Structures of EccB1 and EccD1 from the core complex of the mycobacterial ESX-1 type VII secretion system
© Wagner et al. 2016
Received: 8 October 2015
Accepted: 23 February 2016
Published: 27 February 2016
The ESX-1 type VII secretion system is an important determinant of virulence in pathogenic mycobacteria, including Mycobacterium tuberculosis. This complicated molecular machine secretes folded proteins through the mycobacterial cell envelope to subvert the host immune response. Despite its important role in disease very little is known about the molecular architecture of the ESX-1 secretion system.
This study characterizes the structures of the soluble domains of two conserved core ESX-1 components – EccB1 and EccD1. The periplasmic domain of EccB1 consists of 4 repeat domains and a central domain, which together form a quasi 2-fold symmetrical structure. The repeat domains of EccB1 are structurally similar to a known peptidoglycan binding protein suggesting a role in anchoring the ESX-1 system within the periplasmic space. The cytoplasmic domain of EccD1has a ubiquitin-like fold and forms a dimer with a negatively charged groove.
These structures represent a major step towards resolving the molecular architecture of the entire ESX-1 assembly and may contribute to ESX-1 targeted tuberculosis intervention strategies.
KeywordsEccB EccD Mycobacterium tuberculosis Type VII secretion system ESX
Pathogenic bacteria rely on a variety of secretion systems to transport virulence factors, proteins that mediate host-pathogen interactions, across their hydrophobic cell membranes to sites where they can interact with the host. Gram-positive bacteria need only transport proteins across a single membrane, but Gram-negative bacteria require specialized secretion machinery that spans both inner and outer membranes. Mycobacterium tuberculosis, the causative agent of tuberculosis, was recently re-classified as a diderm bacterium when it was shown to have an outer membrane bi-layer — referred to as the mycomembrane — composed largely of mycolic acids . In order to transport key virulence factors across both membranes M. tuberculosis has evolved specialized Type VII secretion systems (T7SS). The T7SSs were discovered based on attenuated strains of M. tuberculosis deficient in EsxA (ESAT-6, early secreted antigenic target of 6 kDa) secretion and are commonly called ESAT six (ESX) secretion systems [2–4]. In M. tuberculosis there are five gene clusters, named ESX-1 to ESX-5, which encode T7SS. Each gene cluster encodes a number of proteins that are either secreted or are building blocks for the secretion apparatus. ESX-1 is responsible for secretion of the important virulence factors EsxA and EsxB as well as other virulence-associated proteins (e.g., EspB, EspF, EspJ) that are secreted to the cell surface or extracellular milieu based on recognition of a conserved C-terminal signal sequence on the secretion substrates [5–8]. These secreted factors have been linked to mycobacterial virulence through studies of the attenuated BCG strain of M. tuberculosis [2, 4, 9]; in non-pathogenic Mycobacterium smegmatis the orthologous ESX-1 system is involved in conjugation [10, 11]. ESX-3 is critical for mycobacterial survival due to its role in metal acquisition [12–14]. ESX-5 is important for the secretion of many members of the PE/PPE family of proteins that also play a role in virulence and cell wall integrity [15–17]. The functional role of ESX-2 and ESX-4 is still unknown although ESX-4 appears to be the ancestral system from which the other ESX systems have evolved .
All ESX gene clusters contain at least three or four ESX conserved components (Ecc), named EccB, EccC, and EccD, with EccE being present in all ESX systems with the exception of ESX-4 . Multiple copies of each core protein as well as other T7SS-associated proteins are present in the core complex resulting in a large ~1500 kDa particle . The function of some core components is known, for example, EccC is a member of the FtsK/SpoIIIE-like ATPase family and provides the energy to transport proteins across the mycobacterial membrane(s) [21, 22]. EccD contains an N-terminal cytoplasmic domain followed by 11 predicted transmembrane helices, and may form the cytoplasmic membrane channel through which cargo proteins are secreted. The functions of EccB and EccE within the secretion apparatus are less clear. These proteins both have N-terminal transmembrane elements and large C-terminal regions predicted bioinformatically to be localized in the periplasm, but their molecular structures and interacting partners remain unknown.
Understanding the T7SS architecture is critical for development of new antitubercular agents. Currently, no structural data is available for three of the four conserved components EccB, EccD, and EccE. In this study we report the molecular structures of the periplasmic domain of EccB1 and the cytoplasmic domain of EccD1 from the ESX-1 cluster. The structures reveal probable functional surfaces of EccB1, and an unexpected dimerization by EccD1. Here we describe these structures in detail and how they might fit into the larger context of the T7SS.
Results and discussion
Structure of EccB1
EccB1 does not bear significant sequence similarity to any protein of known structure, and Dali searches using the complete EccB1 structures revealed no proteins with significant structural homology. However, Dali searches using only EccB1mt repeat 1 (S74–P124) revealed weak homology (r.m.s.d. 2.7 Å and Dali Z-score of 5.0) to the N-terminal domain of PlyCB (PDB 4 F87, residues 14–70) from streptococcal C1 bacteriophage . Eight PlyCB monomers assemble into a ring that associates with the bacterial cell wall and facilitate phage egress by tethering the degradative PlyCA subunit to the bacterial cell wall. The structural similarity between the two proteins and a common localization of both to bacterial cell envelope structures is intriguing but no clues to EccB1 function are apparent from our examination of PlyCB.
Structure of EccD1mt
EccD1mt (Rv3877) is a 54 kDa protein containing an ~110 amino acid (aa) N-terminal ubiquitin-like domain followed by a 30 aa linker and 11 closely spaced transmembrane helices at its C-terminus. The ubiquitin-like domain of EccD1 classifies it as a member of the YukD family within the Pfam database. Based on the characteristics of the transmembrane regions the N-terminal portion of EccD1 is predicted to be localized in the cytoplasm.
Interestingly, the asymmetric unit of both crystal forms contains two EccD1 molecules and in both crystal forms the two EccD1 molecules are arranged as a head-to-tail homodimer stabilized by an extensive interface. The interface is formed by interlocking side chains from β strands 1 and 2 and the N-terminal α-helix of both EccD1 molecules (Fig. 4) and ~650 Å2 of each EccD1 molecule (13 % of the total surface) is buried in the interface as calculated with the PISA webserver . The interaction is stabilized by 4 hydrogen bonds and a cluster of buried hydrophobic residues including Met1, Val54, and Val58 resulting in a solvation energy of −13.9 kcal/mol and a Complex Significance Score of 1.0 calculated by the PISA server. The extensive nature of the interface and its re-occurrence in both crystal forms, with or without the MBP fusion, suggests that EccD1 is a natural homodimer.
Putative function of EccB1 and EccD1
Mutations in EccB3 of the ESX-3 secretion system have been shown to confer drug resistance in M. tuberculosis . The mutations found to confer resistance (Arg14Leu, and Asn24His) occur in the small cytoplasmic domain preceding the transmembrane element of EccB3, a region not present in our EccB1 constructs which contain the soluble periplasmic domain. The fact that mutations in this region confer drug resistance indicates an important function for this short region perhaps in mediating interactions with other cytoplasmically exposed components of the T7SS. The elongated shape and continuous hydrophobic core of EccB1 suggest that it may serve a structural role – perhaps forming part of a structure that spans the inner and outer membrane components of the ESX secretion system. The structural similarities between PlyCB, the viral cell wall binding protein complex, and EccB1 hints that EccB1 may also bind elements of the peptidoglycan layer, but there is not yet any experimental data to support this idea. However, post-translational modification of secreted bacterial proteins with O-linked polysaccharides has been shown to be important for solubility or maintaining subcellular localization to the cell wall [28, 29]. EccB1 contains 24 putative glycosylation sites, as predicted by the NetOGlyc webserver , and many of these are surface-exposed in the EccB1 structures (including Ser143, Thr144, Ser351, and Ser356). While this manuscript was under preparation, the ATPase activity of EccB1 has been reported . Further studies are needed to define the precise role of EccB1 in the context of a functional ESX-1 secretion complex.
The dimerization of the cytoplasmic domain of EccD1 raises interesting possibilities regarding the nature of the transmembrane pore. Each EccD1 monomer has 11 transmembrane elements thus a dimer would have a total of 22 transmembrane elements. Each monomer may form an independent pore resulting in a pair of closely associated channels, or the transmembrane elements may comprise a single, larger, transmembrane channel. The cytoplasmic domain itself is connected to the first transmembrane element by a 30 amino acid linker that may facilitate protein-protein interactions, either with the cytoplasmic EccD1 domain or other components of the secretion system, or it may simply form an extended tether allowing increased mobility of the ubiquitin-like domains.
The negatively charged groove of the EccD1 dimer indicates that it should associate with a positively charged partner(s). It may act to recruit other T7SS components or secretion substrates with positively charged patches into the system, or it may be part of a gating element required to close the channel during periods of inactivity. The residues contributing to the negatively charged groove are not conserved in EccD1 homologs from other ESX systems indicating that they may serve a system-specific role. Indeed, the ESX-1 locus encodes a variety of secretion substrates not found in the paralogous M. tuberculosis ESX systems and thus it is likely that the ESX-1 system has structural adaptations to enable the secretion of these substrates [6–8, 32]. As more structures of ESX-1 components are determined likely partners for interaction with the EccD1 dimer may be revealed.
In summary, we have determined the structures of soluble domains of two integral, conserved components, EccB1 and EccD1, of the ESX-1 secretion channel. Given the importance of the ESX-1 secretion system to mycobacterial virulence, our structures provide crucial information about the molecular makeup of this important protein complex that will aid future drug development efforts.
Expression and purification of EccB1mt
A construct for expression of the periplasmic domain of EccB1mt (residues 72–463) was designed based on predicted transmembrane helix using the TOPCONS server , secondary structure prediction using the JPred4 server , and the sequence alignment of EccB1 orthologs (Additional file 1: Figure S1). The DNA fragment was PCR-amplified from M. tuberculosis H37Rv genomic DNA using primers EccB1_F72_Nco 5′–CACCATGGGCACCAGCCTGTTCACCGACC and EccB1_RS463_Hind 5′–GCAAGCTTACAGCGTGTCGTGCTCGAGCAG, and cloned into a modified pET-22b(+) vector (Novagen), which contains the Escherichia coli DsbA signal sequence, a hexahistidine tag and a tobacco etch virus (TEV) protease cleavage sequence.
EccB1mt was expressed in E. coli Rosetta2(DE3) strain using LB media and 0.5 mM IPTG for induction. Cells were harvested after 4 h incubation at 18 °C, resuspended in 20 mM Tris–HCl pH 8.0, 300 mM NaCl buffer, and lysed using microfluidizer (Avestin). EccB1mt was purified via a Ni-NTA affinity column, incubated with TEV protease to remove the hexahistidine tag, and passed over a Ni-NTA column to remove uncleaved protein, and further purified by size exclusion chromatography using a Superdex 200 column (GE Healthcare). Protein was flash-frozen using liquid nitrogen and stored at −80 °C.
Crystallization and structure determination of EccB1mt
Crystals were grown using the sitting drop vapor diffusion method with precipitant containing 0.1 M Tris–HCl pH 5.6, 15 % PEG2000 MME, 10 mM NiCl. Crystals were transferred to crystallization solution supplemented with 20 % glycerol, or with 20 % glycerol and 0.5 NaI , and flash-frozen in liquid nitrogen.
Data were collected at the 22-ID beamline at the Advance Photon Source, Argonne National Laboratory, and processed using XDS  and HKL-3000 . Iodide ion positions were determined using SHELXD  as implemented in HKL-3000, and phases were calculated using SHARP . The model was built using Buccaneer  and Coot , and refined by REFMAC5  using TLS groups defined by the TLSMD server . The final structure includes residues 74–458.
Expression and purification of EccB1ms
The periplasmic domain (residues S73-G479) of the MSMEG_0060 gene was PCR-amplified from M. smegmatis mc2155 genomic DNA with the gene-specific primers MsEccB1.For. 5′-AACCTGTATTTCCAGAGTAGTGACCAGCTGCTGGTGG and MsEccB1.Rev. 5′-TTCGGGCTTTGTTAGCAGTTAGCCCTCCCCGCTCG and cloned into the pMAPLe4 expression vector , which appends a TEV protease cleavable hexahistidine tag to the N-terminus of the target protein, using the Gibson ISO cloning method . The sequence of the expression clone was verified by DNA sequencing (Genewiz, Piscataway, NJ).
Recombinant protein was overexpressed in E. coli BL21(DE3) by inducing protein expression, of 1 L Terrific broth cultures, at an OD600 of 1.0 with the addition of IPTG to 0.5 mM. Cell growth was continued overnight at 18 °C. The following day the cells were harvested by centrifugation and resuspended in Buffer A (20 mM Tris, pH 8.0, 300 mM NaCl, 10 % Glycerol) containing 10 mM imidazole, 1 mM EDTA and Complete protease inhibitor and lysed by sonication. The lysate was clarified by centrifugation (15,000 × g, 30 min, 4 °C) and the supernatant was loaded on a Ni-NTA affinity column equilibrated in Buffer A. After extensive washing the bound protein was eluted with Buffer B (Buffer A containing 250 mM imidazole). The target protein was further purified by size exclusion chromatography using a Sephacryl S-100 column (GE Healthcare) equilibrated in Buffer A.
Crystallization and structure determination of EccB1ms
Crystals of EccB1ms were grown using the hanging drop vapor diffusion method by mixing protein at a 1:1 ratio of protein to reservoir solution (14 % PEG 8000, 200 mM NaCl, 100 mM phosphate-citrate pH 4.2). Crystals were cryoprotected by a brief soak in reservoir solution containing 20 % propylene glycol. Data from a single crystal was collected at beamline 24-ID-C at the Advanced Photon Source, Argonne National Laboratory. The data were processed with XDS  and the structure solved by molecular replacement using the program Phaser  and a homology model, prepared with the Phyre2 web server , based on the structure of M. tuberculosis EccB1 (PDB ID 4KK7). The structure was refined with BUSTER .
Expression and purification of cyto-EccD1mt
A construct for expression of the cytoplasmic domain of EccD1mt (residues 21–109) was designed based on predicted ubiquitin-like domain using the HHpred server . The DNA fragment was PCR-amplified from M. tuberculosis H37Rv genomic DNA using primers EccD1_F21_Nco 5′–CACCATGGCCACCACCCGGGTGACGATC and EccD1_R109_SpeEcoR 5′–GGGAATTCACTAGTCATGACACCAGAGTCAGCAGTGAC, and cloned into a modified pET-Duet1 vector, which contains an N-terminal hexahistidine tag and TEV protease cleavage sequence. To create a maltose-binding protein (MBP) fusion construct, the same DNA fragment was cloned into a modified pET-22b(+) vector, which contains an N-terminal hexahistidine tag and TEV protease cleavage sequence followed by MBP sequence. Both cyto-EccD1mt and MBP-cyto-EccD1mt proteins were expressed and purified as described for EccB1mt. 5 mM maltose was included in the size-exclusion buffer during purification of MBP-cyto-EccD1mt variant to obtain ligand-bound MBP .
Crystallization and structure determination of MBP-cyto-EccD1mt and cyto-EccD1mt
Diffraction data collection and refinement statistics
a, b, c (Å)
31.70, 110.63, 110.51
74.41, 74.41, 280.60
46.72, 46.72, 279.02
125.68, 125.68, 124.49
α, β, γ, (°)
90, 90, 90
90, 90, 120
90, 90, 120
90, 90, 120
No. reflections (total/free)
R work/R free
Bond lengths (Å)
Bond angles (°)
Ramachandran distribution (%)c
Data were collected at the 22-ID beamline at the Advance Photon Source, Argonne National Laboratory, and processed using XDS . The structure of MBP-cyto-EccD1mt was solved by molecular replacement using Phaser  and an MBP structure as a search model (PDB ID 1ANF) . The electron density modification was performed using Parrot , and the model was extended using Buccaneer and Coot. The fragment corresponding to cyto-EccD1mt from the structure of MBP-cyto-EccD1mt was used as a search model to solve the structure of cyto-EccD1mt alone using Phaser. The structures were refined using REFMAC5 and TLS groups defined by the TLSMD server.
Availability of supporting data
The structure factors and atomic coordinates have been deposited in the Protein Data Bank under accession codes 4KK7 (EccB1mt), 5CYU (EccB1ms), 4KV2 (cyto-EccD1mt), and 4KV3 (MBP-cyto-EccD1mt).
type VII secretion system
early secreted antigenic target of 6 kDa
ESX conserved component
maltose binding protein
root mean square deviation.
We thank Maksymilian Chruszcz, University of South Carolina, for assistance and advice on data processing. We thank the staff of the UCLA-DOE Institute Protein Expression Technology Center, supported by the U.S. Department of Energy, Office of Biological and Environmental Research (BER) program under Award Number DE-FC02-02ER63421, and the UCLA Crystallization Core for assistance in protein purification and crystallization screening. Authors thank staff members of beamline 24-ID-C, and Southeast Regional Collaborative Access Team (SER-CAT) at the Advanced Photon Source, Argonne National Laboratory, for assistance during data collection. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. Work performed in the laboratory of D.E. is supported by the Howard Hughes Medical Institute and National Institutes of Health grants 23616-002-06 F3:02, TBSGC P01 (AI068135), and TBSGC P01 (AI095208). Research reported in this publication was partially supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant numbers P20GM103486 and P30GM110787, and by the National Institute of Allergy and Infectious Diseases grant number R01AI119022 to KVK.
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