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Keywords:

  • Rv3868;
  • tetratricopeptide repeat;
  • TPR domain;
  • AAA+ ATPase;
  • type VII secretion system

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
  8. Supporting Information

EccA1 is an important component of the type VII secretion system (T7SS) that is responsible for transport of virulence factors in pathogenic mycobacteria. EccA1 has an N-terminal domain of unknown function and a C-terminal AAA+ (ATPases associated with various cellular activities) domain. Here we report the crystal structure of the N-terminal domain of EccA1 from Mycobacterium tuberculosis, which shows an arrangement of six tetratricopeptide repeats that may mediate interactions of EccA1 with secreted substrates. Furthermore, the size and shape of the N-terminal domain suggest its orientation in the context of a hexamer model of full-length EccA1. Proteins 2014; 82:159–163. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
  8. Supporting Information

Mycobacterium tuberculosis employs an army of secreted proteins to subvert the immune system during infection. To transport these virulence factors across an unusual two-membrane cell envelope mycobacteria use the specialized type VII secretion system (T7SS).[1] The T7SS machinery is an ∼1500 kDa complex composed of a membrane pore and associated proteins including membrane-associated and cytosolic ATPases that are thought to provide the energy for transporting protein cargoes across the membrane. In the genome of M. tuberculosis there are five homologous T7SS clusters named ESX-1 to ESX-5. The most intensely studied region, ESX-1, includes the Rv3868 gene encoding the AAA+ ATPase EccA1. Like other AAA+ ATPases EccA1 forms oligomers, possibly hexamers, and hydrolyzes ATP in vitro.[2] Furthermore, its ATPase activity promotes virulence in vivo through increased mycolic acid synthesis.[3] In addition EccA1 has been shown to be essential in vivo for specific targeting and secretion of EspC and other cosecreted virulence factors such as ESAT-6/CFP-10 through the ESX-1 system.[4]

Sequence analysis of EccA1 indicates that it contains a C-terminal ATPase domain and a tetratricopeptide repeat (TPR) containing N-terminal domain of unknown function [Fig. 1(A)]. However, there is not yet any structure of an AAA+ ATPase containing a TPR domain, nor is there any reported structure with significant primary sequence identity with the EccA1 N-terminal domain. To gain insight into the function of EccA1 and related T7SS ATPases, we solved the structure of the N-terminal TPR domain of EccA1 from M. tuberculosis.

image

Figure 1. EccA1 N-terminal domain structure. (A) A schematic diagram of EccA1's TPR repeats, β finger insert, and C-terminal AAA+ ATPase domain. TPR motifs 1–6 are colored in rainbow colors from red to blue, whereas residues 237–273 are colored purple to highlight a capping C-terminal α helix or a possible TPR motif that was truncated in this construct. (B) Cartoon representation of EccA1 N-terminal structure. TPR repeat motifs are colored as in (A). The β finger insert is highlighted in gray. (C) Structural conservation of the TPR superhelix motif between EccA1 (alternating red / orange) and PilF (green). The β finger insert is highlighted in gray.

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METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
  8. Supporting Information

Expression, purification, and crystallization

The gene fragment corresponding to the N-terminal domain of EccA1, residues 1–280, was PCR amplified from genomic DNA of M. tuberculosis H37Rv and cloned into a modified pET-28b vector (EMD Millipore) to encode an N-terminal His6-tag followed by a tobacco etch virus (TEV) protease cleavage site. The resulting vector was transformed into Rosetta (DE3) cells (EMD Millipore) for expression. The cells were grown in Luria broth at 37°C to OD600 0.6 and induced for 4 h with 0.5 mM isopropyl-1-thio-β-d-galactopyranoside at 25°C. Cells were harvested by centrifugation and resuspended in buffer containing 20 mM Tris-HCl pH 8.4, 300 mM NaCl, and 20 mM imidazole. The resuspended cells were lysed using an EmulsiFlex-C5 microfluidizer (Avestin) and EccA1 was purified from the soluble fraction of the lysed cells using Ni-nitrilotriacetic acid agarose (Qiagen) column followed by His6-tag cleavage with TEV protease. EccA1 was further purified by size-exclusion using a Superdex200 column (GE Healthcare) in buffer containing 10 mM Tris-HCl pH 8.4, 200 mM NaCl. Crystallization screens were performed using the vapor diffusion method in a hanging drop 96-well plate format with JCSG Core Suites I–IV (Qiagen). The optimized EccA1 crystals were grown in sitting drop format using 0.1M sodium citrate pH 5.6, 1.0M lithium sulfate, 0.5M ammonium sulfate.

Data collection and structure determination

Crystals were soaked in crystallization solution supplemented with 20% glycerol and flash cooled in liquid nitrogen. To obtain heavy atom derivatives, the crystals were soaked for brief time periods, 30 s to 2 min, in cryo-protectant solution supplemented with compounds from Heavy Atom Screens (Hampton Research).[5] Data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Data were processed and scaled using XDS and XSCALE.[6]

The structure of EccA1 was determined by the single wavelength anomalous diffraction (SAD) method using data from a single crystal soaked in the presence of KAu(CN)2. Initial Au sites were found using SHELXD[7] at resolution 3.3 Å. Additional Au sites were found and refined using SAD protocol in PHASER.[8] Following density modification in Parrot,[9] an initial model was built using Buccaneer.[10] After manual rebuilding in Coot,[11] the improved model was refined using REFMAC5[12] against a “native” dataset at resolution 2.0 Å. The “native” dataset was collected from a crystal soaked in the presence of samarium acetate. A preliminary data analysis showed a lack of strong anomalous signal in this dataset, however, three partially occupied samarium ions were identified by peaks in anomalous difference maps and were included in the final model. The final rounds of refinement were performed applying eight translation, libration and screw-rotation displacement (TLS) groups determined by the TLSMD server.[13] The final model contains two EccA1 monomers (residues 1–273) in the asymmetric unit, three samarium ions, six sulfate ions and 453 water molecules. The structure was validated using Coot and the Molprobity server (http://molprobity.biochem.duke.edu). Coordinates and structure factors have been deposited at the Protein Data Bank with accession code 4F3V.

Sequence alignments were done using ClustalW2 (http://www.clustal.org) and rendered using the ESPript server (http://espript.ibcp.fr). Structural illustrations were prepared using PyMol (http://www.pymol.org).

RESULTS AND DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
  8. Supporting Information

The three dimensional structure of the N-terminal domain (NTD) of M. tuberculosis EccA1, residues 1–280, was determined to 2.0 Å resolution. The protein crystallized in space group P212121 with two molecules in the asymmetric unit (Table 1). Crystallographic phases were experimentally determined by the single wavelength anomalous diffraction method using a potassium dicyanoaurate derivative. The final model includes residues 1–273 of EccA1 in both molecules. The two monomers adopt highly similar structures with an r.m.s.d. of 0.52 Å between the two chains. In the asymmetric unit the two molecules of EccA1 dimerize via a noncrystallographic two-fold axis through surfaces on the edge of TPR motifs 2 and 3. This dimerization interface probably represents a nonphysiological interaction. Analysis using the PISA server[14] shows that the interface between the two chains buries 2200 Å2 of surface area. However, a calculated PISA score of 0.110 for the interface indicates that it probably does not represent a physiological interaction. Furthermore, Arg62 and Arg63 from each chain form bridging interactions mediated by a sulfate ion from crystallization solution. Because the two chains are nearly identical and probably do not dimerize in the context of full-length EccA1 we limit our discussion to a monomer of EccA1.

Table 1. Data Collection and Refinement Statistics
 Native (PDB 4F3V)KAu(CN)2 derivative
  1. a

    Values in parentheses are for the highest-resolution shell.

  2. b

    Calculated using the Molprobity server (http://molprobity.biochem.duke.edu).

Data collection  
Wavelength (Å)1.00001.0000
Space groupP212121P212121
Cell dimensions  
a, b, c (Å)73.23, 92.51, 105.7174.32, 92.58, 105.76
α β γ (°)90, 90, 9090, 90, 90
Resolution (Å)29.6–2.00 (2.11–2.00)a29.6–2.49 (2.63–2.49)
Rsym0.101 (0.782)0.122 (0.508)
II14.2 (2.5)13.9 (4.4)
Completeness (%)99.8 (99.3)99.4 (96.8)
Multiplicity5.7 (5.7)7.3 (7.2)
Anomalous completeness (%) 99.3 (96.1)
Anomalous multiplicity 3.9 (3.8)
Refinement  
Resolution (Å)29.6–2.00 
No. reflections (total/free)49226/2525 
Rwork/Rfree0.174/0.211 
No. atoms  
Protein4135 
Ligand/ion33 
Water453 
B-factors  
Protein26.2 
Ligand/ion56.4 
Water36.3 
Wilson B30.8 
R.m.s. deviations  
Bond lengths (Å)0.011 
Bond angles (°)1.286 
Ramachandran distribution (%)b  
Favored98.5 
Outliers0.0 

The 12 antiparallel α helices of EccA1 arrange into six tandem TPR motifs of ∼34 residues each (Fig. 1). The TPRs associate through hydrophobic interactions between consecutive helices and together form a right-handed superhelix with a pitch of ∼60 Å, and a width of ∼40 Å that is characteristic of TPR domains.[15] A DALI search ranked the Pseudomonas aeruginosa type IV pili system protein PilF as the closest characterized bacterial homolog of EccA1 with an r.m.s.d of 3.5 Å and 9% sequence identity over 173 aligned residues (PDB 2HO1).[16, 17] Despite low sequence similarity between the two proteins, the TPR helices of EccA1 superpose with PilF remarkably well, however, unlike PilF, EccA1 has a β finger insertion after TPR2 that occupies the concave groove of the TPR superhelix (Fig. 1). This is the same groove commonly used for protein-protein interactions by the TPR motif proteins.[18] PilF contains a conserved Asn ladder within its binding groove that is thought to promote peptide binding through formation of bidentate hydrogen bonds to substrate backbone.[16, 19] However, the Asn ladder is missing in EccA1, and in its place are hydrophobic residues that form a hydrophobic core with β finger insert residues. This hydrophobic core suggests that the insert is a permanent feature within the putative binding groove. It may be that the insert participates in protein–protein interactions by forming β strand complementation interactions with the substrates in an extended conformation within the concave groove. Alternatively, proteins may interact with the side of the TPR bundle instead of the canonical binding groove. This latter binding mechanism by a TPR protein is illustrated by the p67phox - Rac complex structure (PDB 1E96), which contains a similar β finger motif in its concave groove.[20]

EccA1 belongs to the CbxX/CfxQ family of ATPases, however, the homology is limited to the AAA+ domain and the N-terminal TPR domain is unique for the EccA family. The nearest homolog of EccA1 with a known structure is Rubisco activase (PDB 3SYL and 3ZUH),[21] which is 46% identical to the C-terminal ATPase domain of EccA1. Similar to Rubisco activase, EccA1 may adopt a hexameric ring stabilized by contacts between the C-terminal domains [Fig. 2(B)].[2] Modeling the architecture of full-length EccA1 using the structure of Rubisco activase (PDB 3ZUH) to align the EccA1 monomers into a hexamer yielded insight into the possible conformations of the N- and C-terminal domains relative to each other. There is no obvious region of the C-terminal ATPase domain that might fit into the large groove of the N-terminal domain structure. This groove probably remains open and available for interactions with other proteins and/or substrates of the T7SS. Also, the length of the N-terminal TPR domain (∼70 Å) is slightly shorter than the radius of the Rubisco activase hexamer. Thus, it is possible that the N-terminal domain lies across the top of the ATPase domain without occluding the central pore. Importantly, this hexameric conformation could be modeled without occluding any of the ATP binding sites indicating that such a configuration is compatible with ATPase activity [Fig. 2(A)].

image

Figure 2. Model of EccA1 hexamer. (A) View looking down the central pore of full length EccA1 modeled using the hexameric model of Rubisco activase (3ZUH). C-terminal AAA+ ATPase domains are colored blue. N-terminal domain TPR motifs are colored alternating red/orange. Bound ADP is shown as spheres. (B) Same as (A) after rotating the view 90°.

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The sequence alignment of EccA1 homologs from the ESX-1 clusters of mycobacteria shows that EccA1 proteins are conserved with at least 74% pairwise sequence identity between family members (Supporting Information Fig. S1). In contrast, the sequence identity ranges between 29 and 39% for EccA proteins from different ESX clusters: ESX-1, ESX-2, ESX-3, and ESX-5. This may indicate that EccA ATPases interact with diverse components of secretion system or substrates. However, the overall protein architecture with an N-terminal TPR domain and a C-terminal AAA+ domain is clearly present in all EccA proteins. The key elements of the AAA+ proteins including Walker A motif, Walker B motif, and pore loop are conserved in EccA1 family members (Supporting Information Fig. S1). In addition, Tyr439 (M. tuberculosis EccA1 numbering) may serve as sensor 1, whereas Arg519 and Arg522 may serve as sensor 2. Arg429 has been suggested as an Arg finger residue and Arg429Ala substitution affected ATP hydrolysis by the C-terminal domain of EccA1.[2] Our hexamer model identified Arg456 as another candidate Arg finger residue. Indeed, Arg456 is located closer than Arg429 to the ATP-binding site of adjacent subunit.

In summary, the EccA1 N-terminal domain adopts a TPR fold indicating it may mediate protein–protein interactions between the C-terminal ATPase domain and substrate proteins. Like other TPR motif proteins, EccA1 may use its central concave groove to interact with protein cargos, but the presence of a β finger fixed within the groove raises questions about the exact nature of the interaction. Oligomerization interactions were not found in the structure indicating that it is the C-terminal domain that primarily mediates EccA1 hexamerization. Finally, the dimensions of the N-terminal TPR domain suggest that along with the C-terminal domain it can form a compact hexamer while maintaining a central pore that can open and close during ATP hydrolysis. What the mechanism may be for specific protein recognition and energy transfer to substrates passing through the T7SS remains to be investigated.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
  8. Supporting Information

The authors thank Carol Beach for expert assistance with mass spectrometry. They thank staff members of Southeast Regional Collaborative Access Team (SER-CAT) at the Advanced Photon Source, Argonne National Laboratory, for assistance during data collection.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
  8. Supporting Information

Supporting Information

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS AND DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
prot24351-sup-0001-suppinfo.pdb752KSupplementary Information
prot24351-sup-0002-suppinfo.mtz4377KSupplementary Information
prot24351-sup-0003-suppinfo.mpg2256KSupplementary Information
prot24351-sup-0004-suppinfo.pdf2994KSupplementary Information
prot24351-sup-0005-suppinfo.pdf67KSupplementary Information
prot24351-sup-0006-suppinfo.pdf8KSupplementary Information

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