Proteins are made up of chains of amino acids that need to fold into intricate three-dimensional shapes to work correctly. But some proteins also have to change their shape drastically when they work. Mechanical forces that change the shape of a protein can therefore be used to determine how a protein folds and how it changes its structure when working.

Although researchers have developed techniques to analyze the effect of force on single proteins, most studies carried out so far have investigated the effect of stretching (or tensile forces) to understand structural changes that naturally involve an extension within the protein. However, many proteins undergo structural changes that involve a compaction in their shape. How these changes occur remains poorly understood because, for these, methods to apply compressive forces to single proteins are required.

Perfringolysin O (PFO for short) is a protein that is made by a bacterium that causes food poisoning in humans. PFO makes pores in the membrane that surrounds cells. This causes the cell’s contents to leak out, killing the cell. When inserting into the membrane, PFO changes from an elongated “prepore” state to a compact pore-forming state.

Czajkowsky et al. now use a combination of single molecule techniques and computer simulations to investigate how PFO undergoes this compaction. Previous work had identified a mutant PFO protein that arrests at the prepore state. Applying a compressive force to the top of this prepore-trapped PFO as it sits on the membrane transmitted forces across the entire PFO protein. This ultimately produced a compressive force across a distant part of the protein that caused the protein to change from the elongated prepore state to the compact, pore-like shape. If a compressive force was not applied, the PFO protein remained in the prepore state. Czajkowsky et al. further found that this compressive force is naturally produced by distant water-repellent parts of the naturally occurring protein interacting with the cell membrane. Therefore, internal forces can transmit across proteins to drive shape changes in distant regions.

In the future, the methods developed in this study could be applied to analyze other naturally occurring changes in proteins where shape compaction happens when working.

The initial demonstration that single macromolecules could be mechanically stretched with exact knowledge of the associated forces (Smith et al., 1992; Kellermayer et al., 1997; Rief et al., 1997; Tskhovrebova et al., 1997) has enabled unprecedented access to measurements of the physical and chemical inter-molecular interactions (Bippes and Muller, 2011; Bustamante et al., 2000; Fisher et al., 2000; Hu and Li, 2014; Zhuang and Rief, 2003). Moreover, pulling on single molecules with tensile forces has enabled detailed probing of functional and structural transitions within proteins, in particular those that involve a separation of specific structural elements (Puchner and Gaub, 2012; Zhang et al., 2009; del Rio et al., 2009). Yet, tensile forces cannot be used to probe transitions that involve a decrease in the distance between structural elements such as the closing of a substrate access gate, for example, as the force acts in a direction opposite to the structural movement. In these instances, what is needed is the controlled application of a compressive force at the single molecule level.

Perfringolysin O (PFO) is an intriguing pore-forming toxin that undergoes a significant reduction in the distance between two of its domains during pore formation (Gilbert, 2005; Hotze and Tweten, 2012). During the prepore-to-pore transition, the distance between the D1 and D4 domains is reduced as a result of conformational changes in the D2 domain (Figure 1): this domain is elongated in the prepore but collapses into a more compact structure in the pore, largely accounting for the remarkable 40 Å decrease in height relative to the bilayer surface first observed by atomic force microscopy (AFM) (Czajkowsky et al., 2004; Tilley et al., 2005). Also within this prepore-to-pore step, an α-helical bundle (the Trans-Membrane Helices (TMHs)) in the D3 domain converts into an amphipathic β-sheet that inserts in the bilayer and lines the aqueous membrane pore. While these changes in the D2 and D3 domains have been firmly established, the mechanism by which these changes are coordinated remains unresolved. In fact, the physical processes associated with long-distance structural communication in proteins are poorly understood in general (Cui and Karplus, 2008; Whitley and Lee, 2009; Li et al., 2011). Herein, using single molecule compressive force spectroscopy and single molecule AFM together with all-atom molecular dynamics (MD) simulations, we demonstrate that intra-protein stresses are the driving force of the structural coordination between the D2 and D3 domains and, ultimately, the catalyst of the collapse of the D2 domain.

Figure 1

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As the D2 and TMHs directly contact each other in the PFO prepore and are well separated in the pore, it was previously suggested that the loss of these contacts during the prepore-to-pore transition might have precipitated the collapse of an inherently unstable D2 domain (Czajkowsky et al., 2004). However, extensive equilibrium MD simulations (>0.3 µs) of a PFO monomer without the TMHs shows that the D2 domain remains in an extended conformation in the absence of the TMHs interactions, fluctuating in height by only a few Å (2.9 Å RMS height deviation) (Figure 2A). Thus, the loss of contact with the TMHs alone is insufficient to trigger the conversion of the D2 domain into a compact structure, a conclusion consistent with recent work showing that the TMHs in the thermally trapped PFO prepore are unfolded with significant conformational freedom (Sato et al., 2013) while cryo-electron microscopy (cryo-EM) images of a related cytotoxin, pneumolysin, showed that the D2 domain in this state is in its extended conformation (Tilley et al., 2005).

Figure 2 with 5 supplements see all

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Closer inspection of this prepore conformation, in fact, reveals that fully extended TMHs are of a sufficient length to contact the bilayer surface (Figure 2—figure supplement 1). This extended conformation exposes hydrophobic residues in the TMHs to water, and so the membrane contact would generate a bilayer-directed hydrophobic force on the TMHs, driving them toward the membrane interior. Such a hydrophobic force exerted on a water-exposed transmembrane region has been reported in the folding of bacteriorhodopsin (Kessler et al., 2006) and implicated in the translocon-mediated membrane integration of transmembrane helices (Ismail et al., 2012).

To examine the effect of this bilayer-directed force on the conformation of the protein, we employed steered MD simulations, applying a downward constant force specifically on the TMHs. These calculations reveal that the most significant structural effect of this force is the collapse of the D2 domain (Figure 2B), with the rest of the structure remaining largely unchanged (Figure 2—figure supplement 2). That is, the tensile force on the TMHs is effectively transmitted through the protein to generate a compressive stress across the D2 domain that can catalyze its collapse. During the simulations, after modest initial changes in conformation, the protein structure remained relatively stable owing to the presence of an energy barrier that prevented further conformational changes. Finally, this barrier was overcome, and the height dropped rapidly to a value ∼40 Å lower than its initial height (Figure 2B). In this final conformation, the lower portion of the D2 domain is still a β-sheet and lies almost perpendicular on the surface of the D4 domain, pivoting at Lys381. More importantly, the D2 domain remains in this compact conformation even after the force is relieved (for >40 ns) (Figure 2—figure supplement 3), suggesting that this conformation is a relatively stable state. The D2/D4 interface in this conformation covers 842 Å (Kellermayer et al., 1997) similar to that observed for interfacial contacts in oligomeric proteins (Miller et al., 1987), and the buried hydrophobic residues (Phe75, Tyr389, Tyr415) in the interface (Figure 2C) likely contributed to the stability of this compact state. As these residues are highly conserved in the cholesterol-dependent cytolysins (CDC) family (Supplementary file 1), it is possible that this structure is a common feature of other CDC toxins. For these simulations, the applied force was 250 pN; different forces consistently produced similar results, but the time scale was significantly longer at lower forces (Figure 2—figure supplement 4). Overall this collapsed structure is consistent with the electron-density profile of the pneumolysin pore by cryo-EM (Tilley et al., 2005) (Figure 2—figure supplement 5).

To further understand the energy associated with this transition, extensive adaptive biasing force (ABF) simulations (Chipot and Hénin, 2005) yielded the energy landscape along the reaction coordinate, the height of the D3 domain. This landscape (Figure 2D) exhibits a broad minimum at heights associated with the prepore conformation (from −10 to 8 Å) and a local minimum at heights associated with the pore conformation (centered at ∼40 Å), separated by an energy barrier of ∼23 kcal/mol (∼38 k_BT) from the prepore minimum. Thus, the extended structure of the D2 domain is indeed associated with the energetically minimized conformation of PFO. Of note, there is a sharp rise in the energy at ∼25 Å, where the energy increases by ∼17 kcal/mol over a distance of ∼6 Å. Such a barrier height would require a time frame of days to be overcome by thermal fluctuations alone (see 'Materials and methods'), far longer than the observed timescales of the prepore-to-pore transition (Hotze et al., 2001). Thus, this is the primary barrier that must be reduced by the force generated by the bilayer-TMHs interaction in order to drive PFO into its pore conformation.

As the aforementioned steered MD simulations revealed that it is, ultimately, a compressive stress across the D2 domain that is responsible for its collapse, we reasoned that this process could be experimentally probed with compressive forces applied by an AFM probe on individual complexes if the tensile forces owing to the bilayer-TMHs interaction could be prevented. In this regard, a mutant protein, PFO^G57C-S190C, has been identified that spontaneously assembles into prepore complexes on the membrane like the wild-type protein but does not proceed to the pore state owing to a disulfide bridge that inhibits the restructuring of the TMHs (Figure 3A) (Czajkowsky et al., 2004; Hotze et al., 2001). When this constraint is removed by adding Dithiothreitol (DTT), this mutant quickly converts into a native-like pore (Czajkowsky et al., 2004; Hotze et al., 2001). Thus, this mutant enables investigation of just the energy barrier of the D2 collapse under compressive forces, without any complicating effects associated with the D3-bilayer interaction (Figure 3B and Figure 3—figure supplement 1). Steered MD simulations confirmed that under compressive forces mimicking those that would be applied with AFM, the D2 domain of this mutant recapitulates the same collapse as that of the D2 domain under tensile forces applied to the TMHs (Figure 3—figure supplement 2). Indeed, direct application of constant compressive forces with an AFM tip to individual PFO^G57C-S190C prepore complexes reproducibly induced the characteristic height reduction in the absence of any reducing agent, leaving the general morphology of the complexes otherwise unchanged (Figure 3C and additional images are presented in Figure 3—figure supplement 3). We note that this pore-like structure does not revert back to the prepore conformation upon removing the applied force, indicating that this compact structure is relatively stable, consistent with the MD simulations. We further note that this applied force (110 pN/monomer) is >10-fold smaller than the force required to break a covalent bond over this time period (see 'Materials and methods') (Grandbois et al., 1999).

Figure 3 with 4 supplements see all

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As expected for a thermally driven transition, this force-catalyzed conversion is dependent on the magnitude and the duration of the applied force (Figure 3D). Assuming a simple, single barrier model commonly employed in force spectroscopic experiments (Hu and Li, 2014; Evans, 2001; Fernandez et al., 2010), the probability to induce the pore-like state under an applied force, F, for a pulse period, t, is given by

where

and x_β is the reaction coordinate distance to the energy barrier peak from the minimum, ∆G_o is the barrier height, A is the attempt frequency, k_B is Boltzmann’s constant, and T is the temperature. Each 1s and 5s dataset is well described by the same single barrier model (Figure 3—figure supplement 4). Thus, we simultaneously fitted both datasets to the above equations, yielding x_β = 5.1 ± 0.8 Å and ∆G_o = 16.3 ± 0.6 kcal/mol (see 'Materials and methods'). This energy barrier is smaller than that observed in previous single molecule protein domain unfolding experiments (Fernandez et al., 2010; Dietz and Rief, 2008), as might be expected since the D2 domain is not unfolded during this transition. Importantly, there is excellent agreement between the experimental values and the aforementioned ABF calculations (6 Å and 17 kcal/mol, respectively), thereby providing quantitative support for the model.

Since the hydrophobic force generated by the TMHs-bilayer interaction is critical for the prepore-to-pore transition, any weakening of this interaction is expected to cause a measurable reduction in the rate of transition. Therefore, we investigated the prepore-to-pore transition in the presence of a detergent (n-dodecyl-β-D-maltoside, DDM), since detergent binding to hydrophobic residues in the TMHs should reduce this hydrophobic force on the TMHs (Figure 4A). As shown in Figure 4B, incubating DDM with the mutant PFO^G57C-S190C prepore complexes, followed by the addition of DTT, prevents conversion to the pore-like structure. This effect was dependent on the concentration of detergent, with a maximal inhibition observed with nearly 20 µM DDM (Figure 4B), a similar concentration as that observed for binding to exposed hydrophobic regions in other proteins (Kragh-Hansen et al., 2001).

Figure 4

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In summary, the combination of single molecule measurements and MD computations has enabled direct identification of the essential role of intra-protein forces in driving the PFO complex into its final pore conformation (Figure 4C). We speculate that such intra-protein forces may also be a critical factor in driving coordinated conformational changes between distant domains in many other systems, which are presently not fully characterized mechanistically (Cui and Karplus, 2008; Changeux and Edelstein, 2005). These results also demonstrate that single molecule compressive force spectroscopy can be an effective means by which previously inaccessible physical mechanisms of conformational coordination within membrane proteins in particular may now be probed and quantified.

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Author details

1. Daniel M Czajkowsky

   State Key Laboratory of Oncogenes and Related Genes and Bio-ID Center, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China

   Contribution

   DMC, Conceived and designed the experiments, performed most of the experiments, and wrote the manuscript

   Competing interests

   The authors declare that no competing interests exist.

2. Jielin Sun

   State Key Laboratory of Oncogenes and Related Genes and Bio-ID Center, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China

   Contribution

   JS, Contributed to some of the experiments

   Competing interests

   The authors declare that no competing interests exist.

3. Yi Shen

   State Key Laboratory of Oncogenes and Related Genes and Bio-ID Center, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China

   Contribution

   YS, Contributed to some of the experiments

   Competing interests

   The authors declare that no competing interests exist.

4. Zhifeng Shao

   State Key Laboratory of Oncogenes and Related Genes and Bio-ID Center, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China

   Contribution

   ZS, Conceived and designed the experiments, supervised all of the research, and wrote the manuscript

   For correspondence

   zfshao@sjtu.edu.cn

   Competing interests

   The authors declare that no competing interests exist.

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