Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism (original) (raw)
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Purification, crystallization and preliminary X-ray diffraction analysis of an archaeal ABC-ATPase
Acta Crystallographica Section D-biological Crystallography, 2002
In the archaeon Sulfolobus solfataricus glucose uptake is mediated by an ABC transport system. The ABC-ATPase of this transporter (GlcV) has been overproduced in Escherichia coli and puri®ed. Crystals of GlcV suitable for data collection were obtained in the absence of nucleotide by microseeding combined with vapour diffusion from a mixture of PEG polymers and NaCl. Appearing under identical conditions, two crystal forms have been characterized by X-ray diffraction. Both forms diffract to high resolution using synchrotron radiation and both belong to space group P2 1 2 1 2 1 . The related crystal forms A (unit-cell parameters a = 47.0, b = 48.2, c = 182.1 A Ê ) and B (a = 47.0, b = 146.6, c = 178.5 A Ê ) feature one and three GlcV molecules in the asymmetric unit, respectively, with a solvent content of about 50%. Crystals have also been obtained in the presence of sodium iodide. From single-wavelength anomalous diffraction data extending to 2.1 A Ê resolution, an iodide substructure could be resolved.
Molecular cell, 2013
Superfamily ATPases in type IV pili, type 2 secretion, and archaella (formerly archaeal flagella) employ similar sequences for distinct biological processes. Here, we structurally and functionally characterize prototypical superfamily ATPase FlaI in Sulfolobus acidocaldarius, showing FlaI activities in archaeal swimming-organelle assembly and movement. X-ray scattering data of FlaI in solution and crystal structures with and without nucleotide reveal a hexameric crown assembly with key cross-subunit interactions. Rigid building blocks form between N-terminal domains (points) and neighboring subunit C-terminal domains (crown ring). Upon nucleotide binding, these six cross-subunit blocks move with respect to each other and distinctly from secretion and pilus ATPases. Crown interactions and conformations regulate assembly, motility, and force direction via a basic-clamp switching mechanism driving conformational changes between stable, backbone-interconnected moving blocks. Collective structural and mutational results identify in vivo functional components for assembly and motility, phosphate-triggered rearrangements by ATP hydrolysis, and molecular predictors for distinct ATPase superfamily functions.
Biochemical Journal, 2011
Microbial motility frequently depends on flagella or type IV pili. Using recently developed archaeal genetic tools, archaeal flagella and its assembly machinery have been identified. Archaeal flagella are functionally similar to bacterial flagella and their assembly systems are homologous with type IV pili assembly systems of Gram-negative bacteria. Therefore elucidating their biochemistry may result in insights in both archaea and bacteria. FlaI, a critical cytoplasmic component of the archaeal flagella assembly system in Sulfolobus acidocaldarius, is a member of the type II/IV secretion system ATPase superfamily, and is proposed to be bi-functional in driving flagella assembly and movement. In the present study we show that purified FlaI is a Mn 2+dependent ATPase that binds MANT-ATP [2′-/3′-O-(N'-methylanthraniloyl)adenosine-5′-Otriphosphate] with a high affinity and hydrolyses ATP in a co-operative manner. FlaI has an optimum pH and temperature of 6.5 and 75 °C for ATP hydrolysis. Remarkably, archaeal, but not bacterial, lipids stimulated the ATPase activity of FlaI 3-4-fold. Analytical gel filtration indicated that FlaI undergoes nucleotide-dependent oligomerization. Furthermore, SAXS (small-angle X-ray scattering) analysis revealed an ATP-dependent hexamerization of FlaI in solution. The results of the present study report the first detailed biochemical analyses of the motor protein of an archaeal flagellum.
Structural analysis of a prototypical ATPase from the type III secretion system
Nature Structural & Molecular Biology, 2007
The type III secretion system (T3SS) ATPase is the conserved and essential inner-membrane component involved in the initial stages of selective secretion of specialized T3SS virulence effector proteins from the bacterial cytoplasm through to the infected host cell, a process crucial to subsequent pathogenicity. Here we present the 1.8-Å -resolution crystal structure of the catalytic domain of the prototypical T3SS ATPase EscN from enteropathogenic Escherichia coli (EPEC). Along with in vitro and in vivo mutational analysis, our data show that the T3SS ATPases share similarity with the F1 ATPases but have important structural and sequence differences that dictate their unique secretory role. We also show that T3SS ATPase activity is dependent on EscN oligomerization and describe the molecular features and possible functional implications of a hexameric ring model.
Structure of the ATP Binding Domain from the Archaeoglobus fulgidus Cu+-ATPase
Journal of Biological Chemistry, 2006
The P-type ATPases translocate cations across membranes using the energy provided by ATP hydrolysis. CopA from Archaeoglobus fulgidus is a hyperthermophilic ATPase responsible for the cellular export of Cu ؉ and is a member of the heavy metal P 1B-type ATPase subfamily, which includes the related Wilson and Menkes diseases proteins. The Cu ؉-ATPases are distinct from their P-type counterparts in ion binding sequences, membrane topology, and the presence of cytoplasmic metal binding domains, suggesting that they employ alternate forms of regulation and novel mechanisms of ion transport. To gain insight into Cu ؉-ATPase function, the structure of the CopA ATP binding domain (ATPBD) was determined to 2.3 Å resolution. Similar to other P-type ATPases, the ATPBD includes nucleotide binding (N-domain) and phosphorylation (P-domain) domains. The ATPBD adopts a closed conformation similar to the nucleotide-bound forms of the Ca 2؉-ATPase. The CopA ATPBD is much smaller and more compact, however, revealing the minimal elements required for ATP binding, hydrolysis, and enzyme phosphorylation. Structural comparisons to the AMP-PMP-bound form of the Escherichia coli K ؉-transporting Kdp-ATPase and to the Wilson disease protein N-domain indicate that the five conserved N-domain residues found in P 1B-type ATPases, but not in the other families, most likely participate in ATP binding. By contrast, the P-domain includes several residues conserved among all P-type ATPases. Finally, the CopA ATPBD structure provides a basis for understanding the likely structural and functional effects of various mutations that lead to Wilson and Menkes diseases.
PeerJ, 2018
The archaellum, the rotating motility structure of archaea, is best studied in the crenarchaeonSulfolobus acidocaldarius. To better understand how assembly and rotation of this structure is driven, two ATP-binding proteins, FlaI and FlaH of the motor complex of the archaellum of the euryarchaeonPyrococcus furiosus, were overexpressed, purified and studied. Contrary to the FlaI ATPase ofS. acidocaldarius, which only forms a hexamer after binding of nucleotides, FlaI ofP. furiosusformed a hexamer in a nucleotide independent manner. In this hexamer only 2 of the ATP binding sites were available for binding of the fluorescent ATP-analog MANT-ATP, suggesting a twofold symmetry in the hexamer.P. furiosusFlaI showed a 250-fold higher ATPase activity thanS. acidocaldariusFlaI. Interaction studies between the isolated N- and C-terminal domains of FlaI showed interactions between the N- and C-terminal domains and strong interactions between the N-terminal domains not previously observed for A...
VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion
The coupling of ATP binding/hydrolysis to macromolecular secretion systems is crucial to the pathogenicity of Gram-negative bacteria. We reported previously the structure of the ADP-bound form of the hexameric traffic VirB11 ATPase of the Helicobacter pylori type IV secretion system (named HP0525), and proposed that it functions as a gating molecule at the inner membrane, cycling through closed and open forms regulated by ATP binding/ hydrolysis. Here, we combine crystal structures with analytical ultracentrifugation experiments to show that VirB11 ATPases indeed function as dynamic hexameric assemblies. In the absence of nucleotide, the N-terminal domains exhibit a collection of rigid-body conformations. Nucleotide binding `locks' the hexamer into a symmetric and compact structure. We propose that VirB11s use the mechanical leverage generated by such nucleotide-dependent conformational changes to facilitate the export of substrates or the assembly of the type IV secretion apparatus. Biochemical characterization of mutant forms of HP0525 coupled with electron microscopy and in vivo assays support such hypothesis, and establish the relevance of VirB11s ATPases as drug targets against pathogenic bacteria.
Crystal structure of the ϵ subunit of the proton-translocating ATP synthase from Escherichia coli
Structure, 1997
Background: Proton-translocating ATP synthases convert the energy generated from photosynthesis or respiration into ATP. These enzymes, termed F 0 F 1-ATPases, are structurally highly conserved. In Escherichia coli, F 0 F 1-ATPase consists of a membrane portion, F 0 , made up of three different polypeptides (a, b and c) and an F 1 portion comprising five different polypeptides in the stoichiometry α 3 β 3 γδε. The minor subunits γ, δ and ε are required for the coupling of proton translocation with ATP synthesis; the ε subunit is in close contact with the α, β, γ and c subunits. The structure of the ε subunit provides clues to its essential role in this complex enzyme. Results: The structure of the E. coli F 0 F 1-ATPase ε subunit has been solved at 2.3 Å resolution by multiple isomorphous replacement. The structure, comprising residues 2-136 of the polypeptide chain and 14 water molecules, refined to an R value of 0.214 (R free = 0.288). The molecule has a novel fold with two domains. The N-terminal domain is a β sandwich with two fivestranded sheets. The C-terminal domain is formed from two α helices arranged in an antiparallel coiled-coil. A series of alanine residues from each helix form the central contacting residues in the helical domain and can be described as an 'alanine zipper'. There is an extensive hydrophobic contact region between the two domains providing a stable interface. The individual domains of the crystal structure closely resemble the structures determined in solution by NMR spectroscopy. Conclusions: Sequence alignments of a number of ε subunits from diverse sources suggest that the C-terminal domain, which is absent in some species, is not essential for function. In the crystal the N-terminal domains of two ε subunits make a close hydrophobic interaction across a crystallographic twofold axis. This region has previously been proposed as the contact surface between the ε and γ subunits in the complete F 1-ATPase complex. In the crystal structure, we observe what is apparently a stable interface between the two domains of the ε subunit, consistent with the fact that the crystal and solution structures are quite similar despite close crystal packing. This suggests that a gross conformational change in the ε subunit, to transmit the effect of proton translocation to the catalytic domain, is unlikely, but cannot be ruled out.
Journal of Biological Chemistry, 2012
Background: FlaX is essential for archaella assembly in Sulfolobus acidocaldarius and its stability is dependent on FlaI-J. Results: FlaX assembles into ring like oligomers and interacts with the motor ATPase FlaI. Conclusion: FlaX oligomers depend on the presence of its C-terminal domain, which also interacts with FlaI. Significance: The first structural analysis of an archaellum subunit, a rotating type IV pilus structure. Archaella are the archaeal motility structure, which are structurally similar to Gram-negative bacterial type IV pili but functionally resemble bacterial flagella. Structural and biochemical data of archaellum subunits are missing. FlaX, a conserved subunit in crenarchaeal archaella, formed high molecular weight complexes that adapted a ring-like structure with an approximate diameter of 30 nm. The C terminus of FlaX was not only involved in the oligomerization, but also essential for FlaX interaction with FlaI, the bifunctional ATPase that is involved in assembly and rotation of the archaellum. This study gives first insights in the assembly apparatus of archaella.
2020
in-line with multi-angle laser light scattering, TMH: transmembrane helix, VHH: the variable part of the heavy chain only camelid antibody ABSTACT The type II secretion system (T2SS) is a multiprotein machinery spanning the diderm of gram-negative bacteria. T2SS contributes to the virulence of numerous gram-negative pathogens, including the multidrug resistant species Pseudomonas aeruginosa, Acinetobacter baumanii, Klebsiella pneumonia and Vibrio cholerae. Even though the T2SS has been studied extensively over the past three decades, our understanding of the molecular basis of its biogenesis and of its overall structure still remains unclear. Here we show that the core component of the inner membrane platform, the GspLM membrane protein complex, can be isolated as a dimer of dimers. Importantly, the complex is able to bind the T2SS ATPase, GspE, with high affinity. Finally, we have developed single domain VHH camelid antibodies (nanobodies) against the GspLM complex and have identified a nanobody that effectively prevents the cytoplasmic domain of GspL, GspL cyto , from binding to GspE. Our findings suggest that the T2SS ATPase is permanently associated with the inner membrane platform and that the GspELM complex should be considered as a key subassembly for the biogenesis of the T2SS apparatus.