Rapid Report pubs.acs.org/biochemistry
Bidirectional Transformation of a Metamorphic Protein between the Water-Soluble and Transmembrane Native States Koji Tanaka,† Jose M. M. Caaveiro,*,‡ and Kouhei Tsumoto*,†,‡ †
Department of Chemistry and Biotechnology and ‡Department of Bioengineering, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan S Supporting Information *
ABSTRACT: The bidirectional transformation of a protein between its native water-soluble and integral transmembrane conformations is demonstrated for FraC, a hemolytic protein of the family of pore-forming toxins. In the presence of biological membranes, the water-soluble conformation of FraC undergoes a remarkable structural reorganization generating cytolytic transmembrane nanopores conducive to cell death. So far, the reverse transformation from the native transmembrane conformation to the native water-soluble conformation has not been reported. We describe the use of detergents with different physicochemical properties to achieve the spontaneous conversion of transmembrane pores of FraC back into the initial water-soluble state. Thermodynamic and kinetic stability data suggest that specific detergents cause an asymmetric change in the energy landscape of the protein, allowing the bidirectional transformation of a membrane protein.
I
n the past two decades, an increasing number of proteins have been reported to display a single primary sequence but multiple three-dimensional structures,1 challenging the Anfinsen dogma.2 The conformational states of these proteins exist in a reversible equilibrium (thermodynamic control)3,4 or in a latent state governed by kinetic factors.5 Here we report the unprecedented bidirectional transformation between the native water-soluble (WS) state and the native transmembrane (TM) state of the hemolytic protein FraC of the class of pore-forming toxins (PFT). PFT undergo structural changes from the WS state to the TM oligomeric state triggered by biological membranes, where they form stable cytolytic nanopores.6 In the model PFT FraC, the structural changes from the WS state [WS-FraC; molecular weight (MW) of 20 kDa] to the TM octameric state (TMFraC; MW of 160 kDa) occur in the presence of biological membranes containing the lipid sphingomyelin (Figure 1).7 Upon oligomerization mediated by specific residues,8 the conformational changes occurring during the transformation from WS-FraC to TM-FraC involve the N-terminal region, which adopts an amphipathic α-helical structure spanning the cell membrane (Figure S1 of the Supporting Information).7 The pores produced by PFT have important biological functions for cellular warfare,9 apoptosis,10 and the immune response.11 Besides, PFT are increasingly employed for nanotechnological applications such as DNA sequencing,12 © 2015 American Chemical Society
Figure 1. Effect of detergents on TM-FraC. (a) Experimental procedure. (b) Analytical SEC profiles of FraC (control), untreated FraC, and FraC treated with a representative group of detergents showing various kinds of responses. TM-FraC and WS-FraC appeared at 11 and 28 mL, respectively. SEC profiles are also shown in Figure S3 of the Supporting Information.
biosensing,13 and nanotherapy.14 PFT have also inspired the design of membrane-spanning artificial molecules.15 The characterization of mild conditions allowing the bidirectional transformation between the native TM state and the native WS state sheds light on the fundamental mechanisms for the assembly of oligomeric TM proteins. In addition, this study expands the chemical toolkit for the manipulation of PFT for bio- and nanoengineering applications. We tested the possibility of the transformation from the TM state to the WS state with FraC by employing detergents under mild (nondenaturing) conditions. Purified TM-FraC in mild detergent n-dodecyl β-D-maltoside (DDM) was obtained with high purity as described previously.7,16 Next, TM-FraC was treated with a panel of 16 different types of detergents often Received: October 13, 2015 Revised: November 6, 2015 Published: November 6, 2015 6863
DOI: 10.1021/acs.biochem.5b01112 Biochemistry 2015, 54, 6863−6866
Biochemistry
Rapid Report
used for the purification and crystallization of membrane proteins (Figure S2 and Tables S1 and S2 of the Supporting Information).17 TM-FraC (in DDM) was immobilized on a cationic affinity column, where the exchange of detergents took place (Figure 1a). After treatment with detergents, analytical size exclusion chromatography (SEC) was employed to monitor the state of FraC. The elution peaks of octameric TM-FraC and monomeric WS-FraC appeared at well-separated peaks at ∼11 and ∼28 mL, respectively, facilitating their unambiguous identification (Figure 1b and Figure S3 of the Supporting Information). The absence of peaks at intermediate positions demonstrates intermediate oligomeric species are not stably formed. The percentage of TM-FraC and WS-FraC were estimated from the area under the elution peaks (Table 1). Some protein was lost during detergent exchange prior to the SEC column because of protein aggregation.
Figure 2. Yield of transformation from TM-FraC to WS-FraC is influenced by the length of the acyl chain of the detergent employed.
shown that the delipidation ability of the detergents improves when the length of the acyl chain decreases, in a manner independent of the type of detergent and membrane protein employed. Therefore, detergents with short acyl chains may preferentially extract structural lipids essential for the stability of TM-FraC,7,19 thus allowing the dissociation of the pore and its spontaneous conformational change to WS-FraC. No correlation between the percentage of WS-FraC and other physicochemical properties of the detergent such as the chemical identity of the headgroup or the acyl chains, the critical micelle concentration (cmc), the type of detergent (harsh vs mild), or the concentration of detergent was observed (Tables S1 and S2 of the Supporting Information). Harsh detergents of the zwitterionic class were not efficient at inducing WS-FraC (DDAO and LDAO) and instead led to protein aggregation, consistent with the idea that these classes of detergents have a tendency to denature membrane proteins (Figure S4 and Table S2 of the Supporting Information).20 On the other hand, harsh detergents of the nonionic class (OG, OTG, and C8E4) are effective at disassembling TM-FraC and facilitating the appearance of WS-FraC with moderate or little protein aggregation, suggesting different mechanisms for each class of detergents. Next, we characterized the WS-FraC species before and after incubation with the lipids and subsequently treatment with DDM/OG followed by detergent removal, from functional, biochemical, and structural points of view (Figure 3 and Figure S5 of the Supporting Information). These experiments were designed to verify if both WS-FraC species were identical to each other. The SEC elution profiles, hemolysis potency, and circular dichroism spectra of purified WS-FraC before treatment with lipids, or after treatment with lipids and detergents, are indeed very similar. Moreover, their crystal structures are essentially indistinguishable from each other (root-mean-square deviation of 0.22 ± 0.04 Å) (Figure 3d and Table S3 of the Supporting Information), demonstrating that the procedure outlined above recycles FraC to yield the same chemical entity as that in the initial WS state. The rate of hemolysis by the TM-FraC species is slower than that of WS-FraC, reflecting the difficulty that the preformed pore has in penetrating the plasma membrane of red blood cells. We note that TM-FraC undergoes irreversible aggregation when the detergent is absent (Table 1). In this regard, TMFraC behaves like other TM proteins, requiring the presence of detergents to prevent aggregation of its hydrophobic TM region.
Table 1. State (%) of FraC after Treatment with Detergents relative to control sample
a
detergent
acyl-chain length
TM
WS
aggregated
Brij 35 C12E8a Fos-Choline-12 DDM Empigen BB TX-100 DDG OMa LDAO DM no detergent DDAO OTG C10E5 C8E4 CHAPS OG
12 12 12 12 12 na 12 8 12 10 na 10 8 10 8 na 8
99 90/88 86 79 70 60 58 52/49 46 36 21 0 0 13 0 0 0
4 0/0 3 0 9 0 8 9/6 8 6 3 36 74 87 96 97 97
nd 10/12 11 21 22 40 34 39/45 46 58 76 64 26 0 4 3 3
Data from two independent experiments are given.
Of the 16 detergents tested, five were effective or very effective at inducing the transformation to monomeric FraC (WS-FraC ≥ 74%) and one showed intermediate behavior (WS-Frac = 36%). The 10 remaining detergents were ineffective (WS-FraC < 10%). The extent of the transformation is correlated with the length of the acyl chain of the detergent (Figure 2). None of the seven detergents with long acyl chains (12 carbon atoms) significantly favored the appearance of WS species (
