Article pubs.acs.org/biochemistry
Membrane Association of a Protein Increases the Rate, Extent, and Specificity of Chemical Cross-Linking Aruni P. K. K. Karunanayake Mudiyanselage,† Meili Yang,† Lee A.-R. Accomando,† Lynmarie K. Thompson,*,†,‡ and Robert M. Weis†,‡ †
Department of Chemistry and ‡Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003, United States S Supporting Information *
ABSTRACT: Many cellular processes involve interactions between membrane-associated proteins, and those interactions are enhanced by membrane association. We have used cross-linking reactions to compare the extent and specificity of protein interactions in solution versus on a membrane surface. Cysteine mutants of a soluble cytoplasmic fragment (CF) of the aspartate receptor, a transmembrane receptor involved in bacterial chemotaxis, are used in disulfide bond formation with the thiol-specific oxidant diamide and chemical crosslinking reactions with the trifunctional maleimide TMEA. CF binding to membranes is mediated by its N-terminal His tag binding to vesicles containing a nickel-chelating lipid, so cross-linking reactions conducted in the presence and absence of vesicles differ only in whether CF is bound to the vesicles or is free in solution. For multiple Cys throughout the CF, membrane association is shown to increase the rate and extent of these reactions. Cross-linking specificity, which is measured as the preference for cross-linking between Cys near each other in the native structure, is also enhanced by membrane association. These results provide an experimental demonstration that membrane binding enhances protein−protein interactions, an important consideration for understanding processes involving membraneassociated proteins. The experiments further demonstrate the importance of cross-linking conditions for these reactions that are often used to probe protein structure and dynamics and the potential of membrane association to restore native interactions of membrane-associated proteins for cross-linking studies.
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(CF), at sites with different exposure in the structure (buried at the intramonomer or intermonomer interface, or solvent exposed) and spread throughout the 3 subdomains along the length of the CF. Each mutant CF was subjected to both disulfide formation with the thiol-specific oxidant diamide to produce receptor dimers and to cross-linking with the trifunctional maleimide TMEA to produce receptor dimers and trimers. Reactions were conducted in the presence and absence of extruded vesicles containing the nickel-chelating lipid DGS-NTA, which binds the N-terminal His tag of the CF construct.4 Comparison of CF cross-linking in solution and on vesicles was used to show that (i) vesicle association of the CF increases the rate and extent of cross-linking and that (ii) crosslinking reactivity differences among sites reflect differences in dynamics along the length of the CF. Furthermore, diamide cross-linking reactions between different Cys mutants were used to determine the preference for the formation of homodimer cross-links (between monomers with the same Cys residue) vs heterodimer cross-links (between monomers with two different Cys residues), to test whether membrane association of the CF increases the specificity of cross-linking.
umerous processes in the cell take place on the surface of membranes, where the proteins involved often display a reversible interaction with the membrane. Membrane association is expected to significantly enhance protein interactions1 and is a postulated means to generate quaternary interactions.2 In addition, membrane association is expected to increase the specificity of protein interactions, which could lower the rates of some reactions among the membrane-associated species relative to the rate for proteins in solution. We used the vesicle-mediated assembly method developed previously in our lab3−5 to compare the extent and specificity of protein associations in solution versus on a membrane surface. Through the introduction of a unique cysteine, the reactivity of the cysteine in disulfide bond and chemical cross-linking reactions were used to make these comparisons. The approach has been applied to the cytoplasmic domains from the Escherichia coli aspartate and serine receptors, representative members of the large superfamily of chemotaxis receptors.6,7 The structure of the cytoplasmic domain is the defining feature of this receptor superfamily. It is organized as a coiled-coil hairpin (18 heptad repeats in length in the E. coli proteins), which dimerizes to form a four-helix bundle shown in Figure 1.8−10 Cysteine residues were introduced individually at 10 positions throughout the receptor cytoplasmic fragment © 2013 American Chemical Society
Received: June 7, 2013 Revised: July 19, 2013 Published: July 23, 2013 6127
dx.doi.org/10.1021/bi4007176 | Biochemistry 2013, 52, 6127−6136
Biochemistry
Article
of 20 μL of gel loading buffer (without β-mercaptoethanol), samples were boiled for 3 min. Permanents were prepared, and plasmids were isolated for the colonies that showed the expected disulfide dimer or truncation by SDS−PAGE . The presence of the mutation in these plasmids was verified by sequencing (GENEWIZ, www.genewiz.com, South Plainfield, New Jersey). Sequence results were analyzed by alignment in the CLUSTALW program (http://www.ebi.ac.uk/Tools/msa/ clustalw2/). Protein Purification. Mutated or truncated CF purification was conducted according to published protocols using nickel affinity chromatography (GE Healthcare).4 CheA, CheW, and CheY were purified as previously described.5,17−19 Protein concentration was determined using a modified Lowry assay kit (Thermo Scientific). Activity Assays. To initiate complex assembly, 580 μM lipid (extruded LUVs of 1:1 DOPC/DGS-NTA-Ni2+) was added to a mixture of 30 μM CF, 10 μM CheW, and 1.2 μM CheA in kinase buffer. Samples were incubated at 25 °C in a water bath for 4 to 5 h prior to activity measurements. Steadystate kinase activities were measured as described previously,3,5 using a coupled ATPase assay in which the rate of ATP consumption was measured spectrophotometrically as the rate of NADH oxidation. For each assay, the assembled complex was diluted 100-fold into kinase buffer containing 55 μM CheY, 2.2 mM phosphoenolpyruvate, 4 mM ATP, 250 μM NADH, and 20 units of PK/LDH enzyme (Sigma-Aldrich, St. Louis, MO) and immediately placed in the spectrophotometer. The background activity of CheY under equivalent conditions without receptor complexes was subtracted prior to the determination of specific activity. Specific activities were calculated from the linear change in absorbance at 340 nm (d[ATP]/dt = −6220[dA340/dt]) over a 1 min time period and normalized to the total CheA concentration. The bound fraction of each protein was determined by sedimentation at 60,000 rpm at 25 °C for 30 min (Beckman, OptimaTLX Ultracentrifuge) to separate bound (pellet) from unbound (supernatant) protein. The bound CheA, CheW, and CF fractions were calculated by analyzing the integrated intensities of Gel-code Blue (Pierce Chem. Co.) stained protein bands from aliquots of total protein (prior to centrifugation) and unbound protein (supernatant) fractions using ImageJ software. Diamide and TMEA Induced Cross-Linking. Tris(2maleimidoethyl)amine (TMEA) was obtained from Thermo Scientific; 1,1′-azobis(N,N-dimethylformamide) (diamide) and N-ethylmaleimide (NEM) were obtained from Sigma. Tris(2carboxyethyl)phosphine (TCEP) was obtained from Thermo Scientific. Purified CF (300 μM) was treated with 10 mM TCEP at 25 °C for 30 min to reduce disulfide-linked dimers that accumulated during purification and storage. TCEP was removed with 7K Zeba Spin desalting columns (Thermo Scientific), using a Beckman Coulter Allegra 6R benchtop centrifuge. The spin column was pre-equilibrated with buffer by centrifuging at 2000 rpm for 2 min at 4 °C three times. The sample was applied to the pre-equilibrated column and centrifuged 3 min at 2000 rpm and 4 °C for TCEP removal. In addition, the reduced CF was subjected to 3 buffer exchanges in 10 kDa MWCO filters (Millipore), by centrifugation at 2000 rpm for 20−30 min at 4 °C using a Beckman Coulter Allegra 6R benchtop centrifuge. Reduction was confirmed by prequenching an aliquot with 10 mM NEM followed by SDS−PAGE analysis on a 10% acrylamide gel.
Suppression of heterodimer formation was observed for CF cross-linking for Cys sites distant from each other in the structure. The results also demonstrated the importance of the choice of cross-linking conditions: stoichiometric cross-linking agent concentrations were important for revealing reactivity differences, and excess cross-linking agent concentrations were important for revealing the partitioning between homo- and heterodimers.
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MATERIALS AND METHODS Vesicle Preparation (LUV). DOPC (1, 2-dioleoyl-snglycero-3-phosphocholine) and the nickel-chelating lipid, DGS-NTA-Ni2+ (1, 2-dioleoyl-sn-glycero-3-[(N-(5-amino-1carboxypentyl) iminodiacetic acid) succinyl), were obtained from Avanti Polar Lipids (Alabaster, Alabama). A suspension of multilamellar vesicles (MLVs) was prepared by hydration of a 1:1 DOPC/DGS-NTA-Ni2+ lipid film in kinase buffer (75 mM Tris and 100 mM KCl, pH 7.5) at room temperature, to give a total lipid concentration of 2 mg/mL. When vesicles were prepared for kinase assays, 5 mM MgCl2 and 5% DMSO were included in the buffer; for assays under reducing conditions, 2 mM TCEP was also included. Large unilamellar vesicles (LUVs) were prepared by extrusion in a small−volume commercial apparatus (Avanti Mini Extruder). The MLV suspension was passed 15 times through a Nucleopore TrackEtch polycarbonate membrane with a 100 nm pore diameter. Construction of Cys Mutants and Truncated Mutants of the Cytoplamic Domain. The histidine-tagged aspartate and serine receptor cytoplasmic domain (TarCF and TsrCF), encoding Glu at the four main sites of methylation (4E), were expressed in the E. coli strain DH5αF’ using plasmids pHTCF11 and pHTCF(tsr).12 Cysteine and stop codon mutations were introduced by site-directed mutagenesis, and reagents were purchased from Agilent Technologies, CA (previously Stratagene) and New England Biolabs, MA. Truncation of the Cterminus of the CF removed ∼3.7 kDa from Tsr and ∼3.3 kDa from Tar; the resulting plasmids expressed Tsr 258−518 and Tar 257−525. Primers were obtained from Integrated DNA Technologies (San Diego, CA). The PCR reaction was carried out in a thermocycler (Mastercycler personal), and Pf u, dNTPs, and DpnI were obtained from Stratagene (Cedar Creek, TX) and New England Bio-Laboratories for some reactions. The PCR products were treated with DpnI at 37 °C for 1 h to digest the template plasmid DNA. Agarose gels were run to verify that the PCR reaction produced a product of the expected length. The PCR product was transformed into DH5αF′ chemically competent cells, and transformants were selected with LBampicillin plates (0.15 mg ampicillin/mL). Colonies were randomly selected and grown at 37 °C with and without IPTG induction for expression analysis. For CF truncation, cultures were screened by SDS−PAGE to determine if a truncated CF (migrating faster than wild type CF) was present. For Cys mutagenesis, cultures were subjected to cross-linking conditions to screen for the presence of the reactive cysteine. Two milliliter cultures were grown at 37 °C to an OD600 of 0.4 to 0.5. These cultures were then induced with IPTG (1 mM) and grown for an additional 1 to 2 h. The cells were harvested by centrifugation at 13,000 rpm for 15 min in a benchtop microcentrifuge (Eppendorf), and the cell pellet was resuspended in 40 μL of water. For each sample, 20 μL was treated with 0.15 mM Cu(II)phenanthroline diluted from 150 mM stock, and 20 μL was not treated.13−16 After the addition 6128
dx.doi.org/10.1021/bi4007176 | Biochemistry 2013, 52, 6127−6136
Biochemistry
Article
Aliquots of the reduced protein were flash frozen in liquid nitrogen and stored at −80 °C. For the TMEA cross-linking reactions, aliquots were prepared in kinase buffer from the TCEP-reduced CF stock solutions to give a final concentration of 30 μM CF in the experimental samples. In the vesicle-assembled CF samples, LUVs were added to give 580 μM total lipid concentration (1:1 DOPC/DGS-NTA-Ni2+) and incubated for 5 min at room temperature. An equivalent volume of buffer was added to CF samples to measure cross-linking in the absence of vesiclemediated assembly. TMEA stock solutions were prepared fresh in 90−100% DMSO. Each reaction was initiated by the addition of the appropriate volume of the TMEA stock solution (DMSO was
