (Novagen) and pQE80L (QIAGEN) plasmids
  16. Tobacco etch virus (TEV) protease (3 mg/ml stock solution, in-house preparation)
  17. Ultra-pure water (> 18 MΩ) filtered with 0.22 µm Express Plus filters (EMD Millipore, catalog number: SCGPT05RE )
  18. Ethanol 95% (Industrial Uruguayan Drugstore)
  19. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 , 17 mg/ml stock solution, stored at -20 °C)
  20. Ampicillin (Sigma-Aldrich, catalog number: A9518 , 100 mg/ml stock solution, stored at -20 °C)
  21. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
  22. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Euromedex, catalog number: EU0008-B , 1 M stock solution, stored at -20 °C)
  23. Lysozyme (Sigma-Aldrich, catalog number: L6876 , 100 mg/ml stock solution)
  24. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  25. Zinc chloride (ZnCl2) (Sigma-Aldrich, catalog number: 229997 )
  26. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  27. Acrylamide/Bis-acrylamide 30% solution (Sigma-Aldrich, catalog number: A3574 )
  28. DNA Ladder GeneRuler 100 bp Plus (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM0321 )
  29. Oligonucleotides for mutagenesis and PCR amplifications (IDT DNA Technologies)
  30. Phusion High Fidelity DNA polymerase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: F530S )
  31. Sodium dodecyl sulfate (Sigma-Aldrich, catalog number: L5750 )
  32. Ammonium persulfate (Sigma-Aldrich, catalog number: 248614 )
  33. N,N,N’,N’-Tetramethylethylenediamine (Sigma-Aldrich, catalog number: T9281 )
  34. Color Protein Ladder Prestained Broad Range (New England Biolabs, catalog number: P7712S )
  35. Precision Plus Protein Standard All Blue (Bio-Rad Laboratories, catalog number: 1610373 )
  36. Brilliant Blue R (Sigma-Aldrich, catalog number: B0149 )
  37. Lithium potassium acetyl phosphate (Sigma-Aldrich, catalog number: A0262 )
  38. Adenosine 5’-triphosphate (ATP) disodium salt hydrate (Sigma-Aldrich, catalog number: A1852 )
  39. β,γ-Methyleneadenosine 5’-triphosphate (AMP-PCP) disodium salt (Sigma-Aldrich, catalog number: M7510 )
  40. Trizma base (Sigma-Aldrich, catalog number: T1503 )
  41. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434 )
  42. Magnesium chloride hexahydrate (MgCl2•6H2O) (Sigma-Aldrich, catalog number: 13152 )
  43. Polyethylene glycol (PEG) 600 (Sigma-Aldrich, catalog number: 87333 )
  44. MES (Sigma-Aldrich, catalog number: M8250 )
  45. Magnesium sulfate (MgSO4)
  46. Glycerol (AppliChem, catalog number: 131339 )
  47. Polyethylene glycol (PEG) 4000 (Sigma-Aldrich, catalog number: 95904 )
  48. Lithium sulfate (Li2SO4) (Sigma-Aldrich, catalog number: 203653 )
  49. Beryllium chloride (BeCl2) (Sigma-Aldrich, catalog number: 201197 )
    Note: This product has been discontinued.
  50. Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: 71519 )
  51. Liquid nitrogen
  52. Polyethylene glycol (PEG) 3350 (Sigma-Aldrich, catalog number: 202444 )
  53. Tri-potassium citrate (Sigma-Aldrich, catalog number: P1722 )
  54. Yeast extract (Sigma-Aldrich, catalog number: Y1625 )
  55. Tryptone plus (Sigma-Aldrich, catalog number: 61044 )
  56. Agar (Sigma-Aldrich, catalog number: A9799 )
  57. EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 11873580001 )
  58. Imidazole (Merck, catalog number: 104716 )
  59. Dithiothreitol (Soltec Ventures, catalog number: M112 )
  60. Hydrochloric acid solution (Sigma-Aldrich, catalog number: 13-1683 )
  61. LB medium (see Recipes)
  62. 2x YT culture medium (see Recipes)
  63. LB agar plates (see Recipes)
  64. Lysis buffer (see Recipes)
  65. Immobilized Metal Affinity Chromatography (IMAC) binding and washing buffer (see Recipes)
  66. IMAC elution buffer (see Recipes)
  67. Dialysis buffer (see Recipes)
  68. Size Exclusion Chromatography buffer for the phosphatase complex (SEC-P buffer)
  69. Size Exclusion Chromatography buffer for the phosphotransferase complex (SEC-PT buffer)

Equipment

  1. Pipetman P2 single channel pipette (Gilson, catalog number: F144801 )
  2. Pipetman P20 single channel pipette (Gilson, catalog number: F123600 )
  3. Pipetman P200 single channel pipette (Gilson, catalog number: F123601 )
  4. Pipetman P1000 single channel pipette (Gilson, catalog number: F123602 )
  5. 250 ml Erlenmeyer flasks (Marienfeld-Superior, catalog number: 4110207 )
  6. 5 L Erlenmeyer flasks (Marienfeld-Superior, catalog number: 4110217 )
  7. HisTrap immobilized metal affinity chromatography (IMAC) 5 ml column with Zn-NTA resin (GE Healthcare, catalog number: 17-5248-01 . In-house preparation)
  8. Superdex S75 26/600 (GE Healthcare, catalog number: 28989334 )
  9. Stirring magnet (Sigma-Aldrich)
  10. Thermomixer C (Eppendorf, model: ThermoMixer® C , catalog number: 4053-8223)
  11. Digital Ultrasonics Sonifier S-450 cell disruptor/homogenizer (Emerson, Branson Ultrasonics, model: S-450 )
  12. Multitron Standard incubation shaker (Infors HT)
  13. Minispin centrifuge (Eppendorf, model: MiniSpin® , catalog number: 5452000018)
  14. Refrigerated 5424R Centrifuge (Eppendorf, model: 5424 R , catalog number: 36-102-3795)
  15. Refrigerated 5810R Centrifuge (Eppendorf, model: 5810 R , catalog number: 5811000010)
  16. Refrigerated Sorvall Lynx4000 centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall Lynx4000 , catalog number: 75006580)
  17. Minipuls 3 peristaltic pump (Gilson, catalog number: F117604 )
  18. Chromatography instrument ÄKTA purifier (GE Healthcare, model: ÄKTAxpress , catalog number: 18664501)
  19. EPS-301 power supply (GE Healthcare, model: EPS 301, catalog number: 18-1130-01 )
  20. Cary 50 Bio UV-visible spectrophotometer (Varian, model: Cary® 50 )
  21. Alchemist DT (Rigaku, model: Alchemist DT )
  22. X-ray generator MicroMax-007HF (Rigaku, model: MicroMax-007 HF )
  23. Multilayer X-ray mirrors Varimax-HF (Rigaku, model: VariMax HF )
  24. Image plate area detector MAR345® (marXperts, model: mar345 )
  25. SZX16 microscope (Olympus, model: SZX16 )
  26. EVOLT E-330 digital camera (Olympus, model: E-330 )
  27. LG-PS2 light source (Olympus, model: LG-PS2 )
  28. Linux Computer workstation with OS Centos 7.0

Software

  1. Produced diffraction datasets:
    1. Complex DesKC:DesR-REC in phosphotransferase state, low Mg2+:
      http://dx.doi.org/10.15785/SBGRID/399
    2. Complex DesKC:DesR-REC in phosphotransferase state, high Mg2+:
      http://dx.doi.org/10.15785/SBGRID/401
    3. Complex DesKC:DesR-REC in phosphotransferase state, high Mg2+ and BeF3-:
      http://dx.doi.org/10.15785/SBGRID/408
    4. Complex DesKC:DesR-REC in phosphatase state:
      http://dx.doi.org/10.15785/SBGRID/400
  2. Produced atomic coordinate models:
    1. Complex DesKC:DesR-REC in phosphotransferase state, low Mg2+:
      http://www.rcsb.org/pdb/explore/explore.do?structureId=5IUJ
    2. Complex DesKC:DesR-REC in phosphotransferase state, high Mg2+:
      http://www.rcsb.org/pdb/explore/explore.do?structureId=5IUK
    3. Complex DesKC:DesR-REC in phosphotransferase state, high Mg2+ and BeF3-:
      http://www.rcsb.org/pdb/explore/explore.do?structureId=5IUL
    4. Complex DesKC:DesR-REC in phosphatase state:
      http://www.rcsb.org/pdb/explore/explore.do?structureId=5IUN
  3. Computational crystallography software utilized:
    1. autoPROC (https://www.globalphasing.com/autoproc)
    2. BUSTER (https://www.globalphasing.com/buster)
    3. CCP4 (http://www.ccp4.ac.uk)
    4. Phaser (http://www.phaser.cimr.cam.ac.uk)
    5. Coot (https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot)
    6. MolProbity (http://molprobity.biochem.duke.edu)
    7. PyMol (https://www.pymol.org)

Procedure

  1. The use of DesKC mutants to stabilize the histidine-kinase either in its phosphatase or its phosphotransferase state
    1. The plasmid pACYC-DesKCH188E:DesRREC encoding for the phosphomimetic DesKC variant and for the receiver domain of DesR (DesRREC), both fused to a His-tag and a TEV protease site to cleave the tag, was already available (Trajtenberg et al., 2014). This plasmid pACYC-DesKCH188E:DesRREC is thus used to co-express the following two proteins:
      1. DesKCH188E:
        MGSSHHHHHHGIHMENLYFQGRKERERLEEKLEDANERIAELVKLEERQRIARDLEDTLGQKLSLIGLKSDLARKLIYKDPEQAARELKSVQQTARTSLNEVRKIVSSMKGIRLKDELINIKQILEAADIMFIYEEEKWPENISLLNENILSMCLKEAVTNVVKHSQAKTCRVDIQQLWKEVVITVSDDGTFKGEENSFSKGHGLLGMRERLEFANGSLHIDTENGTKLTMAIPNNSK
        Theoretical MW = 27.3 kDa (24.9 after TEV-cleavage)
        Theoretical absorbance (280 nm) of a 1 mg/ml solution after TEV-cleavage = 0.56
      2. DesRREC:
        MRGSHHHHHHGSGSENLYFQGSGSMISIFIAEDQQMLLGALGSLLNLEDDMEVVGKGTTGQDAVDFVKKRQPDVCIMDIEMPGKTGLEAAEELKDTGCKIIILTTFARPGYFQRAIKAGVKGYLLKDSPSEELANAIRSVMNGKRIYAPELMEDLYSEA
        Theoretical MW = 17.4 kDa (15.2 after TEV-cleavage)
        Theoretical absorbance (280 nm) of a 1 mg/ml solution after TEV-cleavage = 0.40
    2. Within the sequences listed in the previous point, His-tags are highlighted with blue fonts, in green, the TEV-recognition sites (cleaved proteins start at the final Gly, including it). Underlined in red, the phosphorylation sites: DesRREC bears the wild-type Asp, while DesKCH188E displays instead a Glu replacing the native His.
    3. The phosphatase-stabilized variant of DesKC, DesKCSTAB, is insoluble when expressed by itself in Escherichia coli. This difficulty is solved by co-expressing DesKCSTAB with DesRREC, resulting in excellent yields of both proteins. A co-expression plasmid is generated (pACYC-DesKCSTAB:DesRREC), by sub-cloning DesKCSTAB from pHPKS/Pxyl-desKSTA (Saita et al., 2015) into pACYC-DesKCH188E:DesRREC (Trajtenberg et al., 2014) using restriction-free cloning (Unger et al., 2010), using primers STAB_F (5’-CCTGTATTTTCAGGGATCCGGTATTATAAAACTTCGCAAG-3’) and STAB_R (5’-GTCAGACACTGTAATCACAACTTCCTTCCAG-3’). Both recombinant proteins encoded in pACYC-DesKCSTAB:DesRREC include a His-tag and a TEV protease cleavage site.
    4. pACYC-DesKCSTAB:DesRREC co-expresses the following two proteins:
      1. DesKCSTAB:
        MGSSHHHHHHGSGSENLYFQGSGIIKLRKEIERLEEKLEDANERIAELVKLEERQRIARDLHDTLGQKLSLIGLKSDLARKLIYKDPEQAARELKSVQQTARTSLNEVRKIVSSMKGIRLKDELINIKQILEAADIMFIYEEEKWPENISLLNENILSMCLKEAVTNVVKHSQAKTCRVDIQQLWKEVVITVSDDGTFKGEENSFSKGHGLLGMRERLEFANGSLHIDTENGTKLTMAIPNNSK
        Theoretical MW = 27.8 kDa (25.6 after TEV-cleavage)
        Theoretical absorbance (280 nm) of a 1 mg/ml solution after TEV-cleavage = 0.55
      2. DesRREC:
        MRGSHHHHHHGSGSENLYFQGSGSMISIFIAEDQQMLLGALGSLLNLEDDMEVVGKGTTGQDAVDFVKKRQPDVCIMDIEMPGKTGLEAAEELKDTGCKIIILTTFARPGYFQRAIKAGVKGYLLKDSPSEELANAIRSVMNGKRIYAPELMEDLYSEA
        Theoretical MW = 17.4 kDa (15.2 after TEV-cleavage)
        Theoretical absorbance (280 nm) of a 1 mg/ml solution after TEV-cleavage = 0.40
        Note: the theoretical absorbance (280 nm) of a 1 mg/ml solution of the DesKC:DesR protein complex is approximately 0.5, which corresponds to both protein sequences together.
    5. Within the sequences listed in the previous point, His-tags are highlighted with blue fonts, in green, the TEV-recognition sites (cleaved proteins start at the final Gly, including it). Underlined in orange, the three stabilizing substitutions within the coiled-coil motif of DesK’s DHp domain. Underlined in red, the phosphorylation sites.

  2. Over-expression of DesKC:DesR complexes
    1. The co-expression plasmid pACYC-DesKCSTAB:DesRREC (and similarly for pACYC-DesKCH188E:DesRREC), is transformed into 25 µl of competent Escherichia coli BL21 (DE3) cells (approximately 108 cells/ml) using heat shock for 2 min at 42 °C. A pQE80L plasmid including the DesRREC sequence (Trajtenberg et al., 2014) is co-transformed to produce a stoichiometric excess of DesRREC in the cells. 100 μl of transformed cells are spread on LB agar plates containing 100 μg/ml ampicillin and 17 μg/ml chloramphenicol, and grown overnight at 37 °C.
    2. Between 5 and 10 single colonies are picked and inoculated in 3 ml LB culture media (to be used as pre-cultures in the next step), 100 μg/ml ampicillin and 17 μg/ml chloramphenicol, and further grown at 37 °C for 16 h with agitation (220 rpm).
    3. Pre-cultures are inoculated 1/500 in 250 ml Erlenmeyer flasks containing 50 ml LB media, 100 μg/ml ampicillin and 17 μg/ml chloramphenicol, and further grown at 37 °C for 16 h with agitation (220 rpm).
    4. Larger-scale cultures are launched using three 5 L Erlenmeyer flasks, each containing 1 L 2x YT culture media, 2 mM magnesium sulfate, 100 μg/ml ampicillin, 17 μg/ml chloramphenicol and a 1/100 inoculum of pre-cultured bacteria. Cultures are grown at 37 °C with agitation (220 rpm) until absorbance at wavelength 660 nm (Abs660 nm) reaches 0.7-1 (approximately 3 h). Cultures are quickly shifted to a shaker that has been pre-equilibrated at 20 °C, and further growth is continued for 30 min with agitation (220 rpm).
    5. IPTG is added (0.5 mM final concentration) into the cultures. Induction of over-expression is achieved at 30 °C for 4 h (in the case of DesKCSTAB:DesRREC) or at 20 °C for 16 h (for DesKCH188E:DesRREC), with agitation (220 rpm) in both cases.
    6. Cells are harvested by centrifugation at 4,600 x g for 20 min at 4 °C in 1 L polypropylene bottles.
    7. Pellets are thoroughly resuspended in lysis buffer (see Recipes) (5 ml per g wet weight pellet) in 50 ml Falcon tubes.
    8. Lysozyme (0.5 mg/ml final concentration) and Triton X-100 (1% vol/vol final concentration) are added to resuspended pellets, and incubated at room temperature for 30 min, until the extract is viscous due to DNA release. Extracts are then transferred to -80 °C and stored for at least 2 h.
    9. Frozen extracts are thawed in a water bath at 37 °C. Thawed extracts are sonicated with pulses of 1 sec (set to 30% amplitude) and resting intervals of 3 sec, completing a total timespan of 4 min. This operation is repeated 3-4 times, until complete reduction of viscosity. During sonication the Falcon tube is kept refrigerated with ice. This is considered the total extract, and 40 μl are stored in electrophoresis sample buffer with β-mercaptoethanol (SBβ) for later SDS-PAGE analyses.
    10. Total extracts are centrifuged at 12,000 x g and 4 °C for 45 min. This step separates soluble species (supernatant fraction) from insoluble ones (pellet) such as inclusion bodies, non-lysed cells, insoluble proteins, etc. The supernatants are filtered through a 0.45 μm syringe filter to a new Falcon tube, and imidazole added to 40 mM final concentration (to avoid nonspecific binding to the column during IMAC). 40 μl of supernatants are stored in SBβ for SDS-PAGE analysis. Supernatants are hereafter used as the source of soluble DesKCSTAB (or DesKCH188E) and DesRREC, for structural studies.

  3. Purification of DesKC:DesR complexes
    1. Zinc is used as the immobilized metal, during the first purification step by IMAC. Nickel is avoided to preclude cysteine oxidation, which previously hampered crystallization due to resulting heterogeneity in DesR samples. The IMAC column is connected to the ÄKTA purifier and pre-equilibrated with binding buffer (see Recipes) (10 column volumes or CV). Samples are injected at a flow rate of 5 ml/min, and target proteins are bound to the column through their N-terminal His-tags.
    2. IMAC washing is achieved with 15-20 CV binding buffer, until a stable Abs280 baseline is obtained. Aliquots of the flow-through material are stored in SBβ for SDS-PAGE analysis, to monitor for potential column saturation.
    3. IMAC elution is achieved with a linear 0-100% gradient of elution buffer (see Recipes) (30 CV), at 5 ml/min. Absorbance at wavelength 280 nm is used to monitor protein elution peaks. DesKCSTAB, DesKCH188E and DesRREC typically elute at approximately 65 mM imidazole and are collected in 96-deep-well plates. 40 μl of eluted peaks are stored in SBβ for SDS-PAGE analysis.
    4. The elution peaks of selected proteins are pooled and incubated with TEV protease (1:40 w/w TEV/target ratio). Proteolysis is performed overnight, in dialysis bags. Dialyses are performed against 200-300 volumes of dialysis buffer (see Recipes) with gentle stirring.
    5. Samples are recovered from the dialysis bag, and filtered with 0.45 μm syringe filters. A second IMAC zinc column is used, attached to a peristaltic pump and pre-equilibrated with binding buffer (10 CV). Samples are injected into the column at a flow rate of 5 ml/min. The flow-through is now carefully collected. The TEV protease itself includes an N-terminal His-tag, hence typically excluded from the flow-through. 20 CV of elution buffer is applied to the column to elute TEV (and potentially non-digested target protein, normally absent if the procedure worked correctly) to be monitored by SDS-PAGE.
    6. The second IMAC flow-through sample is concentrated by ultra-filtration in Vivaspin centrifuge devices at 6,300 x g and 15 °C for 20 min to a final volume of 10 ml. This is immediately filtered through a 0.22 μm syringe filter, and injected into a size exclusion chromatography (SEC) column Superdex S75 26/600, at a flow rate of 1 ml/min. The SEC column is previously equilibrated with 2 CV SEC buffer (see Recipes).
    7. The SEC is eluted isocratically at 1 ml/min with SEC-P buffer in the case of the DesKCSTAB:DesRREC (phosphatase) complex, or with SEC-PT buffer in the case of the DesKCH188E:DesRREC (phosphotransferase) complex. Eluted fractions are collected in 96-deep-well plates. Protein elution is monitored with 280 nm wavelength absorbance (Figures 1A and 2A). 40 μl of eluted peaks are stored in SBβ for SDS-PAGE analysis.
      The calculated molecular weight (MW) of the complex is approximately 65 kDa taking into account that DesKC is a homo-dimer, and that one monomer of DesRREC could be binding to DesKC. If instead the DesKC:DesRREC ratio is 2:2, the MW is anticipated to increase to ~80 kDa. According to the calibration curves, the SEC chromatograms reveal a MW of 53.5 kDa in the case of DesKCSTAB:DesRREC, and 44.9 kDa for DesKCH188E:DesRREC. The difference with the theoretical MW figures is likely due to tertiary and quaternary structure features deviating from the ideal assumptions for globular species and their hydrodynamic radii. That these SEC peaks correspond indeed to the targeted DesKC:DesR complexes is afterwards confirmed by mass spectrometry analyses and then 3D structure determination. The final yields approximated 12 mg protein per L of cell culture (or 1-2 mg protein per g of wet weight pellet), for both complexes.


      Figure 1. Size-exclusion chromatography and crystallization of the DesKCSTAB:DesRREC complex. A. Elution profile of the DesKCSTAB:DesRREC protein complex in a Superdex S75 pg 26/600 size exclusion chromatography column. The peak at 140.9 ml corresponds to the complex, according to SDS-PAGE analysis. The peak at 201.5 ml corresponds to monomeric DesRREC in excess. B. Trigonal crystal of the DesKCSTAB:DesRREC complex.


      Figure 2. Size-exclusion chromatography and crystallization of the DesKCH188E:DesRREC complex. A. Elution profile of the DesKCH188E:DesRREC protein complex in a Superdex S75 pg 26/600 size exclusion chromatography column. The peak at 148.5 ml corresponds to the complex, according to SDS-PAGE analysis. The peak at 198.0 ml corresponds to monomeric DesRREC in excess. B. Monoclinic crystal of the DesKCH188E:DesRREC complex.

      Selected elution peaks from SEC are pooled and concentrated with Vivaspin centrifuge devices at 6,300 x g and 15 °C for 20 min. Final concentrations of 16 mg/ml for the DesKCSTAB:DesRREC complex, and 19 mg/ml for the DesKCH188E:DesRREC complex, are typically achieved. Protein concentration is determined by UV spectrophotometry at 280 nm and calculated extinction coefficients are derived from ProtParam (ExPASy, SIB Bioinformatics Resource Portal: http://www.expasy.org/tools). Proteins are stored in 25, 50 and 100 µl aliquots at -80 °C.
    8. Prior to crystallization, the purity and integrity of samples are checked by SDS-PAGE. Samples in SBβ are heated at 100 °C for 5 min and separated by electrophoresis in 12% polyacrylamide gels ran at 200 V.

  4. Crystallization of DesKC:DesR complexes
    1. DI: The DesKCSTAB:DesRREC (phosphatase) complex
      1. The protein stock solution of pure DesKCSTAB:DesRREC complex is prepared according to a recipe where the order in the addition of the reagents is critical. First MIXA is obtained by combining 7.6 μl 100 mM AMP-PCP, 3.8 μl 1 M Tris pH 8.5 and 44.6 μl buffer SEC-P. Then 95 μl of DesKCSTAB:DesRREC complex (at ~16 mg/ml in buffer SEC-P) is added to the 56 μl of MIXA. The tube is centrifuged at 16,000 x g and 4 °C for 10 min and the supernatant used for further manipulations. In this manner ~150 µl of DesKCSTAB:DesRREC complex stock solution (at ~10 mg/ml final concentration) is obtained, containing approximately 5 mM AMP-PCP, 43.7 mM Tris pH 8.5, 462.2 mM NaCl and 9.25 mM MgCl2.
      2. Microseeding is used to speed up the process of crystallogenesis: 2 μl are drawn from drops containing previously grown crystals, and these are crushed by vigorous pipetting with P2 micropipette tips. This is used as the source of microseeds, adding 50 μl of 30% (v/v) PEG 600, 0.1 M MES pH 6, 0.15 M MgSO4 and 5% (v/v) glycerol. This seed stock solution is diluted 1/400 into an additive solution previously prepared containing 27% (v/v) PEG 600, 0.1 M MES pH 6, 0.15 M MgSO4 and 5% (v/v) glycerol.
      3. Hanging drop crystallizations are done at 20 °C in Linbro plates. The crystallization drops contain 0.8 μl of mother liquor (30% [w/v] PEG 4000, 0.1 M Tris-HCl pH 8.5, 0.2 M Li2SO4), 2 μl of stock protein solution, and 1.2 μl of additive solution with seeds. Three drops are set on a cover slide to seal each reservoir well. Crystals typically appear in 3-4 days, growing to suitable sizes (0.5 µm) in 10 days (Figure 1B).
      4. Crystals are cryo-protected by slowly adding 4 μl of cryo-protection solution: 32% (w/v) PEG 4000, 0.1 M Tris-HCl pH 8, 0.2 M Li2SO4, 20 mM MgCl2, 18 mM BeF3-, 5 mM AMP-PCP and 15% glycerol. BeF3- is prepared by mixing 50 μl of a 0.9 M stock solution of NaF with 9 μl of a 1 M stock solution of BeCl2, rendering a stock solution of 152 mM BeF3-. Be is extremely toxic; all solutions containing it are handled with particular caution and wastes treated appropriately, according to safety rules. Finally, crystals are briefly soaked in 100% cryoprotection solution, fished out of the drops using cryo-loops of approximately the same size as the selected specimen, flash-frozen in liquid nitrogen and stored in cryo-vials under liquid nitrogen for further use.
    2. DII: The DesKCH188E:DesRREC (phosphotransferase) complex
      1. The protein stock solution of pure DesKCH188E:DesRREC complex is prepared according to a recipe where the order in the addition of the reagents is critical. First MIXA is obtained by combining 5.5 μl 100 mM AMP-PCP, 2.75 μl 1 M Tris pH 8.5, 2.2 μl 1 M MgCl2 and 43.35 μl buffer SEC-PT. Then 48 μl of DesKCH188E:DesRREC complex (at ~19 mg/ml in buffer SEC-PT) are added to 8.2 μl DesRREC (at 30 mg/ml in buffer SEC-PT) and the 53.8 μl of MIXA. The tube is centrifuged at 16,000 x g and 4 °C for 10 min and the supernatant used for further manipulations. In this manner ~110 µl of DesKCH188E:DesRREC complex stock solution (at ~8.3 mg/ml final concentration) is obtained, containing approximately 5 mM AMP-PCP, 43.1 mM Tris pH 8-8.5, 271.5 mM NaCl and 20 mM MgCl2.
      2. Hanging drop crystallizations are done at 20 °C in Linbro plates. The crystallization drops contain 2 μl of mother liquor (18% PEG 3350, 0.3 M tri-potassium citrate) and 2 μl of stock protein solution. Two drops are set on a cover slide to seal each reservoir well. Crystals appear typically in 5-6 days, growing to suitable sizes (0.5 µm) in 15 days (Figure 2B).
      3. Crystals are cryo-protected by quick soaking in 20% PEG 3350, 0.3 M tri-potassium citrate, 5 mM AMP-PCP, 25% glycerol, 20 or 150 mM MgCl2, and 0 or 5 mM BeF3-. Crystals are fished out of the drops using cryo-loops of approximately the same size as the selected specimen, flash-frozen in liquid nitrogen, and stored in cryo-vials under liquid nitrogen for further use.

  5. X-ray diffraction data collection.
    1. Single crystal X-ray diffraction experiments are carried out with an in-house copper rotating-anode source (Protein Crystallography Facility, Institut Pasteur Montevideo), or with synchrotron radiation (Soleil, France).
      1. DesKCSTAB in complex with DesRREC (PDB Id 5IUN), crystallizes in the trigonal space group P3121. Crystals are measured in the synchrotron (Beamline Proxima I, Soleil, France), collecting 180 images with 1° oscillation range and 30 sec exposure time per image (Figure 3A). The raw data is deposited in the SBGrid Data Bank (DOI: 10.15785/SBGRID/400).


        Figure 3. X-ray diffraction of DesKCSTAB:DesRREC crystals and resulting electron density map. A. Representative frame showing the X-ray diffraction from a single DesKCSTAB:DesRREC crystal (space group P3121). B. Zoom-in on the mid-sector of the DHp domain of the DesKCSTAB:DesRREC (phosphatase) complex, illustrated in stick representation (all atoms for the kinase, and only the Cα trace for the regulator). Oxygen atoms are colored red, nitrogens in blue, and carbons distinguished according to protein and chain: green and yellow, for the two chains in the DesKCSTAB dimer, and orange and magenta for the two bound DesRREC moieties.

      2. DesKCH188E in complex with DesRREC with high Mg2+ and BeF3- (PDB Id 5IUL), is collected in a rotating anode X-ray generator (Protein Crystallography Facility, Institut Pasteur de Montevideo, Uruguay). 593 images are collected with 0.3° oscillation range and 10 min exposure per image (Figure 4A). The raw data is deposited in the SBGrid Data Bank (DOI: 10.15785/SBGRID/408).


        Figure 4. X-ray diffraction of DesKCH188E:DesRREC crystals and resulting electron density map. A. Representative frame showing the X-ray diffraction from a single DesKCH188E:DesRREC crystal (space group P21). B. Zoom-in on the mid-sector of the DHp domain of the DesKCH188E:DesRREC (phosphotransferase) complex, illustrated in stick representation (all atoms for the kinase, and only the Cα trace for the regulator). Oxygen atoms are colored red, nitrogens in blue, and carbons distinguished according to protein and chain: green and yellow, for the two chains in the DesKCH188E dimer, and magenta for the single asymmetrically bound DesRREC molecule.

        DesKCH188E in complex with DesRREC with high Mg2+ (PDB Id 5IUK), crystallizes in the monoclinic space group P21. Crystals are measured in the synchrotron (Beamline Proxima I, Soleil, France), collecting two different sets of 1,000 frames each. The oscillation range is 0.2° and exposure time is 0.2 sec per image. The raw data is deposited in the SBGrid Data Bank (DOI: 10.15785/SBGRID/401).
      3. DesKCH188E in complex with DesRREC with low Mg2+ (PDB Id 5IUJ), crystallizes in the monoclinic space group P21. Crystals are measured in the synchrotron (Beamline Proxima II, Soleil, France), collecting 110 images with 1° oscillation range and 30 sec exposure per image. The raw data is deposited in the SBGrid Data Bank (DOI: 10.15785/SBGRID/399).

Data analysis

  1. Data sets are processed using the automatic pipeline autoPROC (Vonrhein et al., 2011), which uses XDS (Kabsch, 2010) for indexing/integration, and Pointless/Aimless (Evans, 2006; Evans, 2011) for data reduction and scaling, with the following comments for each case:
    1. DesKCSTAB in complex with DesRREC (PDB Id 5IUN): the best processing statistics are achieved by integrating all images.
    2. DesKCH188E in complex with DesRREC with high Mg2+ (PDB Id 5IUK): the best processing strategy is to integrate and scale the first 800 frames from the first data set, merging it with frames 1-530 and 650-910 from the second set.
    3. DesKCH188E in complex with DesRREC with high Mg2+ and BeF3- (PDB Id 5IUL): all images are eventually integrated, but successful indexing is achieved by selecting frames 10-40 and 200-240 and using the 1,000 strongest reflections.
    4. DesKCH188E in complex with DesRREC with low Mg2+ (PDB Id 5IUJ): the best processing statistics are achieved by integrating all images.

  2. To solve the structure of DesKCSTAB in complex with DesRREC (Figure 3B), molecular replacement is used, as previously reported (Trajtenberg et al., 2016). The search probe is a model of a DesKC:DesRREC complex generated in silico (Trajtenberg et al., 2014) by superposition of a distantly related complex from Termothoga maritima (PDB Id 3DGE) (Casino et al., 2009), and partial truncation of DesKC’s DHp domain, only keeping the invariant region (residues 190-230). It must be highlighted that the in silico modeling strategy produces hundreds of models (Trajtenberg et al., 2014). Molecular replacement is performed using Phaser (McCoy et al., 2007), searching for one copy of the in silico-generated DHp-DesRREC model of the complex, and repeating this automatically with hundreds of different initial candidates as search probes. Eventually only a handful of them are good to be placed in the asymmetric unit, giving clear signals in the rotation and the translation functions calculated with default settings. Starting with the first refinement cycles, the remaining domains and a second hemi-complex are clearly visible in the electron density maps, allowing for manual model building of the whole molecules using Coot (Emsley et al., 2010). The strategy of using in silico-generated DHp-DesRREC models as search probes is critical for molecular replacement to succeed, and the final refined model proves indeed that the selected probes were similar enough to allow for molecular replacement to succeed, providing with solutions within the radius of convergence for refinement procedures (Figure 5). Using instead crystallographic models of individual partners or domains instead is ineffective, likely due to insufficient scattering mass of small search probes and/or conformational differences between isolated vs. complexed proteins.
    The structures corresponding to the phosphotransferase complex are also solved by molecular replacement with Phaser as before, using the DHp-DesRREC model as search probe, and then completing as described above, guided by the electron density maps. Once more, we refer the reader to our previous report for full details of crystallographic structure determination procedures (Trajtenberg et al., 2016).
    1. Structure refinement is performed with Buster-TNT (Bricogne et al., 2009) using standard procedures, including non-crystallographic symmetry (NCS) restraints and Translation/Libration/Screw (TLS) descriptions in each model (documented in detail within the header of each atomic coordinates file available in the PDB).
    2. Structures are validated throughout the refinement and towards the end, using MolProbity tools (Chen et al., 2010).


      Figure 5. Structural superposition of the initial model of DesK:DesR used as Molecular Replacement probe and the final refined structure. Superposition of the Cα traces of the in silico-generated model of DesKDHp:DesRREC (orange:cyan) (Trajtenberg et al., 2014), onto the final refined crystal structure of DesKCSTAB:DesRREC (green:magenta). The entire proteins for the latter are observed in the crystal structure and could thus be manually built (indicated in grey). Of note, among the hundreds of in silico-generated models to be used for molecular replacement procedures, the few that proved useful in solving the structure, are close enough to the final structure as readily seen in this illustration, explaining their utility. Nonetheless, the actual experimental structure displays substantial changes, readily detectable in this view mostly along the DesRREC domains.

Recipes

  1. LB medium
    5 g/L NaCl
    5 g/L yeast extract
    15 g/L tryptone plus
  2. 2x YT culture medium
    5 g/L NaCl
    10 g/L yeast extract
    16 g/L tryptone plus
  3. LB agar plates
    300 ml LB medium
    4.5 g agar
  4. Lysis buffer
    50 mM Tris-HCl pH 8
    500 mM NaCl
    EDTA-free cocktail of protease inhibitors
  5. Immobilized Metal Affinity Chromatography (IMAC) binding and washing buffer
    50 mM Tris-HCl pH 8
    500 mM NaCl
    40 mM imidazole
    10% glycerol
  6. IMAC elution buffer
    50 mM Tris-HCl pH 8
    500 mM NaCl
    500 mM imidazole
    10% glycerol
  7. Dialysis buffer
    50 mM Tris-HCl pH 8
    300 mM NaCl
    0.5 mM dithiothreitol
  8. Size Exclusion Chromatography buffer for the phosphatase complex (SEC-P buffer)
    20 mM Tris-HCl pH 8
    500 mM NaCl
    10 mM MgCl2
  9. Size Exclusion Chromatography buffer for the phosphotransferase complex (SEC-PT buffer)
    20 mM Tris-HCl pH 8
    300 mM NaCl

Acknowledgments

These protocols are adapted from previous work reported by our group (Trajtenberg et al., 2016). We are grateful to the staffs at synchrotron beamlines Proxima I and II, Soleil (France), especially William Shepard; and Daniela Albanesi for providing plasmid pHPKS/Pxyl-desKSTA. This work was supported by grants from Agencia Nacional de Investigación e Innovación (ANII), Uruguay (FCE2009_1_2679;FCE2007_219); Agence Nationale de la Recherche (ANR), France (PCV06_138918); Centro de Biología Estructural del Mercosur (www.cebem-lat.org) and Fondo para la Convergencia Estructural del MERCOSUR (COF 03/11). We are also grateful to the Institut Pasteur International Network for institutional support through the IMiZA International Joint Unit.

References

  1. Albanesi, D., Mansilla, M. C. and de Mendoza, D. (2004). The membrane fluidity sensor DesK of Bacillus subtilis controls the signal decay of its cognate response regulator. J Bacteriol 186(9): 2655-2663.
  2. Albanesi, D., Martin, M., Trajtenberg, F., Mansilla, M. C., Haouz, A., Alzari, P. M., de Mendoza, D. and Buschiazzo, A. (2009). Structural plasticity and catalysis regulation of a thermosensor histidine kinase. Proc Natl Acad Sci U S A 106(38): 16185-16190.
  3. Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart, O. S., Vonrhein, C. and Womack, T. O. (2009). BUSTER version 2.8.0. Global Phasing.
  4. Casino, P., Rubio, V. and Marina, A.(2009). Structural insight into partner specificity and phosphoryl transfer in two-component signal transduction. Cell 139(2): 325-336.
  5. Chen, V. B., Arendall, W. B., 3rd, Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. and Richardson, D. C. (2010). MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66(Pt 1): 12-21.
  6. de Mendoza, D. (2014). Temperature sensing by membranes. Annu Rev Microbiol 68: 101-116.
  7. Emsley, P., Lohkamp, B., Scott, W. G. and Cowtan, K. (2010). Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66(Pt 4): 486-501.
  8. Evans, P. (2006). Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr 62(Pt 1): 72-82.
  9. Evans, P. R. (2011). An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr D Biol Crystallogr 67(Pt 4): 282-292.
  10. Gao, R. and Stock, A. M. (2009). Biological insights from structures of two-component proteins. Annu Rev Microbiol 63: 133-154.
  11. Kabsch, W. (2010). Xds. Acta Crystallogr D Biol Crystallogr 66(Pt 2): 125-132.
  12. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. and Read, R. J. (2007). Phaser crystallographic software. J Appl Crystallogr 40(Pt 4): 658-674.
  13. Saita, E., Abriata, L. A., Tsai, Y. T., Trajtenberg, F., Lemmin, T., Buschiazzo, A., Dal Peraro, M., de Mendoza, D. and Albanesi, D. (2015). A coiled coil switch mediates cold sensing by the thermosensory protein DesK. Mol Microbiol 98(2): 258-271.
  14. Trajtenberg, F., Albanesi, D., Ruetalo, N., Botti, H., Mechaly, A. E., Nieves, M., Aguilar, P. S., Cybulski, L., Larrieux, N., de Mendoza, D. and Buschiazzo, A. (2014). Allosteric activation of bacterial response regulators: the role of the cognate histidine kinase beyond phosphorylation. MBio 5(6): e02105.
  15. Trajtenberg, F., Imelio, J. A., Machado, M. R., Larrieux, N., Marti, M. A., Obal, G., Mechaly, A. E. and Buschiazzo, A. (2016). Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action. Elife 5.
  16. Unger, T., Jacobovitch, Y., Dantes, A., Bernheim, R. and Peleg, Y. (2010). Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. J Struct Biol 172(1): 34-44.
  17. Vonrhein, C., Flensburg, C., Keller, P., Sharff, A., Smart, O., Paciorek, W., Womack, T. and Bricogne, G. (2011). Data processing and analysis with the autoPROC toolbox. Acta Crystallogr D Biol Crystallogr 67(Pt 4): 293-302.

简介

我们已经开发了产生组氨酸激酶DesK及其同源反应调节物DesR的位点特异性变体的方案,有助于捕获蛋白质的不同信号状态。两个合作伙伴在大肠杆菌中的共表达,确保调节剂过量,对于DesK:DesR复合物的可溶性生产和进一步纯化是至关重要的。通过使用分子置换的X射线晶体学解决了捕获在磷酸转移酶和磷酸酶反应步骤中的复合物的3D结构。该解决方案不是微不足道的,我们发现在用作搜索探针的硅片生成的模型中,有助于将大部分复合物放置在不对称单元中。电子密度图就足够清楚了,可以进行人工建模,获得完整的原子模型。这些方法有助于解决细菌信号领域的主要挑战,即获得稳定的激酶:调节复合物,具有不同的构象状态,适用于高分辨率晶体学研究。
【背景】关于细菌信号复合物,特别是双组分系统(TCS)的结构信息仍然很少(Casino et al。,2009; Gao and Stock,2009)。 TCS包含几乎所有细菌中的感觉组氨酸激酶(HK)和响应调节剂(RR)配偶体,它们允许细胞感知环境并通过适应性反应相应地反应。尽管在信号传输中这种切换机制的重要性(Trajtenberg等,2016),结构信息对于采用不同功能状态的TCS复合体甚至更为有限。我们研究了DesK-DesR途径(de Mendoza,2014),一种来自枯草芽孢杆菌的TCS,其参与调节细胞膜组成以适应降低双层流动性的线索,如冷休克。
   我们开发的方案旨在克服主要的技术瓶颈,包括复杂的纯化,结晶和X射线结构测定。这些障碍中的大多数可能来自特征TCS蛋白质的内在灵活性和异质性。为了在定义的信号步骤中捕获DesK:DesR复合体,根据我们实验室的以前发现,回顾一些细节是有用的。已经开发了这些方案与DesKC(一种包含DesK的整个细胞质区域的截短的DesK变体)一起使用,没有跨膜感觉结构域,其具有催化能力以磷酸转移到DesR,并且使磷酸化脱磷酸酯(Albanesi等)等等,2004)。对于响应调节器合作伙伴DesR,我们选择使用截断形式,包括整个受体结构域(REC),适合所有DesK介导的磷酸转移反应(Trajtenberg等,2014),但缺少C-末端DNA结合域,从而最小化潜在的域间灵活性问题。
   为了在信号通路的磷酸转移步骤中捕获DesKC:DesR复合物,我们选择使用磷酸模拟点突变体DesKC-His188Glu。当不与DesR结合时,该变体采用非常类似于野生型DesKC的磷酸化形式的结构构象(Albanesi等人,2009),因此是在转移反应之前模拟磷酸化HK的有吸引力的模板,也是避免有效转移发生。
   另一方面,为了在去磷酸化步骤中捕获DesKC:DesR复合物,以前的工作通过揭示DesK的切换机制,在“活性”(激酶启动/磷酸酶脱落)和“无活性”(磷酸酶启动/激酶脱离特性)激酶状态(Albanesi等人,2009)。简而言之,DesK从其激酶活性到抑制形式的构象转换牵涉到中心二聚化和His-磷酸转移(DHp)结构域内的卷曲螺旋结构的组装,一个盘绕的线圈,否则“激酶是活性的。 DHp是全螺旋结构域,将跨膜传感器与催化ATP结合(CA)结构域连接,因此鉴定的DHp的构象切换在通过远程变构重排的信号传输中起关键作用。随后这种机械性的见解导致构建稳定磷酸酶本构形式的关键位置(Ser150Ile,Ser153Leu和Arg157Ile)上的点突变(Saita et al。,2015)的线圈螺旋超稳定变体(DesaSTA)(Saita等,2015) et al。,2015)。具有跨膜结构域截短(DesKCSTAB)的相应的可溶性构建体确实显示磷酸酶捕获的3D结构(Trajtenberg等,2016)。 DesKCSTAB用于在去磷酸化步骤中捕获DesKC:DesR复合物,如本方案所述。

关键字:信号蛋白, 蛋白磷酸化, 捕获构象重排, 基于结构的诱变, X射线晶体学, 蛋白质工程

材料和试剂

  1. P2,P200和P1000微量吸头,高压灭菌(Gilson,目录号:F161630,F161930和F161670)
  2. 1.5ml Eppendorf管(Eppendorf,目录号:022364111)
  3. 15和50毫升Falcon管(康宁,目录号:352097和352098)
  4. Minisart 0.45μm注射器过滤器(Sartorius,目录号:16555-K)
  5. 96孔清澈V底2ml聚丙烯深孔板(Corning,目录号:3960)
  6. Minisart0.22μm注射器过滤器(Sartorius,目录号:16532-K)
  7. 1升聚丙烯瓶(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:3140-1002)
  8. SnakeSkin透析管3.5K MWCO,35mm干I.D.,35英尺(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88244)
  9. Vivaspin 6 ml 10,000 MWCO离心浓缩装置(Sartorius,目录号VS0601)
  10. Vivaspin 20 ml 10,000 MWCO离心浓缩装置(Sartorius,目录号VS2001)
  11. Linbro 24孔板(MP Biomedicals,目录号:CPL-101)
  12. 冷冻循环(HAMPTON RESEARCH,目录号:HR4-955)
  13. 封面幻灯片
  14. 大肠杆菌BL21(DE3)和TOP10F'菌株储存在-80°C
  15. pACYCDuet-1(Novagen)和pQE80L(QIAGEN)质粒
  16. 烟草蚀刻病毒(TEV)蛋白酶(3mg / ml储备溶液,内部制备)
  17. 用0.22μmExpress Plus过滤器(EMD Millipore,目录号:SCGPT05RE)过滤的超纯水(> 18MΩ)
  18. 乙醇95%(工业乌拉圭药店)
  19. 氯霉素(Sigma-Aldrich,目录号:C0378,17mg / ml储备溶液,储存于-20℃)
  20. 氨苄青霉素(Sigma-Aldrich,目录号:A9518,100mg / ml储备溶液,储存于-20℃)
  21. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M7506)
  22. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Euromedex,目录号:EU0008-B,1M储备溶液,储存于-20℃)
  23. 溶菌酶(Sigma-Aldrich,目录号:L6876,100mg / ml储备溶液)
  24. Triton X-100(Sigma-Aldrich,目录号:T9284)
  25. 氯化锌(ZnCl 2)(Sigma-Aldrich,目录号:229997)
  26. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  27. 丙烯酰胺/双丙烯酰胺30%溶液(Sigma-Aldrich,目录号:A3574)
  28. DNA Ladder GeneRuler 100 bp Plus(Thermo Fisher Scientific,Thermo Scientific TM,目录号:SM0321)
  29. 用于诱变和PCR扩增的寡核苷酸(IDT DNA技术)
  30. Phusion高保真DNA聚合酶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:F530S)
  31. 十二烷基硫酸钠(Sigma-Aldrich,目录号:L5750)
  32. 过硫酸铵(Sigma-Aldrich,目录号:248614)
  33. N,N,N',N' - 四甲基乙二胺(Sigma-Aldrich,目录号:T9281)
  34. 彩色蛋白梯子预染广泛(New England Biolabs,目录号:P7712S)
  35. Precision Plus Protein Standard All Blue(Bio-Rad Laboratories,目录号:1610373)
  36. Brilliant Blue R(Sigma-Aldrich,目录号:B0149)
  37. 乙酸磷酸钾钾(Sigma-Aldrich,目录号:A0262)
  38. 腺苷5'-三磷酸(ATP)二钠盐水合物(Sigma-Aldrich,目录号:A1852)
  39. β,γ-亚甲基腺苷5'-三磷酸(AMP-PCP)二钠盐(Sigma-Aldrich,目录号:M7510)
  40. Trizma碱(Sigma-Aldrich,目录号:T1503)
  41. 氯化钠(NaCl)(Sigma-Aldrich,目录号:31434)
  42. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:13152)
  43. 聚乙二醇(PEG)600(Sigma-Aldrich,目录号:87333)
  44. MES(Sigma-Aldrich,目录号:M8250)
  45. 硫酸镁(MgSO 4)
  46. 甘油(AppliChem,目录号:131339)
  47. 聚乙二醇(PEG)4000(Sigma-Aldrich,目录号:95904)
  48. 硫酸锂(Li 2 SO 4)(Sigma-Aldrich,目录号:203653)
  49. 氯化铍(BeCl 2)(Sigma-Aldrich,目录号:201197)
    注意:本产品已停产。
  50. 氟化钠(NaF)(Sigma-Aldrich,目录号:71519)
  51. 液氮
  52. 聚乙二醇(PEG)3350(Sigma-Aldrich,目录号:202444)
  53. 柠檬酸三钾(Sigma-Aldrich,目录号:P1722)
  54. 酵母提取物(Sigma-Aldrich,目录号:Y1625)
  55. 胰蛋白胨加(Sigma-Aldrich,目录号:61044)
  56. 琼脂(Sigma-Aldrich,目录号:A9799)
  57. 不含EDTA的蛋白酶抑制剂鸡尾酒片(Roche Diagnostics,目录号:11873580001)
  58. 咪唑(Merck,目录号:104716)
  59. 二硫苏糖醇(Soltec Ventrues,目录号:M112)
  60. 盐酸溶液(Sigma-Aldrich,目录号:13-1683)
  61. LB培养基(参见食谱)
  62. 2x YT培养基(参见食谱)
  63. LB琼脂平板(参见食谱)
  64. 裂解缓冲液(见配方)
  65. 固定金属亲和色谱(IMAC)结合和洗涤缓冲液(见配方)
  66. IMAC洗脱缓冲液(参见食谱)
  67. 透析缓冲液(见配方)
  68. 磷酸酶复合物的大小排阻色谱缓冲液(SEC-P缓冲液)
  69. 磷酸转移酶复合物的大小排阻色谱缓冲液(SEC-PT缓冲液)

设备

  1. Pipetman P2单通道移液器(Gilson,目录号:F144801)
  2. Pipetman P20单通道移液器(Gilson,目录号:F123600)
  3. Pipetman P200单通道移液器(Gilson,目录号:F123601)
  4. Pipetman P1000单通道移液器(Gilson,目录号:F123602)
  5. 250ml锥形瓶(Marienfeld-Surperior,目录号:4110207)
  6. 5升锥形瓶(Marienfeld-Surperior,目录号:4110217)
  7. HisTrap固定化金属亲和层析(IMAC)5 ml柱用Zn-NTA树脂(GE Healthcare,目录号:17-5248-01。内部制备)
  8. Superdex S75 26/600(GE Healthcare,目录号:28989334)
  9. 搅拌磁铁(Sigma-Aldrich)
  10. Thermomixer C(Eppendorf,型号:ThermoMixer C,目录号:4053-8223)
  11. 数字超声波超声波S-450细胞破碎机/均质器(艾默生,布兰森超声波,型号:S-450)
  12. Multitron标准孵化器(Infors HT)
  13. Minispin离心机(Eppendorf,型号:MiniSpin ®,目录号:5452000018)
  14. 冷藏5424R离心机(Eppendorf,型号:5424 R,目录号:36-102-3795)
  15. 冷藏式5810R离心机(Eppendorf型号:5810 R,目录号:5811000010)
  16. 冷冻Sorvall Lynx4000离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorvall Lynx4000,目录号:75006580)
  17. Minipuls 3蠕动泵(Gilson,目录号:F117604)
  18. 色谱仪ÄKTA净化器(GE Healthcare,型号:ÄKTAxpress,目录号:18664501)
  19. EPS-301电源(GE Healthcare,型号:EPS 301,目录号:18-1130-01)
  20. Cary 50生物紫外可见分光光度计(Varian,型号:Cary ® 50)
  21. 炼金术士DT(Rigaku,型号:炼金术士DT)
  22. X射线发生器MicroMax-007HF(Rigaku,型号:MicroMax-007 HF)
  23. 多层X射线镜Varimax-HF(Rigaku,型号:VariMax HF)
  24. 图像板区域检测器MAR345 ®(marXperts,型号:mar345)
  25. SZX16显微镜(Olympus,型号:SZX16)
  26. EVOLT E-330数码相机(Olympus,型号:E-330)
  27. LG-PS2光源(奥林巴斯,型号:LG-PS2)
  28. 具有OS Centos 7.0的Linux计算机工作站

软件

  1. 生成的衍射数据集:
    1. 复合物DesKC:磷酸转移酶状态下的DesR-REC,低Mg 2 + :
      http://dx.doi.org/10.15785/SBGRID/399 < / a>
    2. 复合物DesKC:磷酸转移酶状态下的DesR-REC,高Mg 2 + :
      http://dx.doi.org/10.15785/SBGRID/401 < / a>
    3. 复合物DesKC:磷酸转移酶状态下的DesR-REC,高Mg 2+,和BeF 3 - :
      http://dx.doi.org/10.15785/SBGRID/408 < / a>
    4. 复合物DesKC:磷酸酶状态下的DesR-REC:
      http://dx.doi.org/10.15785/SBGRID/400 < / a>
  2. 产生原子坐标模型:
    1. 复合物DesKC:磷酸转移酶状态下的DesR-REC,低Mg 2 + :
      http://www.rcsb.org/ pdb / explore / explore.do?structureId = 5IUJ
    2. 复合物DesKC:磷酸转移酶状态下的DesR-REC,高Mg 2 + :
      http://www.rcsb.org/ pdb / explore / explore.do?structureId = 5IUK
    3. 复合物DesKC:磷酸转移酶状态下的DesR-REC,高Mg 2 + 和BeF 3 - :
      http://www.rcsb.org/ pdb / explore / explore.do?structureId = 5IUL
    4. 复合物DesKC:磷酸酶状态下的DesR-REC:
      http://www.rcsb.org/ pdb / explore / explore.do?structureId = 5IUN
  3. 使用的计算结晶学软件:
    1. autoPROC( https://www.globalphasing.com/autoproc
    2. BUSTER( https://www.globalphasing.com/buster
    3. CCP4( http://www.ccp4.ac.uk
    4. Phaser( http://www.phaser.cimr.cam.ac。 uk
    5. Coot( https://www2.mrc- lmb.cam.ac.uk/personal/pemsley/coot
    6. MolProbity( http://molprobity.biochem.duke.edu
    7. PyMol( https://www.pymol.org

程序

  1. 使用DesKC突变体在其磷酸酶或其磷酸转移酶状态下稳定组氨酸激酶
    1. 编码磷酸化DesKC变体的质粒pACYC-DesKC H188E:DesR&gt;编码DesRC变体和DesR(DesR&lt; REC&gt;)的接收者结构域,均融合到His标签和TEV蛋白酶位点来切割标签,已经可用(Trajtenberg等人,2014)。因此,使用该质粒pACYC-DesKC H188E:DesR&lt; REC&gt;共表达以下两种蛋白质:
      1. DesKCH188E:
        MGSS HHHHHH GIHM ENLYFQG RKERERLEEKLEDANERIAELVKLEERQRIARDL E 电讯 理论MW = 27.3kDa(TEV裂解后24.9)
        TEV裂解后1 mg / ml溶液的理论吸光度(280 nm)= 0.56〜
      2. DesR REC :
        GSGS HHHHHH GSGS ENLYFQG SGSMISIFIAEDQQMLLGALGSLLNLEDDMEVVGKGTTGQDAVDFVKKRQPDVCIM D 电子邮件 理论MW = 17.4kDa(TEV裂解后15.2)
        TEV裂解后1 mg / ml溶液的理论吸光度(280 nm)= 0.40
    2. 在前面列出的序列中,His标签用蓝色字体突出显示,绿色,TEV识别位点(切割的蛋白质从最后的Gly开始,包括它)。下划线为红色,磷酸化位点:DesR >RAR 具有野生型Asp,而DesKC H188E 显示代替Glu替代原生His。
    3. 当在大肠杆菌中表达时,DesKC,DesKC STAB 的磷酸酶稳定化变体是不溶的。通过用DesR RET结合共表达DesKC STAB 来解决这个困难,导致两种蛋白质的优异产率。通过从pHPKS / Pxyl-desK中克隆DesKC STAB ,产生共表达质粒(pACYC-DesKC STAB DesR> REC ) (Saita等人,2015年)转化为pACYC-DesKC H188E:DesR&lt; REC&gt;(Trajtenberg 使用无限制克隆(Unger等人,2010),使用引物STAB_F(5'-CCTGTATTTTCAGGGATCCGGTATTATAAAACTTCGCAAG-3')和STAB_R(5'-GTCAGACACTGTAATCACAACTTCCTTCCAG) 3' )。编码于pACYC-DesKC STAB 的重组蛋白质包括His标签和TEV蛋白酶切割位点。
    4. pACYC-DesKC STAB :DesR&lt; REC&lt; / sub&gt;共同表达以下两种蛋白质:
      1. DesKC STAB :
        MGSS HHHHHH GSGS ENLYFQG SG IK L RKE ERLEEKLEDANERIAELVKLEERQRIARDL > H DTLGQKLSLIGLKSDLARKLIYKDPEQAARELKSVQQTARTSLNEVRKIVSSMKGIRLKDELINIKQILEAADIMFIYEEEKWPENISLLNENILSMCLKEAVTNVVKHSQAKTCRVDIQQLWKEVVITVSDDGTFKGEENSFSKGHGLLGMRERLEFANGSLHIDTENGTKLTMAIPNNSK
        理论MW = 27.8kDa(TEV裂解后25.6)
        TEV切割后1 mg / ml溶液的理论吸光度(280 nm)= 0.55〜
      2. DesR REC :
        GSGS HHHHHH GSGS ENLYFQG SGSMISIFIAEDQQMLLGALGSLLNLEDDMEVVGKGTTGQDAVDFVKKRQPDVCIM D 电子邮件 理论MW = 17.4kDa(TEV裂解后15.2)
        TEV裂解后1 mg / ml溶液的理论吸光度(280 nm)= 0.40 注意:DesKC:DesR蛋白复合物的1mg / ml溶液的理论吸光度(280nm)为约0.5,其对应于两个蛋白质序列。 br />
    5. 在前面列出的序列中,His标签用蓝色字体突出显示,绿色,TEV识别位点(切割的蛋白质从最后的Gly开始,包括它)。在橙色下划线,在DesK的DHp结构域的盘绕线圈基序内的三个稳定取代。以红色表示磷酸化位点。

  2. DesKC:DesR复合物的过表达
    1. 共表达质粒pACYC-DesKC STAB (同样地用于pACYC-DesKC H188E:DesR REC )在42℃下使用热休克转化为25μl的感受态大肠杆菌BL21(DE3)细胞(约10μg/ ml细胞/ ml)2分钟。包含DesRREC序列(Trajtenberg等人,2014)的pQE80L质粒被共转化,以产生化学计量过量的DesRREC细胞。将100μl转化细胞铺在含有100μg/ ml氨苄青霉素和17μg/ ml氯霉素的LB琼脂平板上,并在37℃下生长过夜。
    2. 挑取5至10个单个菌落并接种于3ml LB培养基(用作下一步中的预培养物),100μg/ ml氨苄青霉素和17μg/ ml氯霉素中,并在37℃下进一步生长搅拌16小时(220rpm)。
    3. 将前培养物接种在含有50ml LB培养基,100μg/ ml氨苄青霉素和17μg/ ml氯霉素的250ml锥形瓶中的1/500中,并在37℃下搅拌(220rpm)生长16小时。
    4. 使用三个5L锥形瓶,每个包含1L 2x YT培养基,2mM硫酸镁,100μg/ ml氨苄青霉素,17μg/ ml氯霉素和1/100预培养细菌接种物,发射更大规模的培养物。培养物在37℃下通过搅拌(220rpm)生长直到波长660nm(Abs <660nm)的吸光度达到0.7-1(约3小时)。将培养物快速转移到已经在20℃下预平衡的摇床上,并继续生长持续搅拌30分钟(220rpm)。
    5. 向培养物中加入IPTG(0.5mM终浓度)。在30℃下达到过表达的诱导4小时(在DesKC STAB DesR> REC 的情况下)或在20℃下达到16小时(对于DesKC在两种情况下,搅拌(220rpm)。
      &gt;&lt; H188E&gt;:DesR&lt; REC&gt;
    6. 通过在4升聚丙烯瓶中4℃下以4,600×g离心20分钟收集细胞。
    7. 将颗粒在50ml Falcon管中彻底重悬于裂解缓冲液(参见食谱)(每ml湿重颗粒5ml)。
    8. 将溶菌酶(0.5mg / ml终浓度)和Triton X-100(1%体积/体积终浓度)加入到重悬浮的小丸中,并在室温下温育30分钟,直到由于DNA释放而提取物粘稠。然后将提取物转移至-80℃并储存至少2小时。
    9. 冷冻的提取物在37℃的水浴中解冻。解冻的提取物用1秒(设置为30%振幅)和3秒的休息间隔的脉冲进行超声处理,完成4分钟的总时间。该操作重复3-4次,直至粘度完全降低。超声处理时,Falcon管与冰保持冷藏。这被认为是总提取物,并将40μl储存在具有β-巯基乙醇(SBβ)的电泳样品缓冲液中用于随后的SDS-PAGE分析。
    10. 将总提取物以12,000xg和4℃离心45分钟。该步骤将可溶性物质(上清液级分)与不溶性物质(沉淀物)如包涵体,非裂解细胞,不溶性蛋白质等分离。将上清液通过0.45μm注射器过滤器过滤到新的Falcon管中,并将咪唑加入到40mM终浓度(以避免IMAC期间与柱的非特异性结合)。将40μl上清液储存在SBβ中用于SDS-PAGE分析。以上用作结构研究的可溶性DesKC STAB(或DesKC&lt; H188E&gt;)和DesR&gt; REC&lt; / sub&gt;的来源。

  3. DesKC:DesR复合物的纯化
    1. 在IMAC的第一次纯化步骤中,使用锌作为固定化金属。避免镍排除半胱氨酸氧化,其先前阻碍了由于在DesR样品中的异质性导致的结晶。 IMAC柱连接到ÄKTA净化器,并用结合缓冲液预先平衡(参见食谱)(10个柱体积或CV)。以5ml / min的流速注射样品,靶蛋白通过N末端His标签与柱结合。
    2. 使用15-20V CV结合缓冲液实现IMAC洗涤,直到获得稳定的Abs280基线。流通材料的等分试样储存在SBβ中进行SDS-PAGE分析,以监测柱饱和度。
    3. 用洗脱缓冲液的线性0-100%梯度(参见食谱)(30CV)以5ml / min实现IMAC洗脱。使用波长280nm的吸光度来监测蛋白质洗脱峰。 DesKC STAB ,DesKC&gt; H188E&gt;和DesR&gt; REC 通常在约65mM咪唑洗脱并收集在96-深孔板中。将40μl洗脱的峰存储在SBβ中用于SDS-PAGE分析。
    4. 合并所选蛋白质的洗脱峰,并用TEV蛋白酶(1:40w / w TEV /目标比)孵育。蛋白水解在透析袋中进行一夜。对于200-300体积的透析缓冲液进行透析(参见食谱),并轻轻搅拌。
    5. 将样品从透析袋中回收,并用0.45μm注射器过滤器过滤。使用第二个IMAC锌柱,连接到蠕动泵并用结合缓冲液(10 CV)预平衡。将样品以5ml / min的流速注入柱中。现在仔细收集流通。 TEV蛋白酶本身包括N末端His标签,因此通常排除流通。将洗脱缓冲液的20 CV应用于柱,以洗脱TEV(和潜在的未消化的靶蛋白,如果程序正常工作通常不存在)通过SDS-PAGE监测。
    6. 将第二个IMAC流通样品在Vivaspin离心机中以6,300 x g和15℃超滤20分钟浓缩至终体积为10 ml。将其立即通过0.22μm注射器过滤器过滤,并以1ml / min的流速注入尺寸排阻色谱(SEC)柱Superdex S75 26/600。 SEC列预先用2 CV SEC缓冲液平衡(参见食谱)。
    7. 在DesKC STAB (DesR)REC(磷酸酶)复合物的情况下,用SEC-P缓冲液或SEC-PT缓冲液,SEC以1ml / min的速度洗脱SEC在DesKC&lt; H188E&gt;:DesR&lt; REC&gt;(磷酸转移酶)复合物的情况下。将洗脱的级分收集在96孔深孔板中。用280nm波长吸光度监测蛋白质洗脱(图1A和2A)。将40μl洗脱的峰存储在SBβ中用于SDS-PAGE分析。
      考虑到DesKC是同二聚体,并且DesRREC的一个单体可以与DesKC结合,所以复合物的计算分子量(MW)约为65kDa。如果DesKC:DesR&lt; REC&lt; / sub&gt;比为2:2,则预计MW增加到〜80kDa。根据校准曲线,SEC色谱图显示在DesKC STAB 的情况下为53.5kDa的MW,而DesKC H188E为44.9kDa,子>:DESR <子> REC 。与理论MW数值的差异可能是由于三级和四分体结构特征偏离了球状物种及其流体动力学半径的理想假设。这些SEC峰值确实对应于靶向的DesKC:DesR复合物之后通过质谱分析证实,然后通过3D结构测定确认。对于两种络合物,最终产率接近每L细胞培养物(或每1g湿重颗粒中1-2mg蛋白质)12mg蛋白质。


      图1.尺寸排阻色谱法和DesKC的结晶 STAB :DesR REC 复合物。 A. SuperDex S75第26页中的DesKC :DesR / 600尺寸排阻色谱柱。根据SDS-PAGE分析,140.9ml的峰对应于复合物。在201.5ml处的峰对应于过量的单体DesRREC。 B. DesKC STAB 的双重晶体:DesR 复合体。


      图2.尺寸排阻色谱法和DesKC的结晶 H188E :DesR REC 复合物。A. Superdex S75第26页中的DesKC H188E :DesR 蛋白复合物的洗脱曲线/ 600尺寸排阻色谱柱。根据SDS-PAGE分析,148.5ml的峰对应于复合物。 198.0ml的峰对应于过量的单体DesRREC。。 B. DesKC H188E的单斜晶体:DesR&gt; REC 复合体。

      合并来自SEC的选定的洗脱峰,并用Vivaspin离心机在6,300xg和15℃浓缩20分钟。 DesKC STAB 复合物的最终浓度为16mg / ml,DesKC H188E的最终浓度为19mg / ml:DesR REC 复合体,通常是实现的。蛋白质浓度通过280nm的紫外分光光度法测定,计算的消光系数来源于ProtParam(ExPASy,SIB Bioinformatics资源门户: http://www.expasy.org/tools )。蛋白质以-25℃,50和100μl等分试样储存在-80℃
    8. 在结晶之前,通过SDS-PAGE检查样品的纯度和完整性。将SBβ中的样品在100℃下加热5分钟,并在200V下运行的12%聚丙烯酰胺凝胶中通过电泳分离。

  4. DesKC的结晶:DesR复合物
    1. DI:DesKC STAB :DesR REC (磷酸酶)复合物
      1. 根据添加试剂的顺序是关键的配方制备纯DesKC STAB :DesR&gt; REC 复合物的蛋白质储备溶液。通过组合7.6μl100mM AMP-PCP,3.8μl1M Tris pH 8.5和44.6μl缓冲液SEC-P获得第一混合物A 。然后将95μl的DesKC STAB :DesR&gt; REC 复合物(在缓冲液SEC-P中〜16mg / ml)加入到56μlMIXA /子>。将管以16,000xg和4℃离心10分钟,并将上清液用于进一步操作。以这种方式,获得含有约5mM AMP-PCP的150μlDesKC STAB :DesR&gt; REC 复合储备溶液(〜10mg / ml终浓度) 43.7mM Tris pH 8.5,462.2mM NaCl和9.25mM MgCl 2。
      2. 微步骤用于加速晶体发生过程:从含有先前生长的晶体的液滴中抽取2μl,并通过用P2微量移液管吸头的剧烈移液将其粉碎。将其用作微量来源,加入50μl30%(v / v)PEG 600,0.1M MES pH 6,0.15M MgSO 4和5%(v / v)甘油。将该种子储备溶液稀释至预先制备的含有27%(v / v)PEG 600,0.1M MES pH 6,0.15M MgSO 4和5%(v / v)的添加剂溶液中)甘油。
      3. 悬挂液滴结晶在Linbro平板上在20℃下进行。结晶液含有0.8μl母液(30%[w / v] PEG 4000,0.1M Tris-HCl pH 8.5,0.2M Li 2 SO 4), 2μl蛋白质溶液,1.2μl添加剂与种子溶液。在盖板上设置三滴以密封每个储存器。晶体通常在3-4天出现,在10天内生长至合适的尺寸(0.5μm)(图1B)
      4. 通过缓慢加入4μl低温保护溶液冷冻保护晶体:32%(w / v)PEG 4000,0.1M Tris-HCl pH8,0.2M Li 2 SO 4 20mM MgCl 2,18mM BeF 3,5mM AMP-PCP和15%甘油。通过将50μl0.9M NaF储备溶液与9μl的1M ClCl 2的储备溶液混合来制备BeF 3 / ,提供152mM BeF 3的储备溶液。是非常有毒的;根据安全规定,包含其中的所有解决方案都应特别小心处理,并妥善处理。最后,将晶体短暂浸泡在100%冷冻保护溶液中,使用与所选样品大致相同大小的冷冻循环从液滴中取出,在液氮中冷冻冷冻并在液氮下冷冻储存在冷冻瓶中供进一步使用。
    2. DII:DesKC H188E :DesR (磷酸转移酶)复合物
      1. 根据添加试剂的顺序是关键的配方制备纯DesKC H188E:DesR&gt; REC&lt;&gt;复合物的蛋白质储备溶液。通过组合5.5μl100mM AMP-PCP,2.75μl1M Tris pH8.5,2.2μl1M MgCl 2和43.35μl缓冲液SEC-PT,获得第一混合物A 。然后将48μl的DesKC H188E:DesR&gt; REC 复合物(在缓冲液SEC-PT中〜19mg / ml)加入到8.2μlDesR REC REC中>(在缓冲液SEC-PT中为30mg / ml)和53.8μlMIXA。将管在16,000xg和4℃下离心10分钟,并将上清液用于进一步操作。以这种方式,获得〜110μlDesKC H188E:DesRREC 复合储备溶液(〜8.3mg / ml终浓度),其含有约5mM AMP-PCP, 43.1mM Tris pH8-8.5,271.5mM NaCl和20mM MgCl 2 。
      2. 悬挂液滴结晶在Linbro平板上在20℃下进行。结晶液含有2μl母液(18%PEG 3350,0.3M柠檬酸三钾)和2μl储备蛋白溶液。在盖子滑块上设置两滴以密封每个储存器。晶体通常出现5-6天,在15天内生长至合适的尺寸(0.5μm)(图2B)
      3. 通过在20%PEG 3350,0.3M柠檬酸三柠檬酸钾,5mM AMP-PCP,25%甘油,20或150mM MgCl 2,和0或5mM中快速浸泡,将晶体冷冻保护BEF <子> 3 - 。使用与所选样品大致相同大小的冷冻循环将晶体从液滴中捞出,在液氮中冷冻,并在液氮下储存在冷冻小瓶中供进一步使用。

  5. X射线衍射数据收集。
    1. 使用内部铜旋转阳极源(Protein Crystallography Facility,Institut Pasteur Montevideo)或与同步辐射(Soleil,France)进行单晶X射线衍射实验。
      1. 与DesR (PDB Id 5IUN )复合的DesKC STAB 结晶在三角形空间组P3 <1> 。在同步加速器(Beamline Proxima I,Soleil,France)中测量晶体,收集180个图像,每个图像具有1°的振荡范围和30秒的曝光时间(图3A)。原始数据存入SBGrid数据库(DOI: 10.15785 / SBGRID / 400 )。


        图3:DesKC STAB的X射线衍射:DesR&lt; REC&gt;晶体和所得到的电子密度图。 A.代表性的X射线来自单个DesKC STAB 的衍射:DesR&lt; REC&gt;晶体(空间组P3 21)。 B.放大在DesKC STAB :DesR REC (磷酸酶)复合物的DHp结构域的中间部分,以棒表示(激素的所有原子) ,只有调节器的Cα迹线)。氧原子是红色的,蓝色的氮和根据蛋白质和链的绿色和黄色区分的碳:对于DesKC STAB 二聚体中的两个链,对于两个结合的DesR < sub> REC 部分。

      2. 与具有高Mg 2 + 和BeF 3 的DesR&gt; REC 复合的DesKC H188E 10.15785 / SBGRID / 408 )。


        图4.DeKC H188E的X射线衍射:DesR&gt; REC&gt;晶体和所得电子密度图。 :一种。代表性的框架示出了来自单个DesKC H188E:DesR&lt; REC&gt;晶体(空间群P2&lt; 1&gt; 1)的X射线衍射。 B.放大在DesKC H188E的DHp结构域的中间部分:DesR&lt; REC&gt;(磷酸转移酶)复合物,如棒所示表示(激酶的所有原子,只有调节子的Cα迹线)。氧原子是红色的,蓝色的氮和根据蛋白质和链的绿色和黄色区分的碳:对于DesKC H188E 二聚体中的两条链,并且用于单个不对称结合的DesR REC 分子。

        与具有高Mg 2+,(PDB Id <5IUK )的DesR&gt; REC 的复合物中的DesKC H188E结晶在单斜晶空间组P2 <1> 。在同步加速器(Beamline Proxima I,Soleil,France)中测量晶体,收集两组不同的1000个帧。振荡范围为0.2°,曝光时间为每张图像0.2秒。原始数据存入SBGrid数据库(DOI: 10.15785 / SBGRID / 401 )。
      3. 与具有低Mg 2+ /(PDB Id <5IUJ )的DesR 的复合物中的DesKC H188E结晶在单斜晶空间组P2 <1> 。在同步加速器(Beamline Proxima II,Soleil,France)中测量晶体,收集110幅图像,每幅图像具有1°振荡范围和30秒曝光。原始数据存入SBGrid数据库(DOI: 10.15785 / SBGRID / 399 )。

数据分析

  1. 数据集使用XDS(Kabsch,2010)进行索引/集成的自动管道autoPROC(Vonrhein等人,2011)进行处理,而Pointless / Aimless(Evans,2006; Evans,2011 )用于数据缩减和缩放,并对每种情况提供以下注释:
    1. 与DesR (PDB Id 5IUN )复合的DesKC STAB :通过集成所有图像来实现最佳处理统计。
    2. 与具有高Mg 2+的PDR (PDB Id <5IUK )的复合物中的DesKC H188E:最佳处理策略将第一个数据集中的前800个帧进行集成和缩放,并将其与第二组的帧1-530和650-910合并。
    3. 与具有高Mg 2+和/或SupF 3+的DesR Sub REC复合物中的DesKC H188E >(PDB Id 5IUL ):所有图像最终都被集成,但是通过选择帧10-40和200-240并使用1000个最强反射来实现成功的索引。
    4. 与具有低Mg 2 + (PDB Id 5IUJ )的DesR REC 复合的DesKC H188E:最佳处理统计通过集成所有图像来实现。

  2. 为了解决与DesR&lt; REC&gt;(图3B)复合的DesKC STAB 的结构,如先前报道的那样使用分子置换(Trajtenberg等人。 >,2016)。搜索探针是通过叠加远程的计算机,在计算机(Trajtenberg等人,2014)中生成的DesKC:DesR&gt; REC 复合体的模型。 (PDB Id 3DGE)(Casino et al。,2009),以及DesKC的DHp结构域的部分截短,仅保留不变区(残基190- 230)。必须强调的是,电脑 建模策略可以生成数百种模型(Trajtenberg等人,2014)。使用Phaser(McCoy等人,2007)进行分子替换,在计算机上搜索的一个拷贝生成的DHp-DesR REC 复杂的模型,并自动重复这个数百个不同的初始候选作为搜索探针。最终只有少数几个被放置在非对称单位中,给出了旋转中的清晰信号和使用默认设置计算的翻译功能。从第一个细化周期开始,剩余的域和第二个半配合物在电子密度图中清晰可见,允许使用Coot手动建模整个分子(Emsley等人,2010 )。使用电子邮件生成的DHP-DesR&gt; REC 模型作为搜索探针的策略对于分子置换成功至关重要,最终的精制模型确实证明所选择的探针是类似于允许分子替代成功,在精益化程序的收敛半径内提供解决方案(图5)。使用代替的个体合作伙伴或领域的晶体学模型是无效的,可能是由于小型搜索探针的散射质量不足和/或分离蛋白与复合蛋白质之间的构象差异。
    对应于磷酸转移酶复合物的结构也通过如前所述用Phaser进行分子置换来解决,使用DHp-DesR&gt; REC 模型作为检索探针,然后按电子密度图指导完成。再次,我们将读者参考我们以前的报告,了解晶体学结构测定程序的全部细节(Trajtenberg等人,2016年)。
    1. 使用标准程序(包括非晶体对称性(NCS)约束)和每个模型中的翻译/振动/螺旋(TLS)描述,使用Buster-TNT(Bricogne等人,2009)进行结构改进(在PDB中可用的每个原子坐标文件的标题内详细记录)。
    2. 使用MolProbity工具(Chen等人,2010),整个细化和结束都会对结构进行验证。


      图5.用作分子置换探针和最终精制结构的DesK:DesR初始模型的结构叠加。生成模型中Cα痕迹的叠加的DesK :DesR (橙色:青色)(Trajtenberg等人,2014),到DesKC的最终精制晶体结构< sub> STAB :DesR REC (绿色:品红色)。在晶体结构中观察到后者的整个蛋白质,因此可以手动构建(以灰色表示)。值得注意的是,在用于分子替代程序的数百种生成模型中,证明在解决结构中有用的几个模型足够接近于该图中容易看到的最终结构解释其实用性。尽管如此,实际的实验结构显示出实质性的变化,主要是沿着DesR&lt; REC&lt; / sub&gt;域在该视图中可以容易地检测到。

食谱

  1. LB培养基
    5克/升NaCl
    5克/升酵母提取物
    15g / L胰蛋白胨加上
  2. 2x YT培养基
    5克/升NaCl
    10g / L酵母提取物
    16克/升胰蛋白胨加上
  3. LB琼脂板
    300 ml LB培养基 4.5克琼脂
  4. 裂解缓冲液
    50mM Tris-HCl pH 8
    500 mM NaCl
    不含EDTA的蛋白酶抑制剂混合物
  5. 固定金属亲和层析(IMAC)结合和洗涤缓冲液
    50mM Tris-HCl pH 8
    500 mM NaCl
    40 mM咪唑
    10%甘油
  6. IMAC洗脱缓冲液
    50mM Tris-HCl pH 8
    500 mM NaCl
    500毫克咪唑
    10%甘油
  7. 透析缓冲液
    50mM Tris-HCl pH 8
    300 mM NaCl
    0.5 mM二硫苏糖醇
  8. 磷酸酶复合物的大小排阻色谱缓冲液(SEC-P缓冲液)
    20mM Tris-HCl pH 8
    500 mM NaCl
    10mM MgCl 2
  9. 磷酸转移酶复合物的大小排阻色谱缓冲液(SEC-PT缓冲液)
    20mM Tris-HCl pH 8
    300 mM NaCl
对应于磷酸转移酶复合物的结构也通过如前所述用移相器进行分子置换来解决,使用DHP-DESR&GT;再次,我们将读者参考我们以前的报告,了解晶体学结构测定程序的全部细节(Trajtenberg等人,2016年)。
  1. 使用标准程序(包括非晶体对称性(NCS)约束)和每个模型中的翻译/振动/螺旋(TLS)描述,使用克星-TNT(Bricogne等人,2009)进行结构改进(在PDB中可用的每个原子坐标文件的标题内详细记录)
  2. 使用MolProbity工具(陈等人,2010),整个细化和结束都会对结构进行验证。


    图5.用作分子置换探针和最终精制结构的DesK:DesR初始模型的结构叠加。生成模型中Cα痕迹的叠加的DesK :DesR 橙色:青色)(Trajtenberg等人,2014),到达DesKC的最终精细晶体结构 STAB :DesR REC (绿色:品红色)在晶体结构中观察到后者的整个蛋白质,因此可以手动构建(以灰色表示)。值得注意的是,在用于分子替代程序的数百种生成模型中,证明在解决结构中有用的几个模型足够接近于该图中容易看到的最终结构解释其实用性尽管如此,实际的实验结构显示出实质性的变化,主要是沿着DESR&LT。 REC&LT; / sub&gt;域在该视图中可以容易地检测到。

食谱

  1. LB培养基
    5克/升NaCl
    5克/升酵母提取物
    15g / L胰蛋白胨加上
  2. 2x YT培养基
    5克/升NaCl
    10g / L酵母提取物
    16克/升胰蛋白胨加上
  3. LB琼脂板
    300 ml LB培养基 4.5克琼脂
  4. 裂解缓冲液
    50mM Tris-HCl pH 8
    500 mM NaCl
    不含EDTA的蛋白酶抑制剂混合物
  5. 固定金属亲和层析(IMAC)结合和洗涤缓冲液
    50mM Tris-HCl pH 8
    500 mM NaCl
    40 mM咪唑
    10%甘油
  6. IMAC洗脱缓冲液
    50mM Tris-HCl pH 8
    500 mM NaCl
    500毫克咪唑
    10%甘油
  7. 透析缓冲液
    50mM Tris-HCl pH 8
    300 mM NaCl
    0.5 mM二硫苏糖醇
  8. 磷酸酶复合物的大小排阻色谱缓冲液(SEC-P缓冲液)
    20mM Tris-HCl pH 8
    500 mM NaCl
    10mM MgCl 2
  9. 磷酸转移酶复合物的大小排阻色谱缓冲液(SEC-PT缓冲液)
    20mM Tris-HCl pH 8
    300 mM NaCl
  • de Mendoza,D。(2014)。温度感应由膜。 Annu Rev Microbiol 68:101-116。
  • Emsley,P.,Lohkamp,B.,Scott,WG和Cowtan,K.(2010)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/ pubmed / 20383002“target =”_ blank“> Coot的特征和开发。 Acta Crystallogr D Biol Crystallogr 66(Pt 4):486-501。
  • Evans,P.(2006)。缩放和评估数据质量。 Acta Crystallogr D Biol Crystallogr 62(Pt 1):72-82。
  • Evans,PR(2011)。数据简化简介:空间组确定,缩放和强度统计。 Acta Crystallogr D Biol Crystallogr 67(Pt 4):282-292。
  • Gao,R.and Stock,AM(2009)。&nbsp; 来自双组分蛋白质的结构的生物学见解。 Annu Rev Microbiol 63:133-154。
  • Kabsch,W。(2010)。&nbsp; Xds 。 Acta Crystallogr D Biol Crystallogr 66(Pt 2):125-132。
  • McCoy,A.J.,Grosse-Kunstleve,R.W.,Adams,P.D.,Winn,M.D.,Storoni,L.C。和Read,R.J。(2007)。 Phaser晶体学软件。 应用Crystallogr 40(Pt 4):658-674。
  • Saita,E.,Abriata,LA,Tsai,YT,Trajtenberg,F.,Lemmin,T.,Buschiazzo,A.,Dal Peraro,M.,de Mendoza,D.and Albanesi,D。(2015) 卷线圈开关介导热敏蛋白DesK的感冒。 / a> Mol Microbiol 98(2):258-271。
  • Trajtenberg,F.,Albanesi,D.,Ruetalo,N.,Botti,H.,Mechaly,AE,Nieves,M.,Aguilar,PS,Cybulski,L.,Larrieux,N.,de Mendoza,D.and Buschiazzo ,A.(2014)。变构细菌反应调节剂的活化:超过磷酸化的同源组氨酸激酶的作用。 5(6):e02105。
  • Trajtenberg,F.,Imelio,JA,Machado,MR,Larrieux,N.,Marti,MA,Obal,G.,Mechaly,AE and Buschiazzo,A。(2016)。&lt; a class =“ke-insertfile” href =“http://www.ncbi.nlm.nih.gov/pubmed/27938660”target =“_ blank”>激素三维快照显示的信号方向性调节:调节复合物的作用。 > Elife 5.
  • Unger,T.,Jacobovitch,Y.,Dantes,A.,Bernheim,R。和Peleg,Y。(2010)。&lt; a class =“ke-insertfile”href =“http://www.ncbi。 nlm.nih.gov/pubmed/20600952“target =”_ blank“>用于分子操作和蛋白质表达的限制性免疫(RF)克隆程序的应用结构生物 172(1 ):34-44。
  • Vonrhein,C.,Flensburg,C.,Keller,P.,Sharff,A.,Smart,O.,Paciorek,W.,Womack,T。和Bricogne,G。(2011)。使用autoPROC工具箱进行数据处理和分析。 Acta Crystallogr D Biol Crystallogr 67(Pt 4):293-302。
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    Copyright Imelio et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
    引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
    1. Imelio, J. A., Larrieux, N., Mechaly, A. E., Trajtenberg, F. and Buschiazzo, A. A. (2017). Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography. Bio-protocol 7(16): e2510. DOI: 10.21769/BioProtoc.2510.
    2. Trajtenberg, F., Imelio, J. A., Machado, M. R., Larrieux, N., Marti, M. A., Obal, G., Mechaly, A. E. and Buschiazzo, A. (2016). Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action. Elife 5.
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