- Email: [email protected]
Single-Molecule Tour de Force: Teasing Apart a Signaling Complex
Structure
Previews Single-Molecule Tour de Force: Teasing Apart a Signaling Complex Wouter D. Hoff1 and John L. Spudich2,* 1Department
of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, TX 77030, USA *Correspondence: [email protected] DOI 10.1016/j.str.2008.07.004 2Center
The phototaxis receptor sensory rhodopsin II communicates with its transducer in a membrane-embedded complex. The first application of single-molecule force spectroscopy to receptor-transducer interaction by Cisneros et al. (2008) reveals new structural features in the signaling complex. The Sensory Rhodopsin II-Transducer Complex The photoreactive, 7-transmembranehelix, retinal-containing receptor sensory rhodopsin II (SRII) forms a molecular complex with its transducer subunit HtrII in cytoplasmic membranes of haloarchaeal cells (Hoff et al., 1997). Two SRII subunits flank an HtrII dimer, the latter consisting of a membrane-embedded domain and cytoplasmic domains homologous to those of prokaryotic chemotaxis receptors. Bluelight photoisomerization of retinal in the buried photoactive site of SRII activates a histidinyl-kinase bound to the distal end of HtrII. The resulting phosphorylation of a diffusible response regulator, which binds in its phosphorylated form to the motility apparatus, causes the cell to reverse its swimming direction, resulting in an avoidance of blue-light (repellent phototaxis). The relay of the photosignal from SRII to HtrII has attracted much attention as an especially tractable example of protein-protein communication in membranes, about which very little is known. Progress on this complex from X-ray crystallography, time-resolved optical and vibrational spectroscopy, genetics, biochemistry, and microbial physiology has made SRII-HtrII one of the best understood membrane protein complexes, and has begun to resolve a first example of the chemistry of dynamic communication between membrane proteins at the atomic level (Spudich, 2006). The current challenging questions require new methodology to go beyond the limitations of conventional techniques. Cisneros et al., in this issue of Structure, apply single molecule force spectroscopy (SMFS) to examine structural effects of HtrII binding to SRII.
Single Molecule Force Spectroscopy of Protein Unfolding The last decade has brought an explosion of approaches to study proteins at the single molecule level. These can be divided into single molecule detection (often by fluorescence) and single molecule manipulation. SMFS, a powerful class of molecular manipulation experiments, measures the elongation caused by the unfolding of a single protein molecule upon the application of an external force. SMFS combines structural insights, obtained by mapping of the observed unfolding lengths onto known crystal structures, with energetic information derived from the force measurements. Two key approaches to SMFS are the use of optical tweezers, in which single particles are trapped in the focal point of a laser beam, and atomic force microscopy (AFM), in which a sample is immobilized on a surface and probed with an AFM tip. SMFS studies based on AFM have been largely limited to unfolding of water-soluble proteins, but recently have been expanded to protein function (e.g., activation of a soluble photoreceptor) (Zhao et al., 2006). The Mu¨ller group has been instrumental in developing AFMbased SMFS of membrane proteins (Kedrov et al., 2007), and here extend their work to the effect of bound HtrII on the unfolding of SRII. Force-Induced Unfolding of Single Protein Molecules The preferential attachment of the AFM tip to the solvent-exposed C terminus of SRII allows the detection of the ‘‘unraveling’’ of SRII in a directional fashion. In both bulk and SMFS measurements, small water
soluble proteins often exhibit an all-ornothing pattern of unfolding, in which the fully folded state is directly converted into the fully unfolded state without the involvement of detectable partially unfolded states. In striking contrast, the Mu¨ller group has shown that small a-helical membrane proteins unfold in a stepwise fashion. Each unfolding step is detected as a distinct force peak in the SMFS measurement, typically corresponding to the extraction and subsequent unfolding of one or two a helices from the membrane (Figure 1). This striking difference in the unfolding behavior of water-soluble proteins and transmembrane proteins may have its origin in the relatively large stability of a single transmembrane a helix, which is comparable to the stability of an entire water-soluble protein (Kedrov et al., 2007). Different single molecule force-extension curves for the same protein exhibit significant variation, revealing the involvement of multiple different unfolding pathways. Each of these pathways results in a unique pattern of force peaks, complicating analysis of the data. Cisneros et al. provide a novel protocol to address this issue. They fit each force peak in the forcedistance curve of a single protein molecule as a worm-like chain, a model that has been widely used to describe the unfolding of an entire water-soluble protein. This analysis allows two key parameters to be determined: (1) the probability that a force peak is detected at a certain distance, and (2) the average force for the event when it occurs. By multiplying these two values, Cisneros et al. were able to extract the true interaction strength for each barrier on the unfolding energy landscape.
Structure 16, August 6, 2008 ª2008 Elsevier Ltd All rights reserved 1149
Structure
Previews lices F and G (Sudo and Spudich, 2006; Ito et al., 2008). This steric trigger is essential for propagation of the photosignal to HtrII, and engineering the F-G pin into bacteriorhodopsin introduces the steric trigger (Sudo et al., 2007) and converts the proton pump to a signaling form that is able to activate HtrII (Sudo and Spudich, 2006). The region of the SRII-HtrII interface for signal relay has been localized to the membrane-embedded portion of the complex, which contains most of helices F and G (Sasaki et al., 2007). Strengthening of helix F at its extracellular end by HtrII and rigidification of helix G as observed by Cisneros et al. may control helix F movement with respect to helix G, with the pin—altered by isomerization—serving to guide the movement to the signal-receiving site on HtrII in the membrane. Several studies have implicated helix F movement, which is known to occur in bacteriorhodopsin, as likely to occur during signal relay from SRII to HtrII (Spudich, 2006). Cisneros and coworkers’ outstanding contribution have opened the way to future studies of this signal relay process by SMFS. REFERENCES
Figure 1. Probing Interactions Between Sensory Rhodopsin II and Its Signal Transducer HtrII Using Single-Molecule Force Spectroscopy The seven transmembrane helices of SRII and the two transmembrane helices of HtrII are indicated schematically. The peaks in the single-molecule force-distance curve, which is an idealized cartoon based on data from Cisneros et al. (2008), and their corresponding secondary structure elements of SRII are matched in color. The two force peaks that significantly increase upon the binding of HtrII are indicated by arrows. The interhelical hydrogen-bond pin that functions as a steric trigger during photoactivation of the complex (see text) extends between SRII helices F and G near the midmembrane plane.
This approach should be generally applicable to SMFS unfolding data on macromolecules that exhibit multiple different parallel unfolding pathways. Single-Molecule Detection of Interactions Between a Receptor and Its Transducer By performing SMFS on both SRII alone and SRII in complex with its transducer HtrII, Cisneros et al. obtained information on the location and strength of the interactions between these two proteins. Their study reveals that the binding of HtrII significantly stabilizes two existing unfolding units in SRII: its entire helix G and the extracellular end of helix F (Figure 1). This is the first example of the detection of interactions between a receptor and its transducer at the single molecule level. The
results illustrate the power of SMFS to provide both structural and energetic information; for each of the two interaction sites, the strength of the interaction is quantified. The strengthening of helix G is four times greater than that of helix F. The crystal structures of free SRII (Luecke et al., 2001) and HtrII-bound SRII (Gordeliy et al., 2002) are indistinguishable in these regions, indicating that small conformational changes can cause significant and functionally important energetic effects. The two regions in SRII that are stabilized upon HtrII binding are notable in the light of recent findings on the mechanism of SRII signal relay. SRII photoactivation entails a steric conflict of the isomerizing retinal with a pair of residues, Tyr174 and Thr204, which in the dark form a hydrogen-bond ‘‘pin’’ between he-
1150 Structure 16, August 6, 2008 ª2008 Elsevier Ltd All rights reserved
Cisneros, D.A., Oberbarnscheidt, L., Pannier, A., Klare, J.P., Helenius, J., Engelhard, M., Oesterhelt, F., and Muller, D.J. (2008). Structure 16, this issue, 1206–1213. Gordeliy, V.I., Labahn, J., Moukhametzianov, R., Efremov, R., Granzin, J., Schlesinger, R., Buldt, G., Savopol, T., Scheidig, A.J., Klare, J.P., and Engelhard, M. (2002). Nature 419, 484–487. Hoff, W.D., Jung, K.-H., and Spudich, J.L. (1997). Annu. Rev. Biophys. Biomol. Struct. 26, 223–258. Ito, M., Sudo, Y., Furutani, Y., Okitsu, T., Wada, A., Homma, M., Spudich, J.L., and Kandori, H. (2008). Biochemistry 47, 6208–6215. Kedrov, A., Janovjak, H., Sapra, K.T., and Mu¨ller, D.J. (2007). Annu. Rev. Biophys. Biomol. Struct. 36, 233–260. Luecke, H., Schobert, B., Lanyi, J.K., Spudich, E.N., and Spudich, J.L. (2001). Science 293, 1499–1503. Sasaki, J., Nara, T., Spudich, E.N., and Spudich, J.L. (2007). Mol. Microbiol. 66, 1321–1330. Spudich, J.L. (2006). Trends Microbiol. 14, 480–487. Sudo, Y., and Spudich, J.L. (2006). Proc. Natl. Acad. Sci. USA 103, 16129–16134. Sudo, Y., Furutani, Y., Spudich, J.L., and Kandori, H. (2007). J. Biol. Chem. 282, 15550–15558. Zhao, J.M., Lee, H., Nome, R.A., Majid, S., Scherer, N.F., and Hoff, W.D. (2006). Proc. Natl. Acad. Sci. USA 103, 11561–11566.
Previews Single-Molecule Tour de Force: Teasing Apart a Signaling Complex Wouter D. Hoff1 and John L. Spudich2,* 1Department
of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, TX 77030, USA *Correspondence: [email protected] DOI 10.1016/j.str.2008.07.004 2Center
The phototaxis receptor sensory rhodopsin II communicates with its transducer in a membrane-embedded complex. The first application of single-molecule force spectroscopy to receptor-transducer interaction by Cisneros et al. (2008) reveals new structural features in the signaling complex. The Sensory Rhodopsin II-Transducer Complex The photoreactive, 7-transmembranehelix, retinal-containing receptor sensory rhodopsin II (SRII) forms a molecular complex with its transducer subunit HtrII in cytoplasmic membranes of haloarchaeal cells (Hoff et al., 1997). Two SRII subunits flank an HtrII dimer, the latter consisting of a membrane-embedded domain and cytoplasmic domains homologous to those of prokaryotic chemotaxis receptors. Bluelight photoisomerization of retinal in the buried photoactive site of SRII activates a histidinyl-kinase bound to the distal end of HtrII. The resulting phosphorylation of a diffusible response regulator, which binds in its phosphorylated form to the motility apparatus, causes the cell to reverse its swimming direction, resulting in an avoidance of blue-light (repellent phototaxis). The relay of the photosignal from SRII to HtrII has attracted much attention as an especially tractable example of protein-protein communication in membranes, about which very little is known. Progress on this complex from X-ray crystallography, time-resolved optical and vibrational spectroscopy, genetics, biochemistry, and microbial physiology has made SRII-HtrII one of the best understood membrane protein complexes, and has begun to resolve a first example of the chemistry of dynamic communication between membrane proteins at the atomic level (Spudich, 2006). The current challenging questions require new methodology to go beyond the limitations of conventional techniques. Cisneros et al., in this issue of Structure, apply single molecule force spectroscopy (SMFS) to examine structural effects of HtrII binding to SRII.
Single Molecule Force Spectroscopy of Protein Unfolding The last decade has brought an explosion of approaches to study proteins at the single molecule level. These can be divided into single molecule detection (often by fluorescence) and single molecule manipulation. SMFS, a powerful class of molecular manipulation experiments, measures the elongation caused by the unfolding of a single protein molecule upon the application of an external force. SMFS combines structural insights, obtained by mapping of the observed unfolding lengths onto known crystal structures, with energetic information derived from the force measurements. Two key approaches to SMFS are the use of optical tweezers, in which single particles are trapped in the focal point of a laser beam, and atomic force microscopy (AFM), in which a sample is immobilized on a surface and probed with an AFM tip. SMFS studies based on AFM have been largely limited to unfolding of water-soluble proteins, but recently have been expanded to protein function (e.g., activation of a soluble photoreceptor) (Zhao et al., 2006). The Mu¨ller group has been instrumental in developing AFMbased SMFS of membrane proteins (Kedrov et al., 2007), and here extend their work to the effect of bound HtrII on the unfolding of SRII. Force-Induced Unfolding of Single Protein Molecules The preferential attachment of the AFM tip to the solvent-exposed C terminus of SRII allows the detection of the ‘‘unraveling’’ of SRII in a directional fashion. In both bulk and SMFS measurements, small water
soluble proteins often exhibit an all-ornothing pattern of unfolding, in which the fully folded state is directly converted into the fully unfolded state without the involvement of detectable partially unfolded states. In striking contrast, the Mu¨ller group has shown that small a-helical membrane proteins unfold in a stepwise fashion. Each unfolding step is detected as a distinct force peak in the SMFS measurement, typically corresponding to the extraction and subsequent unfolding of one or two a helices from the membrane (Figure 1). This striking difference in the unfolding behavior of water-soluble proteins and transmembrane proteins may have its origin in the relatively large stability of a single transmembrane a helix, which is comparable to the stability of an entire water-soluble protein (Kedrov et al., 2007). Different single molecule force-extension curves for the same protein exhibit significant variation, revealing the involvement of multiple different unfolding pathways. Each of these pathways results in a unique pattern of force peaks, complicating analysis of the data. Cisneros et al. provide a novel protocol to address this issue. They fit each force peak in the forcedistance curve of a single protein molecule as a worm-like chain, a model that has been widely used to describe the unfolding of an entire water-soluble protein. This analysis allows two key parameters to be determined: (1) the probability that a force peak is detected at a certain distance, and (2) the average force for the event when it occurs. By multiplying these two values, Cisneros et al. were able to extract the true interaction strength for each barrier on the unfolding energy landscape.
Structure 16, August 6, 2008 ª2008 Elsevier Ltd All rights reserved 1149
Structure
Previews lices F and G (Sudo and Spudich, 2006; Ito et al., 2008). This steric trigger is essential for propagation of the photosignal to HtrII, and engineering the F-G pin into bacteriorhodopsin introduces the steric trigger (Sudo et al., 2007) and converts the proton pump to a signaling form that is able to activate HtrII (Sudo and Spudich, 2006). The region of the SRII-HtrII interface for signal relay has been localized to the membrane-embedded portion of the complex, which contains most of helices F and G (Sasaki et al., 2007). Strengthening of helix F at its extracellular end by HtrII and rigidification of helix G as observed by Cisneros et al. may control helix F movement with respect to helix G, with the pin—altered by isomerization—serving to guide the movement to the signal-receiving site on HtrII in the membrane. Several studies have implicated helix F movement, which is known to occur in bacteriorhodopsin, as likely to occur during signal relay from SRII to HtrII (Spudich, 2006). Cisneros and coworkers’ outstanding contribution have opened the way to future studies of this signal relay process by SMFS. REFERENCES
Figure 1. Probing Interactions Between Sensory Rhodopsin II and Its Signal Transducer HtrII Using Single-Molecule Force Spectroscopy The seven transmembrane helices of SRII and the two transmembrane helices of HtrII are indicated schematically. The peaks in the single-molecule force-distance curve, which is an idealized cartoon based on data from Cisneros et al. (2008), and their corresponding secondary structure elements of SRII are matched in color. The two force peaks that significantly increase upon the binding of HtrII are indicated by arrows. The interhelical hydrogen-bond pin that functions as a steric trigger during photoactivation of the complex (see text) extends between SRII helices F and G near the midmembrane plane.
This approach should be generally applicable to SMFS unfolding data on macromolecules that exhibit multiple different parallel unfolding pathways. Single-Molecule Detection of Interactions Between a Receptor and Its Transducer By performing SMFS on both SRII alone and SRII in complex with its transducer HtrII, Cisneros et al. obtained information on the location and strength of the interactions between these two proteins. Their study reveals that the binding of HtrII significantly stabilizes two existing unfolding units in SRII: its entire helix G and the extracellular end of helix F (Figure 1). This is the first example of the detection of interactions between a receptor and its transducer at the single molecule level. The
results illustrate the power of SMFS to provide both structural and energetic information; for each of the two interaction sites, the strength of the interaction is quantified. The strengthening of helix G is four times greater than that of helix F. The crystal structures of free SRII (Luecke et al., 2001) and HtrII-bound SRII (Gordeliy et al., 2002) are indistinguishable in these regions, indicating that small conformational changes can cause significant and functionally important energetic effects. The two regions in SRII that are stabilized upon HtrII binding are notable in the light of recent findings on the mechanism of SRII signal relay. SRII photoactivation entails a steric conflict of the isomerizing retinal with a pair of residues, Tyr174 and Thr204, which in the dark form a hydrogen-bond ‘‘pin’’ between he-
1150 Structure 16, August 6, 2008 ª2008 Elsevier Ltd All rights reserved
Cisneros, D.A., Oberbarnscheidt, L., Pannier, A., Klare, J.P., Helenius, J., Engelhard, M., Oesterhelt, F., and Muller, D.J. (2008). Structure 16, this issue, 1206–1213. Gordeliy, V.I., Labahn, J., Moukhametzianov, R., Efremov, R., Granzin, J., Schlesinger, R., Buldt, G., Savopol, T., Scheidig, A.J., Klare, J.P., and Engelhard, M. (2002). Nature 419, 484–487. Hoff, W.D., Jung, K.-H., and Spudich, J.L. (1997). Annu. Rev. Biophys. Biomol. Struct. 26, 223–258. Ito, M., Sudo, Y., Furutani, Y., Okitsu, T., Wada, A., Homma, M., Spudich, J.L., and Kandori, H. (2008). Biochemistry 47, 6208–6215. Kedrov, A., Janovjak, H., Sapra, K.T., and Mu¨ller, D.J. (2007). Annu. Rev. Biophys. Biomol. Struct. 36, 233–260. Luecke, H., Schobert, B., Lanyi, J.K., Spudich, E.N., and Spudich, J.L. (2001). Science 293, 1499–1503. Sasaki, J., Nara, T., Spudich, E.N., and Spudich, J.L. (2007). Mol. Microbiol. 66, 1321–1330. Spudich, J.L. (2006). Trends Microbiol. 14, 480–487. Sudo, Y., and Spudich, J.L. (2006). Proc. Natl. Acad. Sci. USA 103, 16129–16134. Sudo, Y., Furutani, Y., Spudich, J.L., and Kandori, H. (2007). J. Biol. Chem. 282, 15550–15558. Zhao, J.M., Lee, H., Nome, R.A., Majid, S., Scherer, N.F., and Hoff, W.D. (2006). Proc. Natl. Acad. Sci. USA 103, 11561–11566.