Article pubs.acs.org/accounts
Conditional Chaperone−Client Interactions Revealed by Genetically Encoded Photo-cross-linkers Shuai Zhang,† Dan He,† Zhi Lin,† Yi Yang,† Haiping Song,† and Peng R. Chen*,†,‡ †
Beijing National Laboratory for Molecular Sciences, Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China ‡ Academy for Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life Sciences, Beijing 100871, China CONSPECTUS: The cell envelope is an integral and essential component of Gram-negative bacteria. As the front line during host−pathogen interactions, it is directly challenged by host immune responses as well as other harsh extracellular stimuli. The high permeability of the outer-membrane and the lack of ATP energy system render it difficult to maintain important biological activities within the periplasmic space under stress conditions. The HdeA/B chaperone machinery is the only known acid resistant system found in bacterial periplasm, enabling enteric pathogens to survive through the highly acidic human stomach and establish infections in the intestine. These two homologous chaperones belong to a fast growing family of conditionally disordered chaperones that conditionally lose their well-defined three-dimensional structures to exert biological activities. Upon losing ordered structures, these proteins commit promiscuous binding of diverse clients in response to environmental stimulation. For example, HdeA and HdeB are well-folded inactive dimers at neutral pH but become partially unfolded to protect a wide array of acid-denatured proteins upon acid stress. Whether these conditionally disordered chaperones possess client specificities remains unclear. This is in part due to the lack of efficient tools to investigate such versatile and heterogeneous protein−protein interactions under living conditions. Genetically encoded protein photo-cross-linkers have offered a powerful strategy to capture protein−protein interactions, showing great potential in profiling protein interaction networks, mapping binding interfaces, and probing dynamic changes in both physiological and pathological settings. Despite great success, photo-cross-linkers that can simultaneously capture the promiscuous binding partners and directly identify the interaction interfaces remain technically challenging. Furthermore, methods for side-by-side profiling and comparing the condition-dependent client pools from two homologous chaperones are lacking. Herein, we introduce our recent efforts in developing a panel of versatile genetically encoded photo-cross-linkers to study the disorder-mediated chaperone−client interactions in living cells. In particular, we have developed a series of proteomic-based strategies relying on these new photo-cross-linkers to systematically compare the client profiles of HdeA and HdeB, as well as to map their interaction interfaces. These studies revealed the mode-of-action, particularly the client specificity, of these two conditionally disordered chaperones. In the end, some recent elegant work from other groups that applied the genetically encoded photo-cross-linking strategy to illuminate important protein−protein interactions within bacterial cell envelope is also discussed.
1. INTRODUCTION
line during host−pathogen interactions. Revealing the mechanisms of the essential biological systems within the cell envelope will deepen our understanding of host−pathogen interactions, facilitating the development of novel antibiotics. For example, due to the essential role of the periplasm in bacterial physiology and the critical functions of protein quality control (PQC) factors in maintaining proteostasis in this space,5 targeting periplasmic PQC factors could be an attractive direction for antibiotic development. HdeA6 and HdeB7 are two important periplasmic chaperones that maintain protein homeostasis during acid stress. For
The cell envelope of Gram-negative bacteria is a multilayered structure that comprises an outer membrane (OM), a thin peptidoglycan layer, an inner membrane (IM), and the periplasmic space between the OM and IM.1 The periplasm is a unique space that offers the playground for many important biological processes, such as the translocation of proteins, lipopolysaccharide (LPS), and peptidoglycan, as well as the defense against hostile external conditions.2−4 However, due to the highly permeable OM, bacterial periplasm is a particularly vulnerable space to environmental stress. This, in conjunction with the lack of ATP in periplasm renders the mechanism underlying many biological processes within this space mysterious. Notably, cell envelope also serves as the front © 2017 American Chemical Society
Received: December 27, 2016 Published: May 3, 2017 1184
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Accounts of Chemical Research enteric pathogens such as Escherichia coli and Shigella, powerful acid-resistant systems are required to survive through the highly acidic human stomach (pH < 3) in order to establish infection in the intestine. Among many acid protection systems in E. coli, HdeA/B is the only known acid-resistant chaperoning machinery in the periplasm8 (Figure 1). They are well-folded
Figure 1. Acid-resistant machinery in bacterial periplasm.
dimers at neutral pH with no discernible chaperoning activity.9,10 Upon acidification, however, HdeA and HdeB become partially unfolded monomers, gaining chaperoning capability to bind and protect a broad range of acid-denatured periplasmic proteins.7,11,12 The stress-induced unfolding mechanism enables the rapid exposure of HdeA/B’s hydrophobic surfaces to interact with an array of client proteins in a promiscuous fashion,13 which makes them suitable to serve as the first line of defense against acid stress. In fact, this unfolding-mediated activation may be ubiquitous among a growing category of proteins termed conditionally disordered proteins (CDPs).14,15 By conditionally losing their well-defined three-dimensional structures when exerting biological activities, these disordered proteins play diverse roles. In contrast to proteins with well-defined structures, the mode-of-action for many CDPs remains elusive, especially under living conditions. Increasing evidence indicates that chaperones such as HdeA and HdeB belong to the rapidly growing family of conditionally disordered chaperones (CDCs) that work independently of ATP and undergo order-to-disorder transitions to gain physiological activities upon environmental stimulations.15 However, the unique disorder-triggered interactions between CDCs and their clients make direct characterization of the recognition mechanisms very challenging. Although intensive attempts have been made to illustrate these disorder-mediated protein−protein interactions (PPIs) in vitro, whether these observations can be extrapolated to the living systems remains controversial.16,17 Genetically encoded photo-cross-linking technology has emerged as a powerful strategy to discover and characterize native PPIs in living cells.18−20 Unnatural amino acids bearing photoaffinity groups can be site-specifically incorporated into proteins via genetic code expansion technology by using orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs.21−27 A series of phenylalanine or pyrrolysine analogues have been developed (Figure 2A), containing different photoaffinity groups for specific PPI analyses and binding interface mapping.21−27 Upon photoactivation, these photo-cross-linkers become highly reactive and covalently bind to neighboring molecules, thus allowing trapping of intracellular PPIs. By transforming the noncovalent protein interactions into covalent bonds, one can capture even weak or transient interactions under harsh conditions where most CDCs function. This strategy can present a “snapshot” of the PPI network involving a specific region of the target protein in a time-resolved
Figure 2. Structures of genetically encoded photo-cross-linkers. (A) Commonly used phenyalanine and pyrrolysine-based photo-crosslinkers. (B−D) Structures and functional features of DiZPK, DiZSeK and DiZHSeC.
manner. Further mass spectrometry (MS) analysis of the crosslinked complexes can provide information regarding the interaction partners. To this end, genetically encoded photocross-linkers have shown great potential in analyzing the protein−protein interactome, mapping binding interfaces and probing dynamic changes under both native and non-native settings. Applying the genetically encoded photo-cross-linking strategy to the HdeA/B machinery provides an intriguing model system to study the disorder-triggered functional roles of CDCs under living conditions. The development and applications of some traditional photocross-linkers have been extensively reviewed previously.18−20 In this Account, we focus on our recently developed proteomic strategies based on the new genetically encoded photo-crosslinkers and their recent applications to study PPIs in the cell envelope. In particular, we have systematically examined the unique disorder-triggered interactions between HdeA/B and their clients, highlighting the versatile applications of photocross-linking strategies for identifying CDC−client interactions under stress conditions. To this end, three generations of photo-cross-linkers have been developed26,28,29 for (i) capturing the native interactions between HdeA/B and their clients under different conditions to identify their native client pools,26 (ii) revealing the distinct in vivo client specificities between HdeA and HdeB,30 and (iii) mapping the binding interfaces between HdeA/B and their clients under living conditions (Figure 2B,C,D).29 Based on these results, we illustrated the ATP-independent cooperation among periplasmic PQC factors as well as the pH-regulated specific chaperone−client interactions during bacteria acid-resistance. Besides these 1185
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Figure 3. Mapping the chaperoning regions on (A) HdeA (Reproduced with permission from ref 26. Copyright 2011 Nature Publishing Group) and (B) HdeB via protein photo-cross-linking. Two hydrophobic regions involved in client binding on HdeA and HdeB are colored in cyan.
Figure 4. ATP-independent chaperone−chaperone cooperation mechanism between housekeeping PQC factors (e.g., DegP, SurA) and acid chaperones (e.g., HdeA/B) in E. coli periplasm.
Since the photo-cross-linking method is powerful to capture PPIs even under extreme conditions, we envisioned that it is particularly suitable for profiling HdeA clients at low pH. In our initial efforts, we tried to use a reported photo-cross-linker pBpa (p-benzoyl-L-phenylalanine) (Figure 2A) to capture HdeA−client complexes. However, due to the disordered structure of the activated HdeA at low pH, the short and rigid benzyl phenol photoaffinity group on pBpa showed low photocross-linking efficiency. A more flexible photo-cross-linker with a larger reactivity radius might be more suitable for this study. We thus developed a diazirine-bearing photo-cross-linker, 3-(3methyl-3H-diazirine-3-yl)-propaminocarbonyl-N ε - L -lysine (DiZPK), that is structurally similar to pyrrolysine (Figure 2B). Upon incorporation into the dimer interface of HdeA by the genetic code expansion technique, it showed significantly higher photo-cross-linking efficiency than pBpa.26 Similarly, recent research showed that diazirine was a preferred photo-
main focuses, we also introduce some recent elegant work from other groups that used the genetically encoded photo-crosslinking strategy to illuminate important PPIs within bacterial cell envelope.
2. DIAZIRINE-BEARING PHOTO-CROSS-LINKER DiZPK: EFFICIENT CLIENT CAPTURE AND IDENTIFICATION HdeA has been found to support acid-resistance in the periplasm of many enteric pathogens.31,32 It was found to effectively suppress the aggregation of some model proteins as well as the periplasmic extracts in vitro.10,12 Upon neutralization, many client proteins are released in the non-native and aggregation-sensitive form,11,33 suggesting that additional factors may be needed to help refolding or clearing the released clients during the acid recovery process. Nevertheless, characterizing its native clients and possible facilitating factors in vivo remains challenging. 1186
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Figure 5. Releasable photo-cross-linker DiZSeK and CAPP-DIGE strategy for direct dissection of the client specificities between HdeA and HdeB. Reproduced with permission from ref 30. Copyright 2016 National Academy of Sciences. (A) A schematic illustration of the CAPP strategy; (B) CAPP-DIGE analysis of client pools of HdeA and HdeB at pH 2.
interaction information or are incompatible with harsh conditions. On the other hand, the highly abundant bait proteins after photo-cross-linking by using traditional probes such as DiZPK and pBpa can cause high backgrounds that will interfere with identification and comparison processes.28 Therefore, a strategy that can first trap the direct prey−bait interactions and then exclude the bait proteins from the isolated cross-linked complexes is desired. Based on these considerations, we developed a releasable photo-cross-linker, DiZSeK, by replacing the γ-carbon of DiZPK with a selenium (Se) atom, which can undergo the oxidative β-elimination reaction to generate a dehydroanaline and a selenenic acid,41 allowing the separation of preys and baits (Figure 2C). Because the Se-containing cleavable linker is small in size and chemically inert inside cells, the geneticincorporation efficiency and photo-cross-linking efficiency for DiZSeK were similar to those of DiZPK. This new probe allowed us to develop the cleavage after protein photo-crosslinking (CAPP) strategy, which can decrease the false-positive rate generated by nonspecific interactions with the bait proteins28,42−44 (Figure 5A). After photo-cross-linking and in situ cleavage, the obtained pure client pools from HdeA and HdeB were subject to two-dimensional difference gel-electrophoresis (2D-DIGE) analysis,45 which offers a powerful, versatile, and straightforward proteomic procedure. Together, the combination of CAPP strategy and 2D-DIGE technique created a new comparative proteomic method, termed CAPPDIGE.30 For proof-of-concept, we applied this strategy for proteomic profiling and comparison of the native clients between HdeA and HdeB. In brief, DiZSeK was genetically encoded into HdeA and HdeB, followed by photo-cross-linking at pH 2 to trap the chaperone−client complexes inside E. coli cells. After affinity purification, the isolated chaperone−client complexes were oxidatively cleaved and the released client pools from HdeA and HdeB were fluorescent-labeled by Cy3 and Cy5, respectively. Equal amounts of the labeled client pools were then combined and subject to 2D-PAGE analysis. An overlay of fluorescent images can be obtained with the clients of HdeA and HdeB shown in red and green, respectively, while their common clients appeared in yellow (Figure 5B). This CAPP-DIGE strategy revealed that, although HdeA and HdeB shared many common clients, they did have distinct clients. Subsequent MS analysis uncovered a total of 47 common
cross-linking group over aryl azide because it is more efficient and less nonspecific for capturing PPIs.34 By systematically varying the DiZPK incorporation sites on HdeA followed by photo-cross-linking at pH 2 in live E. coli cells, two hydrophobic regions were identified as its clientbinding interfaces (Figure 3A). Similarly, this strategy was applied to HdeA’s homologue protein HdeB, and two smaller hydrophobic regions were identified (Figure 3B). These results support previous functional studies that HdeA and HdeB may share a similar acid-resistance mechanism, with HdeA possessing a stronger chaperoning activity than HdeB.7 To characterize the native clients of HdeA, the cross-linked complexes were isolated by affinity purification and analyzed by a gel-based proteomic strategy. A total of 32 client proteins were identified for the first time under acid stress conditions. Among them, two periplasmic PQC factors, SurA and DegP, caught our attention, and further analysis showed that both DegP and SurA were able to assist HdeA-mediated client refolding during the neutralization process.26 Based on these results, we proposed the ATP-independent “chaperone− chaperone cooperation” mechanism in supporting protein homeostasis in the ATP-deprived E. coli periplasm under acid stress (Figure 4). Our photo-cross-linker DiZPK offers a robust tool for identifying PPIs involving conditionally disordered proteins.
3. RELEASABLE PHOTO-CROSS-LINKER DiZSeK: COMPARATIVE PROTEOMIC ANALYSIS As mentioned above, in addition to hdeA, hdeB is also located in the acid fitness island gene cluster that is highly induced upon exposure to low pH.35 However, the physiological role of HdeB is less explored. To examine whether functional differences exist between HdeA and HdeB in facilitating bacterial acidresistance, we adopted a comparative proteomic strategy to side-by-side compare their client profiles in the entire periplasmic proteome. Current methods for profiling and comparing the conditionspecific PPIs from two homologous proteins are rather limited.36,37 For example, genetic deletion38,39 and coimmunoprecipitation (co-IP)40 are traditional strategies for isolating the clients or chaperone−client mixtures from the complex cellular context, enabling the subsequent proteomic and MS analysis. However, these strategies either lack direct 1187
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Accounts of Chemical Research clients as well as additional 5 and 6 unique clients for HdeA and HdeB, respectively (Table 1). Among them, many important Table 1. Identified HdeA and HdeB Preferred Clients30 name
MW (kD)
DppA OppA MalE BamB MetQ
60 60 43 42 30
RbsB DsbA YfgM EmtA
31 23 22 22
ecotin OsmE
18 12
function HdeA Preferred Client Proteins periplasmic dipeptide transport protein periplasmic oligopeptide transport protein maltose-binding periplasmic protein outer membrane protein assembly factor D-methionine-binding lipoprotein metQ HdeB Preferred Client Proteins D-ribose-binding periplasmic protein thiol:disulfide interchange protein dsbA UPF0070 protein YfgM endotype membrane-bound lytic murein transglycosylase A serine protease inhibitor ecotin osmotically inducible protein OsmE
periplasmic PQC factors were found to be protected by HdeA or HdeB or both, including all three main protein-folding catalysts (DsbA, DsbC, DsbG), two of the four proline isomerases (SurA and PpiD), and four proteases (DegP, DegQ, Tsp, and PtrA). In particular, a network of essential periplasmic PQC factors are protected by HdeA/B, which further highlighted the critical roles of these chaperones in coping with acid stress. Moreover, these results indicate a potential linkage between the HdeA/B acid chaperone machinery and the broader bacterial protein homeostasis system (Figure 4). For example, examining the activity of the protease DegP during neutralization processes showed that this key PQC factor regained its proteolytic activity when the pH was still as low as 5.30 This funding indicates that the proteolytic activity of DegP may work in conjunction with HdeA/B for better protein quality control by eliminating the aggregation-prone proteins that fail to refold properly during acid recovery. Together, our releasable photo-cross-linker DiZSeK and CAPP-DIGE strategy permit the direct dissection of client specificities between two seemingly redundant chaperones within their native cellular context.
Figure 6. Cleavable photo-cross-linkers to illustrate PPI interface. Reproduced with permission from ref 29. Copyright 2016 Nature Publishing Group. (A) Oxidative cleavable photo-cross-linker DiZSeK and DiZHSeC. (B) Interaction interfaces of HdeA−DegP mapped by IMAPP strategy. (C) A proposed model illustrating the interactions between HdeA and DegP at low pH.
interface. Notably, this MS label can improve the specificity, fidelity, and robustness during target identification at the same time. Based on DiZHSeC, we developed a MS-label assisted IMAPP strategy (in situ cleavage and MS-label transfer after protein photo-cross-linking) to directly capture and identify PPI interfaces within living cells. In brief, the search for MSlabels was conducted after photo-cross-linking, affinity purification, in-gel oxidative cleavage, and MS analysis so that only the modified peptides from the clients were analyzed. We used this strategy to successfully map the HdeA−client interaction interfaces with a wide range of client proteins at pH 2 in living E. coli. In particular, the HdeA−DegP interaction interfaces were closely inspected, and the results indicate that HdeA binds to the protease domain and the PDZ1 domain but not the PDZ2 domain on DegP (Figure 6B,C). Together, the DiZHSeC photo-cross-linker and IMAPP strategy enabled simultaneous profiling of protein−protein interactome and mapping of binding interfaces, which makes it a powerful and versatile strategy for discovering and characterizing native PPIs.
4. MS-LABEL TRANSFERABLE PHOTO-CROSS-LINKER DiZHSeC: MAPPING THE PPI INTERFACE In addition to capturing PPIs, the direct mapping of protein interaction interfaces under living conditions is highly desired.18 For example, due to the promiscuous nature of HdeA’s client binding, little is known about the binding interfaces of the HdeA−client complexes, which is crucial for understanding the client recognition mechanism of HdeA. To address this issue, we integrated a transferable, MS-identifiable label (MS-label) into the DiZPK probe, which can in situ generate a MS-label on the captured client proteins after photo-cross-linking and cleavage.29 This DiZHSeC photo-cross-linker can undergo oxidation-mediated C−Se bond cleavage to produce the N(4,4-bis-substituted-pentyl)acrylamide (NPAA, C8H13NO) modification on prey proteins, which is a stable and readily identifiable MS-label (Figure 6A and 2D). The resulting clientderived peptides bearing such MS-labels are readily distinguishable from the complex biological samples during MS analysis, which provides direct information regarding the binding
5. pH-DEPENDENT PHOTO-CROSS-LINKING: UNCOVERING THE WORKING CYCLE OF ACID CHAPERONES It is still unclear how CDCs such as HdeA use the same hydrophobic chaperoning regions to specifically recognize diverse client proteins.15 We hypothesized that the client specificity might result from some external environmental 1188
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Figure 7. Overview of the mode-of-action of the HdeA/B machinery in coping with bacterial acid stress. Reproduced with permission from ref 30. Copyright 2016 National Academy of Sciences.
Finally, the physiological relevance of this pH-regulated dualchaperone model was examined, and the results highlighted the indispensable role of the weaker chaperone HdeB as well as the functional complementarity of this dual-chaperone machinery in E. coli periplasm. In summary, relying on our versatile photocross-linking tools, we systematically investigated the mode-ofaction of the HdeA/B chaperone machinery, which has been evolved as an efficient, economical, flexible, and fine-tuned PQC system to fight against acid stress in an ATP-independent manner. The molecular mechanism we uncovered from such study may be applicable to other CDC systems such as small heat shock proteins in mammalian cells47 and chaperones ERD10 and ERD14 in plants,48 which all undergo conditiontriggered order-to-disorder transitions to exert chaperoning functions in a seemingly redundant or overlapping fashion.
factors. For example, as a direct activation trigger, acid is a potential stimulus that regulates the client specificities of HdeA and HdeB. To test this hypothesis, we conducted a pHdependent photo-cross-linking experiment during the entire acidification process (pH from 7 to 2) in E. coli cells expressing HdeA-DiZPK or HdeB-DiZPK.30 The results showed that HdeA and HdeB had distinct pH-dependent client interaction profiles, which was consistent with their different pHdependent unfolding behaviors as monitored by biochemical assays in vitro. HdeB starts to unfold and bind to its clients when pH drops to 4.5, while HdeA will not be engaged in client interactions until pH drops to below 3.5. Comparing the client profiles between WT-HdeA and an earlier activated variant HdeA-D20A11,46 using our CAPP-DIGE strategy further confirmed that the different pH responses could result in different client specificities. Indeed, our further analysis showed that HdeB-preferred clients such as RbsB, ecotin, and DsbA significantly exposed their hydrophobic surfaces when pH dropped to around 4.5, whereas the HdeA-preferred clients such as DppA and OppA were less acid-sensitive and would not expose their hydrophobic surfaces until pH dropped below 3.5. Together, this evidence supports that the unfolding and activation of HdeA/B are fine-tuned by pH stimuli, leading to their interactions with different clients with different acid sensitivity. Therefore, pH serves as the environmental cue to regulate the client specificities of the acid-sensitive CDCs HdeA and HdeB. The pH-dependent photo-cross-linking was also conducted on HdeA/B during the acid recovery process (neutralization from pH 2 to pH 7), which showed that the clients would not be completely released by their respective chaperones until pH was restored to above 5. This is in direct contrast to the aforementioned photo-cross-linking results during acidification and thus generates an “asymmetric chaperoning window” for HdeA and HdeB (Figure 7). These observations are in line with the previously demonstrated “fast-binding and slow-release” mechanism33 for HdeA in vitro. Further examinations of the interactions between HdeA/B and their common clients indicate that the clients will not be released until they become folding competent. Therefore, in addition to client binding, pH also functions as an environmental cue to regulate the client release process of HdeA/B, which warrants efficient refolding of the released clients.
6. THE PROTEIN INTERACTOMES ILLUSTRATED BY PHOTO-CROSS-LINKERS IN BACTERIAL CELL ENVELOPE Besides PQC factors for protein homeostasis, the photo-crosslinking strategy has been utilized extensively to illustrate the mechanism underlying important biological processes such as outer membrane protein biogenesis/translocation49,50 and lipopolysaccharide (LPS) translocation.51−53 Here we introduce two recently reported work that revealed dynamic protein interactions during material transfer processes in bacterial cell envelope. DegP, SurA, and Skp are three ATP-independent chaperones that are essential for periplasmic protein homeostasis as well as outer membrane protein (OMP) biogenesis.54 Different from SurA and Skp, DegP also possesses protease activity that can degrade misfolded OMPs.55 Chang and co-workers sitespecifically incorporated DiZPK into DegP and identified βbarrel OMPs as the major native substrates of DegP in E. coli.49 More recently, the same group incorporated pBpa into SurA at various sites and mapped the interaction surfaces between SurA and its different partner proteins.50 This study found that SurA interacted with nascent OMPs on the N-domain of its β-barrel. However, for the β-barrel assembly machine protein BamA, SurA mainly interacted with its satellite P2 domain, suggesting the formation of a ternary complex involving β-barrel OMP, SurA, and BamA. The authors further introduced pBpa into the β-barrel OMPs and BamA and found that OMPs interacted with SurA through their N- and C-terminal regions. In contrast, 1189
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Figure 8. PPIs in cell envelope uncovered by genetically encoded photo-cross-linkers. (A) Interactions among PQC factors in facilitating the OMP biogenesis. (B) Transport of LPS through E. coli periplasm via a multiprotein scaffold. Reproduced with permission from ref 3. Copyright 2016 Nature Publishing Group.
hydrolysis of ATP. Together, the genetically encoded photocross-linking strategy has been thoroughly applied to trap the transient, dynamic, and cascade interactions between these essential cell envelope factors and their interaction partners, which revealed the biological processes and the underlying mechanisms in great details.
the folded BamA interacted with SurA on a region around position K135 in the POTRA 2 domain. More interestingly, when pBpa was simultaneously incorporated into both BamA and OmpF, they identified the photo-cross-linked OmpF− SurA−BamA tricomplex. These results shed light on the molecular mechanism underlying β-barrel OMP biogenesis. In conjunction with other biochemical analysis, this work implicates a “super-complex” that may span through the entire periplasmic space and connect the inner and outer membranes for OMP biogenesis (Figure 8A). A series of elegant work from Kahne and co-workers using genetically encoded photo-cross-linkers have unveiled the dynamic interaction interfaces between LPS transport (Lpt) proteins during LPS translocation51−53 (Figure 8B). As a large amphipathic molecule that contains many fatty acyl chains and sugars,56 LPS are synthesized in bacterial cytoplasm, transported across IM and the periplasm and eventually assembled into the OM. At least seven essential proteins are required for this process:57 four inner membrane proteins (LptB, LptC, LptF, LptG), a periplasmic protein (LptA), and two outer membrane proteins (LptD, LptE). Although the architecture of the Lpt proteins has been proposed, how these components physically interact with each other and the detailed molecular mechanism underlying this LPS transport cascade remains poorly understood. The Kahne group site-specifically introduced pBpa into a series of positions in LptC, LptA, and LptD and defined the interaction sites between the OstA domains of these proteins through photo-cross-linking.51 The results strongly supported the proposed “Lpt bridge model” in which the IM Lpt complex LptB2FGC and the OM Lpt complex LptDE were connected by LptA and mediated by the homologous OstA domains in LptC, LptA, and LptD. Furthermore, by introducing pBpa into LptE and LptD at different positions, the authors defined the unique dynamic interaction between LptD and LptE and illustrated a plug-andbarrel conformation.52 More interestingly, this strategy was also used to detect the interaction intermediates between Lpt proteins and LPS in membrane vesicles with native orientations. By trapping the LPS−Lpt protein interactions through photo-cross-linking under different conditions such as in the presence and absence of specific Lpt proteins or ATP, the authors tracked the different LPS transport intermediates and analyzed the ATP-dependent LPS transport with high temporal resolution.53 These photo-cross-linking results suggest the existence of a continuous stream of LPS from IM to OM, with underlying energy provided by the cytoplasmic
7. CONCLUSIONS AND OUTLOOK This Account summarizes our recent work on developing genetically encoded multifunctional photo-cross-linkers to facilitate the investigation of CDC−client interactions. The introduction of a Se-based cleavable linker into the photo-crosslinking probes expanded the current toolkit, which facilitated the development of new strategies such as CAPP-DIGE and IMAPP to profile the native clients of HdeA/B as well as to characterize their client specificities and interaction interfaces. The genetically encoded photo-cross-linking strategy was also employed by other researchers to investigate the transient or dynamic PPIs within bacterial cell envelope. This work uncovered versatile and dynamic PPIs that play important roles in biological processes. Since a growing number of proteins have been shown to interact with their binding partners under disordered conditions,58−60 the advantages for simultaneous client identification and binding interface mapping with our newly developed multifunctional probes will be highly valuable in studying these proteins. However, the genetically encoded photo-cross-linking strategy is not without limitations. For example, it may not be suitable for trapping very fast PPI changes, and such a covalent-capture method offers limited information regarding the interaction affinity. Nevertheless, considering that our photo-cross-linkers are highly compatible with various proteomic strategies, we expect these probes to find broader applications in diverse areas. The addition of other novel functional groups to the photo-crosslinkers may further expand the versatility of this toolbox. Finally, the unique Se-bearing cleavable linker may be valuable for tool development in other fields that also require small-sized but highly biocompatible and chemically cleavable linkages.
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AUTHOR INFORMATION
Corresponding Author
*Peng Chen, e-mail:
[email protected]. ORCID
Dan He: 0000-0002-9544-5672 Peng R. Chen: 0000-0002-0402-7417 1190
DOI: 10.1021/acs.accounts.6b00647 Acc. Chem. Res. 2017, 50, 1184−1192
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Accounts of Chemical Research Notes
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The authors declare no competing financial interest. Biographies Shuai Zhang received his B.S. in Chemistry from Wuhan University in 2011. He received his Ph.D. from Peking University under the supervision of Professor Peng Chen in 2016. Dan He received her B.S. in Chemistry from Xiamen University in 2012, and she is currently a graduate student in Professor Peng Chen’s group at Peking University. Zhi Lin was born in Shanghai in 1994 and is currently an undergraduate student in Professor Peng Chen’s group at Peking University, with her B.S. expected in 2017. Yi Yang received his B.S. in Chemistry from Shandong University in 2011, and he is currently a graduate student in Professor Peng Chen’s group at Peking University. Haiping Song received her B.S. in Chemistry from Jilin University in 2013, and she is currently a graduate student in Professor Peng Chen’s group at Peking University. Peng Chen graduated with B.S. in Chemistry from Peking University in 2002 and received his Ph.D from the University of Chicago in 2007. He worked as a postdoctoral associate at the Scripps Research Institute between 2007 and 2009 and returned to Peking University as an Investigator in the summer of 2009. He is now Professor and Chairman in Department of Chemical Biology at Peking University.
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ACKNOWLEDGMENTS This work was supported by the National Key Research and Development Program (2016YFA0501500) and the National Natural Science Foundation of China (21225206, 21432002, and 21521003).
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